1
Protein nanomachines
Synthetic large macromolecular assemblies,
virus-based vehicles and artificial signalling networks
Creating synthetic protein complexes and supramolecular
assemblies for biotechnology, biomedicine and basic research
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
2
Short recapitulation of previous lesson
• What is directed evolution?
• Directed evolution: pros and cons
• How to generate gene diversification and library creation?
• How to screen your gene library?
• What is continuous directed evolution? Examples…
• What is semi-rational protein engineering approach?
3
Recapitulation: directed evolution workflow
https://www.kapabiosystems.com/technology/overview
4
Recapitulation: screening is important part of DE cycle
5
Recapitulation: phage-assisted continuous evolution (PACE)
Packer et al., Nature Communications, 8: 956 (2017)
6
Recapitulation: ribosome display vs. mRNA display
7
What will we talk about
• Introduction to protein nanomachines
• Macromolecular protein assemblies
• Synergy between synthetic biology and structural biology
• Resolution revolution, mutual benefits
• Multi-expression technologies
• Strategies, methodology, success stories
• Synthetic virology
• Construction of synthetic virus-based vehicles, examples
• Immunotherapy and gene therapy
• Concepts and applications
8
Design of large protein nanomachines
Concepts
Methods
Applications
9
The anatomy of protein structure
• Proteins are an important class of biological macromolecules which are
polymers of amino acids
• Biochemists have distinguished several levels of structural organization
of proteins
10
Most proteins work in multi-subunit protein complexes
• Protein function and activity are highly regulated within living cells
• To become functional, proteins commonly depend on their interactions with other
molecules. These molecules are known to include other proteins, which readily interact to
form protein complexes
• The majority of cellular processes are dependent on protein-protein interactions (PPI’s)
• An additional layer of complexity is added by the interaction of different protein complexes
in so called super-complexes or metabolons
• A protein complex or multi-protein complex is a group of two or more associated
polypeptide chains. Different polypeptide chains may have different functions
11
A movie of RNA polymerase II transcription
https://www.youtube.com/watch?v=WlMV_l88Lus
Prof. Patrick Cramer
Director at the Max Planck Institute
for Biophysical Chemistry,
Gottingen, DE
12
The ribosome, a large multi-subunit protein-RNA complex
• The ribosomes are protein synthesizers
of the cell
• Made up of rRNAs and distinct
ribosomal proteins
• Arranged into two pieces:
– Small ribosomal subunit
– Large ribosomal subunit
13
Computational design of large protein assemblies
• Self assembling protein materials play critical roles in biology.
• Proteins have many features that make them attractive as building blocks
for the development of completely new classes of advanced functional
materials.
• There are efforts to develop general approaches for creating new self
assembling nanostructures, and using these approaches to develop a next
generation of synthetic biology solutions (nanoreactors, nanocages,
vaccines and drug delivery vehicles).
Science 2012 336 (6085):1171-4. doi: 10.1126/science.1219364
14
General approach to designing self-assembling
protein nanomaterials
Science 2012 336 (6085):1171-4. doi: 10.1126/science.1219364
(A) First, a target symmetric architecture is chosen. Octahedral point group symmetry is used in this example; the threefold
rotational axes are marked here by triangles and shown as black lines throughout. The dashed cube is shown to orient the
viewer. (B) Multiple copies of the building block are symmetrically arranged in the target architecture by aligning their shared
symmetry axes. The pre-existing organization of the oligomeric building block fixes several (in this case four) rigid body degrees
of freedom (DOFs). (C) Symmetrical docking is performed by systematically varying the two DOFs (moves are applied
symmetrically to all subunits) and computing the suitability of each configuration for interface design (red: more suitable; blue:
less suitable). Points corresponding to the docked configurations in panels (B), in which the building blocks are not in contact,
and (D), a highly complementary interface, are indicated (E, F).
15
Computational design of self-assembling protein
nanomaterials: experimental validation
Science 2012 336 (6085):1171-4. doi: 10.1126/science.1219364
(A) A representative negative stain electron
micrograph of O3-33. Selected particles (boxed in
white) that resemble views of the design model along
its 4-fold, 2-fold, and 3-fold rotational axes, shown in
(B), are enlarged at right. (B) The O3-33 design
model, depicted in ribbon format. Each trimeric
building block is shown in a different color. (C) The
density map from a 20 Å resolution cryo-EM
reconstruction of O3-33 clearly recapitulates the
architecture of the design model. (D) The crystal
structure of O3-33 (R32 crystal form). Images in (B) to
(D) are shown to scale along the three types of
symmetry axes present in point group O. (E) The
designed interface in O3-33, highlighting the close
agreement between the crystal structure (green and
magenta) and the design model (white). Oxygen
atoms are red; nitrogens, blue. Hydrogen bonds
between the building blocks are shown as yellow
dashes, and an octahedral 2-fold rotational axis that
passes through the interface is shown as a gray line.
Residues in which substitution disrupted selfassembly
are labeled.
16
Design of multi-component protein materials: overview
a, The T33 architecture comprises four copies each of two distinct trimeric building blocks (green and blue) arranged with
tetrahedral point group symmetry (24 total subunits; triangles indicate three-fold symmetry axes). b, Each building block has
two rigid body degrees of freedom, one translational (r) and one rotational (ω), that are systematically explored during
docking. c–d, The docking procedure, which is independent of the amino acid sequence of the building blocks, identifies large
interfaces with high densities of contacting residues formed by well-anchored regions of the protein structure. e, Amino acid
sequences are designed at the new interface to stabilize the modeled configuration and drive coassembly of the two
components. f, In the T32 architecture, four trimeric (grey) and six dimeric (orange) building blocks are aligned along the
three-fold and two-fold symmetry axes passing through the vertices and edges of a tetrahedron, respectively.
Nature. 2014 Jun 5; 510(7503): 103–108
17
Structures of designed two-component protein nanomaterials
Nature.2014Jun5;510(7503):103–108
18
Accurate design of megadalton-scale two-component
icosahedral protein complexes
Science 2016; 353(6297): 389–394, doi: 10.1126/science.aaf8818
19
Artificial protein dodecahedrons
• The engineered trimeric aldolase from Thermotoga maritima with a modified interface was fused with an industrial biocatalyst
(+)-γ-lactamase from Microbacterium hydrocarbonoxydans and the hybridized protein could be self-assembled into an
organelle-like nanodevice.
• The constructed nanoreactor is readily used for enzymatic resolution of Vince lactam, an important intermediate for synthesis
of carbocyclic nucleoside medicines. Notably, the designed nanoreactors could confer a significant benefit to the biocatalyst
cargo. The encapsulated (+)-γ-lactamase exhibits significantly improved stabilities with respect to heat, organic solvent, and
protease degradation. Moreover, it shows better substrate tolerance than the free enzyme.
• This research demonstrates that bio-designed artificial protein nanocages are an effective way to improve the stability and
strength of biocatalysts and might have broader applications in sustainable catalysis and synthetic biology.
20
Synthetic large protein nanocages to improve drug delivery
Computational models of the 10 successful designs are shown via molecular surface representations
(design names are shown above each model). Each design comprises a pairwise combination of
pentameric (grey), trimeric (blue), or dimeric (orange) building blocks aligned along icosahedral fivefold,
threefold, and twofold symmetry axes, respectively. All models are shown to scale relative to the 30
nanometer scale bar.
21
Encapsulins as nanocontainers for nanoreactor design
• Encapsulins are a new family of microbial proteinaceous compartments that have been engineered for
nanoreactor construction. Typically, encapsulin has an overall size of about 20–40 nm (diameter), which is
very similar to virus
• In fact, encapsulins are naturally occurring nanoreactors that encapsulate specific cargo proteins and are
involved in diverse cell processes including iron mineralization, oxidative and nitrosative stress resistance,
and anaerobic ammonium oxidation
• This example demonstrates that encapsulin compartments could be selected as a general platform for
organelle construction in eukaryotes and has potential for wide application in synthetic biology
22
Membrane protein oligomers by design
Design and characterization of proteins with four transmembrane helices
Science 2018: 359(6379):1042-1046, doi: 10.1126/science.aaq1739
A and B) From left to right, designs and data for
TMHC2 (transmembrane hairpin C2), TMHC2_E
(elongated), TMHC2_L (long span), and TMHC2_S
(short span). (A) Design models with intra- and
extramembrane regions with different lengths.
Horizontal lines demarcate the hydrophobic
membrane regions. Ribbon diagrams are at left,
electrostatic surfaces are at right, and the neutral
transmembrane regions are in gray. (B) Confocal
microscopy images for HEK293T cells transfected
with TMHC2 fused to mTagBFP, TMHC2_E fused to
mTagBFP, TMHC2_L fused to mCherry, and
TMHC2_S fused to enhanced green fluorescent
protein. Line scans (yellow lines) across the
membranes show substantial increase in
fluorescence across the plasma membranes for
TMHC2, TMHC2_E, and TMHC2_L, but less
substantial increase for TMHC2_S. (C)
Representative AUC sedimentation-equilibrium
curves at three different rotor speeds. Each data set
is globally well fit as a single ideal species in solution
corresponding to the dimer molecular weight. “MW
(D)” and “MW (E)” indicate the molecular weight of
the oligomer design and that determined from
experiment, respectively. (D) CD spectra and (inset)
temperature melt. No apparent unfolding transitions
are observed up to 95°C.
23
Membrane protein oligomers by design
Characterization of designs with six and eight membrane-spanning helices
Science 2018: 359(6379):1042-1046, doi: 10.1126/science.aaq1739
(A) Model of designed transmembrane trimer
TMHC3 with six transmembrane helices. Stick
representation from periplasmic side (left) and lateral
surface view (right) are shown. (B) CD
characterization of TMHC3. The design is stable up
to 95°C. (C) Representative AUC sedimentationequilibrium
curves at three different rotor speeds for
TMHC3. The data fit to a single ideal species in
solution with molecular weight close to that of the
designed trimer. (D) Model of designed
transmembrane tetramer TMHC4_R with eight
transmembrane helices. The four protomers are
colored green, yellow, magenta, and blue,
respectively. (E) AUC sedimentation-equilibrium
curves at three different rotor speeds for TMHC4_R
fit well to a single species, with a measured
molecular weight of ~94 kDa. (F) Crystal structure of
TMHC4_R. The overall tetramer structure is very
similar to the design model, with a helical bundle
body and helical repeat fins. The outer helices of the
transmembrane hairpins tilt off the axis by ~10°. (G)
Cross section through the TMHC4_R crystal
structure and electrostatic surface. The HRD forms a
bowl at the base of the overall structure with a depth
of ~20 Å. The transmembrane region is indicated in
lines. (H) Three views of the backbone superposition
of TMHC4_R crystal structure and design model.
24
Design of protein nanomachines is
enabled by advances in structural biology
Concepts
Methods
Applications
25
Structural biology of large macromolecular assemblies
• Determining 3D structure of proteins help protein engineers to understand
molecular mechanism of protein action and its biological function.
Structural biology techniques
• X-ray crystallography
Crystallization required, no size limits,
challenging for highly flexible proteins
• Nuclear magnetic resonance (NMR) spectroscopy
Labelling required, suitable for smaller proteins, capturing protein
motions
• Cryo-electron microscopy
Automation, direct electron detectors, image processing
suitable for large protein complexes
26
Macromolecular crystallography
Prerequisite for successful protein crystallization
• Large quantities (few mgs)
• High purity
• Homogeneity
• Stability
27
Crystal freezing, data collection, processing & model building
28
Examples of macromolecular X-ray structures
RNA polymerase
Ribosome
Membrane
protein
29
Engineered fusion proteins help protein crystallographers
Hai & Christianson, Nat. Chem. Biol.
12:741-747 (2016)
Fusion tags used:
• Thioredoxin (Thx)
• Maltose-binding protein (MBP)
• Glutathione S-transferase (GST)
• Small ubiquitin-like modifier (SUMO)
• Polyhistidenes (6xHis, 12xHis)
30
Cryo-electron microscopy: overview
• Cryo-electron microscopy (cryo-EM) is an electron
microscopy (EM) technique applied on samples cooled
to cryogenic temperatures and embedded in an
environment of vitreous water. An aqueous sample
solution is applied to a grid-mesh and plunge-frozen in
liquid ethane. While development of the technique
began in the 1970s, recent advances in detector
technology and software algorithms have allowed for the
determination of biomolecular structures at near-atomic
resolution.
31
Recent major technical advances in cryo-EM
32
Cryo-EM structures in PDB database
• The number of structures determined by cryo-EM is continuing to grow rapidly
• Computational methods are helping resolve conformational heterogeneity
• Improved biochemical methods are driving advances in cryo-EM of membrane proteins
• Novel approaches for cryo-EM specimen preparation are being explored
33
X-ray crystallography versus cryo-EM
34
The most used cryo-EM techniques
(a) Single-particle cryo-EM: particles are
embedded in a thin layer of amorphous ice.
Resulting representative class averages are
shown as insets on the right. Scale bar, 50 nm. (b)
Single-particle negative-stain EM: particles are
embedded in a layer of heavy metal salts to
increase the weak contrast of biological materials.
Resulting representative class averages are
shown as insets on the right. Scale bar, 50 nm. (c)
Micro-ED: small 3-D crystals are hit with a
focused electron beam and diffraction patterns are
recorded at different tilt angles. Inset shows a
small section of the diffraction image with
individual diffraction spots at higher magnification.
(d) Cryo-ET: the specimen is tilted within the
microscope and images at different angles are
recorded. A tomographic slice shows the cellular
periphery with microtubule bundles (black arrows)
and plasma membrane (green arrows). (e)
Resolution range coverage of various methods in
structural biology. Color code used for the TEMbased
methods corresponds to (a)–(d). Yellow:
single-particle analysis; orange: electron
crystallography/micro-ED; red: electron
tomography
35
Visualizing proteins by cryo-EM
36
Protein sample preparation for cryo-EM
• Cytoplasmic or membrane proteins are
initially expressed in liquid or solid
cultures, and pellets are stored after
harvesting by centrifugation.
• Different physical or chemical cell
disruption methods are utilized for
releasing cytoplasmic proteins into
solution or to obtain cell membrane
extracts.
• Impure cytoplasmic proteins or
solubilized cell membranes containing
the protein of interest are purified by
combination of different fast protein
liquid chromatography (FPLC) methods.
• After protein stability, integrity and
activity is verified by various biophysical
techniques.
• The final sample concentration and
buffer composition are adjusted before
EM grid preparation.
https://doi.org/10.3389/fmolb.2018.00074
37
Membrane protein sample preparation and stabilisation
Protein transmembrane domains are protected by the hydrophobic cell membrane phospholipid acyl chains. Micelles are
spherical vesicles in which the detergent hydrophobic chains face inward and the hydrophilic polar heads face outward.
Bicelles are obtained by a mixture of lipids and short chain detergents. The lipids will interact with the protein to form a lipid
bilayer and the detergent will form the rim of the bicelle. Micelles will form after the solubilization of the membrane protein by
detergents. SMALP (styrene-maleic acid lipid particles) are polymeric nanoparticles that protect the acyl chain of the lipid
bilayer. Nanodiscs are lipid bilayers stabilized by wrapping a belt of amphipathic helix-rich membrane scaffold proteins
(MSPs) around the detergent-solubilized membrane proteins. Amphipol polymers wrap around the hydrophobic patches of
the membrane protein to form a stable complex in solution. Liposomes are artificial spherical lipid membranes where
membrane proteins can assemble.
https://doi.org/10.3389/fmolb.2018.00074
38
Cryo-EM grid preparation
• (A) Examples of a TEM grid with irregular hole size foil
(Lacey) or with defined hole diameter and spacing
(Quantifoil).
• (B) An automated plunge-freezing device is commonly
used for specimen vitrification. Sample is applied with a
pipette at the surface of the cryo-EM grid and sample
excess is removed by blotting with filter paper, followed
by immediate freezing in liquid ethane. The specimen
can be frozen on a grid with (i) or without (ii) a thin
continuous film made of different materials. TEM grids
with different grid mesh, foil and grid support materials
can be used during specimen freezing.
https://doi.org/10.3389/fmolb.2018.00074
39
Breaking cryo-EM resolution barriers
40
Cryo-EM structure of glutamate dehydrogenase
(GDH) at 1.8 Å resolution
(A and B) Projection views of two 3D classes
from cryo-EM analysis of GDH (glutamate
dehydrogenase). In both classes, there is welldefined
density at the core, but it is weakly
defined at the peripheral nucleotide binding
domain (NBD). The two classes display similar
structures in the interior but differ slightly in the
peripheral NBD. The two classes are likely to
be subsets of a continuum of states with
varying orientations for the outer domain
relative to the core.
(C) Ribbon diagrams of the open and closed
structures demonstrating the more extensive
NBD movement associated with substrate
binding and catalytic cleft closure.
(D) A selected region of the cryo-EM map of
the GDH structure, highlighting high-resolution
features such as “holes” in the aromatic rings
of Tyr382, Phe383, and Trp385, water
molecules (shaded yellow), and well-resolved
densities for carbonyl bonds.
https://doi.org/10.1016/j.cell.2016.05.040
41
Density representations for each of the 20 amino acid types from
the 1.8 Å resolution cryo-EM structure of apo-GDH
42
Cryo-EM analysis of isocitrate dehydrogenase (IDH1) in the
absence and presence of the inhibitor ML309
(A) Cryo-EM map of the apo-IDH1 (isocitrate dehydrogenase) dimer, colored by subunit.
(B and C) Selected regions of the IDH1 map demonstrating density for side-chains in an α-helical region (B) and a β sheet
region (C).
(D) Cryo-EM map of IDH1 in complex with ML309 showing density (red) for the inhibitor (inset) close to the dimer interface.
(E) Superposition of the apo- (gray) and ML309-bound (yellow/blue) IDH1 structures shows the outward movement of the
subunits with ML309 binding (density shown in red). The black arrows indicate the direction of the changes in tertiary structure
while the yellow and blue arrows show the overall movements at the level of quaternary structure.
43
Cryo-EM of filamentous proteins
(A) Tobacco mosaic virus is one of the most widespread viruses around the world, being a prime example for plant
pathogens that can have far-reaching consequences for the economy as well as food supply. (B) Microtubules are not only
core components of the cytoskeleton, but are also essential in axonal transport along neurons, constituting the track for
cargo-transporting motor proteins. (C) Tau filaments are neurodegenerative deposits that are found in the brains of AD
patients. (D) The interaction of F-actin and myosin filaments is responsible for muscle contraction. Malfunctions can cause
myopathies.
44Liu et al., PNAS 115: 3362-3367 (2018)
Imaging of small proteins displayed on protein scaffolds
• New electron microscopy (EM) methods are making it possible to
view the structures of large proteins and nucleic acid complexes at
atomic detail, but the methods are difficult to apply to molecules
smaller than approximately 50 kDa.
• This limit can be successfully visualized when it is attached to a
large protein scaffold designed to hold 12 copies of the attached
protein in symmetric and rigidly defined orientations.
45
CEITEC Cryo-Electron Microscopy Core Facility
46
A high-end single particle cryo-EM microscope (3 metres tall)
47
Cryo-EM: take home message
• Cryo-EM can be used to determine structures at native state
• Prepare best sample possible before EM
• Use negative staining: initial screening, homogeneity assessment
• Use the high-tech technologies (microscope, camera & software)
• Be patient! And you will get your high-resolution structure with cryo-EM
48
Structure-based engineering and design of parts
• Until now, structural biology has been
mainly used in synthetic biology
approaches to design new
parts/components and tools
• Many of the characterized components
can be found in the Registry of Standard
Biological Parts (http://partsregistry.org/)
• Of special importance is the recently
developed “SYNZIP protein toolbox”
because it contains a complete biophysical
quantitative description (i.e., affinities) of
synthetic domains
49
Multi-expression technologies
Concepts
Methods
Applications
50
Co-expression of protein complexes in E. coli
• Example of the concatenation of two
vectors of the pET-MCN vector series:
pnEA-His (vector encoding an N-terminal
poly-histidine tag in front of protein A) and
pnCS (vector expressing the native
protein B).
• The pnEA-His (acceptor vector) is
linearized by removing with the restriction
enzymes BglII and SpeI part of its
promoter (T7 promoter and lacO).
• The full promoter of the pnCS (donor
vector) is cut out with the restriction
enzymes BglII and XbaI. After ligation of
the pnCS promoter with the linearized
pnEA-His vector, a new vector is obtained
based on the backbone of the pnEA-His
vector and whose promoter controls the
genes of proteins A and B.
The functional units within cells are often macromolecular complexes rather than single
species. Production of these complexes as assembled homogenous samples is a prerequisite
for their biophysical and structural characterization and hence an understanding of their
function in molecular terms. Co-expression in Escherichia coli can decipher the subunit
composition, assembly, and production of whole protein complexes.
51
ACEMBL system for multiprotein production in E. coli
• ( a ) Donor and acceptor vectors contain
LoxP sequences and a multiple insertion
elements (MIE) for inserting one or
several genes of interest. Acceptors have
regular replicons (BR322). Donors have a
conditional origin of replication derived
from R6Kγ phage. Antibiotic resistance
makers are as follows: Ap ampicillin, Cm
chloramphenicol, Kn kanamycin, and Sp
spectinomycin. Genes of interest are
inserted into acceptor or donor vectors
into the MIEs by SLIC.
• ( b ) Incubation of acceptor and donor
constructs with Cre recombinase results
in all combinations of fusions including
acceptor-donor (AD) and acceptor-donordonor
(ADD) fusions.
• ( c ) The ACEMBL HT pipeline. Genes
are integrated by ligation-independent
methods (SLIC) followed by combinatorial
multi-gene vector generation using CreLoxP
fusion (tandem recombineering,
TR), followed by protein expression and
analysis of purified complex.
52
Production of protein complexes in insect cells
• Insect cells can efficiently express recombinant biologically active proteins and are mostly
used for the development of virus-like particles and vaccines. It has been proven that
insect cells are excellent platforms for the production of recombinant antibodies
• There are mainly three insect expression systems: baculovirus expression vector system
(BEVS), InsectSelect (IS) system and Drosophila expression system (DES)
53
Introduction to baculovirus biology
Slack and Arif (2007)
• Insect-infecting enveloped DNA
viruses
• Baculoviruses are a very diverse
group of viruses with doublestranded,
circular, supercoiled
genomes, with sizes varying from
about 80 to over 180 kb, that encode
between 90 and 180 genes
• The genome is packaged in rodshaped
nucleocapsids that are 230-
385 nm in length and 40–60 nm in
diameter
• In the most well characterized
baculoviruses, the virions are present
as two types, occluded virions (ODV)
and budded virions (BV).
• Although these two types of virions
are similar in their nucleocapsid
structure, they differ in the origin and
composition of their envelopes and
their roles in the virus life cycle
54
Baculovirus morphogenesis: two types of virions
Circular dsDNA genome and associated proteins Circular dsDNA genome and associated proteins
Capsid shell (VP39, etc.) Capsid shell (VP39, etc.)
BV envelope (GP64, F) ODV envelope (P74, ODV-E66, PIFs etc)
Tegument (GP41), occlusion body (Polyhedrin)
Budded virus (BV) Occlusion-derived virus (ODV)
SlackandArif(2007)
55
The baculovirus genome
• Circular, supercoiled dsDNA
genomes, with sizes varying from
about 80 to over 180 kb, that encode
between 90 and 180 genes
• The genome is packaged in rodshaped
nucleocapsids that are 230-
385 nm in length and 40–60 nm in
diameter
56
The MultiBac system
• MultiBac consists of a baculoviral genome
optimized for multigene delivery and protein
complex expression (left).
• The genome is present as a bacterial artificial
chromosome (BAC) in E. coli cells supplying the
Tn7 transposase.
• Expression cassettes are assembled into the
multi-gene expression constructs and inserted
into the MultiBac genome by Tn7 transposition.
• A second entry option into the viral backbone is
provided distal from the Tn7 site, relying on Cre
recombinase catalysed site-specific integration
into a LoxP sequence (circle filled in red).
• Composite MultiBac baculoviral DNA containing
all DNA elements of choice is extracted from E.
coli cultures, followed by transfection into insect
cell cultures to manufacture functional MultiBac
virions.
• These are then used for a wide range of
applications (right), by the infection of insect cell
cultures or transduction of mammalian cells,
tissues and organisms.
Prof. Imre Berger
School of Biochemistry & Bristol Synthetic
Biology Centre, University of Bristol, UK
57
Application of MultiBac system to large complexes
58
Workflow of protein production using BEVS
https://www.intechopen.com/books/new-insights-into-cell-culture-technology/process-optimization-for-recombinant-protein-expression-in-insect-cells
59
The MultiBac system: successful stories
• Chromatin remodeling
enzymes SWR1 (14
subunits) and INO80 (11
subunits)
• The yeast polII DSIF-PAFSpt6
cryo-EM structure
• The CENPN-CENPAnucleosome
complex
• The human CPSF-160–
WDR33–CPSF-30–PAS
RNA quaternary complex
• The E9 polymerase
• The USP18-ISG15
complex
• The Separase-Securin
complex
• The cohesion loader Scc
60
Synthetic virology, immunotherapy
& gene therapy
Concepts
Methods
Applications
61
What are viruses?
• A virus is an infectious particle that reproduces by "commandeering" a host cell and
using its machinery to make progeny viruses
• A virus is made up of a RNA or DNA genome inside a protein shell called a capsid.
Some viruses have an external membrane envelope
• Viruses are very diverse. They come in different shapes and structures, have different
kinds of genomes, and infect different hosts
• Viruses reproduce by infecting their host cells and reprogramming them to become
virus-making "factories"
62
Constructing de novo synthetic viruses for biomedicine
• Synthetic virology aims to reprogram naturally occurring viruses into controllable and
predictable devices
• The field can be divided into two main endeavours:
1) engineering of the virus capsid (immunotherapy, vaccines)
2) engineering of the genetic programs encoded by the viral genome (gene therapy)
Synthetic viruses can be created by
mixing pre-existing viral parts,
resulting in formation of chimeric or
mosaic capsids. Molecular parts, such
as biotin or small molecule drugs, can
be attached to virus capsids to act as
adaptors or to carry out therapeutic
action, respectively. Synthetic parts,
such as man-made polymers and
inorganic nanoparticles, can be
incorporated into viruses to endow
functionalities new to viruses in
general. Genetically encoded peptides
and proteins can be inserted into
viruses, either in rationally chosen
sites or randomly throughout the
capsid, to impart new functions.
Finally, viral properties can be altered
through introduction of point mutations
scattered throughout the capsid or
concentrated in specific capsid
domains.
63
The MultiBac: a factory for synthetic virus-like particles
• A plasmid module comprising expression
cassettes for the capsid-forming influenza
H1N1 M1 protein (colored in grey) and a
fluorescent protein marker, mCherry
(colored in red), was introduced into the
MultiBac baculoviral genome by Cre
recombinase enzyme mediated plasmid
fusion into the LoxP site (circle filled in red,
gradient)
• Co-expression of HA, NA and M1 yields
synthetic influenza virus-like particles
(VLPs) resembling live influenza virus
64
Selected vaccines produced by BEVS
65
Viral capsid-based nanocontainers as nanoreactors
66
Baculovirus-mediated multigene delivery in cells
• HUVEC and REF cells were infected with a MultiPrime
baculovirus encoding EBFP2-Nuc (labelling the nucleus),
mTFP1-FYVE (PI-3-P containing endosomes), tubulinEYFP
(cytoskeleton), Mito-dsRed (mitochondria) and
PLCδ-PH (PI-4,5-P2; plasma membrane)
• Structure of the tested dual promoters. In CMVP10, the
baculoviral very late promoter p10 was inserted
downstream of the CMV promoter. In CMVintP10, the p10
promoter was placed within an intron and is spliced out
from the transcript of the CMV promoter
67
Application of MultiBac in drug discovery
MultiBacMam-BiFC for compound screening
• The MultiBacMam baculovirus is shown (top
left). Genes encoding for vesicular
stomatitis virus glycoprotein (VSV-G) and
mCherry to track virus performance during
manufacturing have been integrated into the
baculoviral backbone, each controlled by
baculoviral late promoters
• MultiBacMam was outfitted in the Tn7 site
with genes encoding for CDK5 and p25,
each fused to complementary fragments of
a split fluorescent protein, which, upon
CDK5-p25 complex formation reconstitute
complete, active fluorophore (bottom left)
• Composite MultiBacMam baculovirus is
produced in insect cells and then used to
transduce mammalian cells with superior
efficacy as compared to plasmid
transfection (top right)
• A selection of chemical compounds
inhibiting the CDK5-p25 PPI is depicted
(bottom right), identified by using our
MultiBac-BiFC cell-based screening assay.
68
The MultiBac system: a perspective
69
Gene therapy comes of age
Dunbar et al.,Science 359: eaan4672 (2018), DOI: 10.1126/science.aan4672
Nearly five decades ago, scientists hypothesized that
genetic modification by exogenous DNA might be an
effective treatment for inherited human diseases. This
“gene therapy” strategy offered the theoretical
advantage that a durable and possibly curative clinical
benefit would be achieved by a single treatment.
Although the journey from concept to clinical
application has been long, gene therapy is now
bringing new treatment options to multiple fields of
medicine.
70
Overview of gene therapy
• Adeno-associated virus (AAV) is a small (25-nm)
virus
Gene therapy is the
insertion of genes into an
individual’s cells to treat
hereditary diseases by
replacing defective
alleles
• Viral vectors are typically used, due to their ability to integrate DNA into the host’s genome
• The process involves removing cells from a patient and using a viral vector to introduce
a functional copy of the defective gene
• When the cells are transplanted back into the patient, they should begin expressing the
missing protein to restore normal health
71
Three essential tools for human gene therapy
• AAV and lentiviral vectors are the basis of several recently approved gene therapies.
• Gene editing technologies (CRISPR/Cas9, TALENs etc.) are in their translational and
clinical infancy but are expected to play an increasing role in the field.
72
AAV biology and vector manufacturing
• Adeno-associated virus (AAV) is a small (25-nm)
virus
• It is composed of a non-enveloped icosahedral
capsid (protein shell) that contains a linear singlestranded
DNA genome of about 4.7 kb
• The AAV genome encodes for several protein
products, namely, four non-structural Rep proteins,
three capsid proteins (VP1–3), and the recently
discovered assembly-activating protein (AAP)
• The AAV genes are required for its biological cycle
and are flanked by two AAV-specific palindromic
inverted terminal repeats (ITRs; 145 bp)
• AAV viruses infect both dividing and non-dividing
cells, and remain latent in the host cell DNA by
integration into specific chromosomic loci (AAVS)
unless a helper virus activates its replication
• AAV viruses naturally infect humans; an exposure
to the wild-type virus occurs at around 1–3 years of
age, and is not associated with any known disease
• Importantly, the timing of human exposure to AAV
viruses determines the host immunological
response to the recombinant AAV vectors
73
Engineering and evolution of AAV vectors by DNA shuffling
Synthetic AAV capsid engineering via DNA
family shuffling and subsequent selection
in cells or in animals.
Example for analysis of a selected AAV
chimera in mouse liver. Clone AAV-DN was
selected on murine hepatoma cells and then
used to produce luciferase-expressing
recombinant vectors. Note that while the
AAV-DN clone gives slightly less overall
expression in the liver (panel I), it is more
specific for this organ since it exhibits
substantially less off-targeting in non-hepatic
tissues (panel II).
74
Baculovirus-mediated production of gene therapy vectors
• In 2002, Prof. Robert Kotin and
colleagues at the US National Heart,
Lung, and Blood Institute first
demonstrated its suitability for AAV
manufacturing
• They infected Sf9 cell lines — derived
from the fall armyworm — with three
different baculoviruses: two containing
essential genes for AAV particle
production (rep and cap), and one
containing the transgene sequence
intended for delivery
• In this manufacturing process, the
baculoviruses play a dual role,
functioning as the ‘helper’ virus normally
required for replication, as well as the
vehicle for AAV genetic material
• In their initial demonstration, Kotin’s
team achieved levels of productivity
comparable with existing AAV
manufacturing approaches — on the
order of 50,000 functional viral particles
per cell
Baculovirus-mediated production of adeno-associated
viral (AAV) vectors as gene therapy vehicles
75
Development of AAV-based gene therapy products
• In 2017, FDA-approval of the first gene therapy product targeting a disease caused
by mutations in a single gene
• This product, LUXTURNA™ (voretigene neparvovec-rzyl; Spark Therapeutics, Inc.,
Philadelphia, PA), delivers a normal copy of the RPE65 gene to retinal cells for the
treatment of RPE65 mutation–associated retinal dystrophy, a blinding disease
• Many additional gene therapy programs targeting both inherited retinal diseases
and other ocular diseases are in development
76
World's most efficient large-scale AAV manufacturing
• Virovek has developed a patented BAC-toAAV
technology that utilizes the baculovirus
expression system to produce AAV vectors in
insect cells under serum-free condition
• The capability to generate over 3e+16vg of
AAV vectors with a single production run,
which is unmatched by any other AAV
production system
77
Synthesis of a functional baculovirus reported in 2017
• So far, most synthetic viruses have been RNA viruses
(<30 kb) and small DNA viruses
• Baculoviruses contain a large circular dsDNA genome
of 80–180 kb and have been used as biocontrol agents
and protein expression vectors
• First synthesis of a baculovirus genome by a
combination of PCR and transformation-associated
recombination in yeast
• The synthetic genome (145,299 bp) comprising the
complete genome of AcMNPV except for the hr4a locus
that was replaced with an 11.5 kb cassette of bacterial
and yeast artificial chromosomal elements and an egfp
gene
• Insect cells were transfected with the synthetic
baculovirus genome and progeny virus was examined
by electron microscopy
• The results showed that the rescued virus had
structural and biological properties comparable to the
parental virus
• This is the largest DNA virus synthesized so far, and its
success will stimulate the fields of other large DNA
viruses such as herpesviruses and poxviruses
https://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00028?journalCode=asbcd6
78
Oncolytic viruses as synthetic platforms for cancer therapy
• Oncolytic viruses (OVs) selectively replicate in and kill cancer cells, and spread within the
tumor, while not harming normal tissue
• In addition to this direct oncolytic activity, OVs are also very effective at inducing immune
responses to themselves and to the infected tumor cells
• OVs encompass a broad diversity of DNA and RNA viruses that are naturally cancerselective
or can be genetically-engineered
79
Cancer therapy: armed oncolytic viruses
Seymour and Fisher
British Journal of Cancer (2016) 114,
357-361 doi:10.1038/bjc.2015.481
Oncolytic viruses can be ‘armed’ to express therapeutic proteins within infected tumour cells, and secrete them into the tumour
microenvironment. This can provide high-level tumour-selective expression of biologics, maximising local activity and minimising
systemic toxicities. If the virus is suitable for systemic delivery, this provides the intriguing concept of localised therapy in
disseminated tumours following intravenous delivery. AntiCTLA4=checkpoint inhibitor antibody; BiTE=Bi-specific T-cell engager,
for modulating activity and tropism of T cells; NIS=sodium iodide symporter (for SPECT imaging of virus activity);
NTR=nitroreductase TNF=tumour necrosis factor.
80
Industrial large-scale production of oncolytic viruses
https://www.fiercebiotech.com/it/synthetic-virus-may-drive-personalized-medicine-into-precision-medicine
Prof. Bruce F. Smith
Auburn University Research Initiative in Cancer
• A collaborative effort involving Auburn University, Gen9 and Autodesk has developed a
synthetic viral genome for bone cancer research and one which may prove revolutionary
in the battle against cancer overall
• The sCAV2 virus, which is the longest functional virus created in oncology research,
targets and destroys selected tumour cells while not impacting healthy cells
• “This could change the way we fight cancer. It is that revolutionary,” states Dr. Bruce
Smith, a professor in the department of pathobiology and director of the Auburn University
Research Initiative in Cancer, in the announcement. “Our concept is taking personalized
medicine to precision medicine. The technology to create a new virus by synthesizing it is
a huge leap, but the ability to then make a customized virus tailored to the specific needs
of each patient will be transformative.”
81
https://andrewhessel.com/
82
Industrial large-scale production of oncolytic viruses
https://www.halix.nl/vectors-and-vaccines/?gclid=EAIaIQobChMIsJvE_6Wc5QIVRrTtCh174QBlEAAYASAAEgKpFPD_BwE
83
Questions
84
Supplementary materials
85
Design of cellular signalling modules
Concepts
Methods
Applications
86
Structural data help to design cellular signalling circuits
• Over last decades, discovery of macromolecular protein structures and complexes has
played a major role in advancing a more systems-oriented view of protein interaction and
signalling networks
• The design of biological systems often employs structural information or structure-based
protein design to successfully implement synthetic signalling circuits or for rewiring
signalling flows
• There are two ways of using structural information and protein design to analyze and
engineer signalling networks:
• One is to modify existing proteins to either eliminate interactions or to change
kinetic or binding constants, with the aim of probing the network behaviour
For example, the structure-based design of mutations with altered binding or kinetic
constants
• The other way is to design and engineer new parts that can be directed and
therefore perturb a signal transduction pathway in a controlled way
For example, structure-based engineering and design of new interchangeable parts
and signalling modules
87
Combining structural biology with synthetic biology to
provide insights into cell signalling
• Structural information provides a
valuable tool in engineering synthetic
signalling devices
• Combined with additional information
from quantitative biochemistry and
proteomics, gene evolution, and
mathematical modelling, this can provide
insights into signalling modules and the
general design principles of cell
signalling
• Altogether, this will improve our
understanding of cell- and tissue-typespecific
signal transduction
• In the future, this knowledge can be the
basis for understanding the molecular
mechanisms of disease by predicting the
effect of disease mutation using
mathematical modelling
88
Flow chart of combining different disciplines to study cellular
design principles and cell-type-specific signalling
• Structural information is a key
component in proposing network
modules, predicting the
localization and effect of disease
mutations, and designing
perturbed protein complexes to
be used in synthetic biology
approaches
89
Synthetic biology approaches to improving immunotherapy
• One of the major drivers of cancer
immunotherapy interest is CAR-T
(CAR stands for Chimeric Antigen
Receptor and T is for T-cells) cell
therapy.
• This is a cell-based therapy in which
T-cells are taken from the patient and
then modified to have specific
receptors that help it recognize and
attack the cancer.
• The chimeric antigen receptor works
by fusing a target-binding domain that
recognizes the cancer and an
activation domain that turns on the Tcell
into attack mode against the
cancer.
• Plenty of these modified cells are then
grown up in the lab and returned to
the patient.
• The first CAR-T treatment was
approved by FDA in 2017 and many
more are in clinical trials.
90
Computational design of self-assembling protein
nanomaterials with atomic level accuracy
• Protein building blocks are docked together symmetrically to identify complementary
packing arrangements, and low-energy protein-protein interfaces are then designed
between the building blocks in order to drive self-assembly.
• Trimeric protein building blocks were used to design a 24-subunit, 13-nm diameter
complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with
tetrahedral symmetry.
• The designed proteins assembled to the desired oligomeric states in solution, and the
crystal structures of the complexes revealed that the resulting materials closely match the
design models.
• The method can be used to design a wide variety of self-assembling protein
nanomaterials.
Science 2012 336 (6085):1171-4. doi: 10.1126/science.1219364
91
The replication cycle of baculovirus
• Baculovirus is a member of the insect-borne virus family and was originally used as a biopesticide with a
large double-stranded circular DNA genome. The virus is enveloped, rod-shaped particles, ranging from 30
to 60 nm in diameter and from 250 to 300 nm in length.
• The natural hosts for these viruses are insects, and Autographa californica (AcMNPV ) is the most studied
member of the family, which was developed into a recombinant baculovirus vector and is still under
application today.
• AcMNPV and other baculoviruses are able to generate ‘‘polyhedra’’ or ‘‘occlusion bodies’’ in the infected
cells of insects. In the late phase of infection, dozens of polyhedra are formed in the host cell nuclei, which
accommodate progeny virions encased by a protective paracrystalline array comprised of virus-encoded
protein-polyhedrin.
92
In 1959 Richard Feynman delivered what many consider the first lecture on
nanotechnology. This lecture, presented to the American Physical Society at the
California Institute of Technology, prompted intense discussion about the
possibilities, or impossibilities, of manipulating materials at the molecular level.
Although at the time of his presentation, the manipulation of single molecules and
single atoms seemed improbable, if not impossible, Feynman challenged his
audience to consider a new field of physics, one in which individual molecules and
atoms would be manipulated and controlled at the molecular level (Feynman 1960).
As an example of highly successful machines at the “small scale,” Feynman
prompted his audience to consider the inherent properties of biological cells. He
colorfully noted that although cells are “very tiny,” they are “very active, they
manufacture various substances, they walk around, they wiggle, and they do all
kinds of wonderful things on a very small scale” (Feynman 1960). Of course, many
of these “wonderful things” that he was referring to are a result of the activities of
proteins and protein complexes within each cell.
The field of nanotechnology has indeed emerged and blossomed since Feynman's
1959 lecture, and scientists from many disciplines are now taking a careful look at
the protein “machines” that power biological cells (Drexler 1986). These “machines”
are inherently nanoscale, ranging in width from a few nanometers (nm) to over 20
nm, and have been carefully refined by millions of years of evolution.
93
Examples of biomedically relevant cryo-EM structures
(A) Cryo-EM structure of the trimeric envelope glycoprotein of HIV. It was solved in complex with two neutralizing antibody
Fab fragments. [EMD-3308]. (B) Cryo-EM structure of the anthrax protective antigen pore from Bacillus anthracis. [EMD-
6224]. (C) Cryo-EM structure of TcdA1 from Photorhabdus luminescens in its prepore state. [EMD-3645]. (D) Cryo-EM
structure of the ryanodine receptor RyR1. Map at low threshold (transparent) is shown to visualize the nanodisc (blue
arrow), which stabilizes the transmembrane helices of RyR1. [EMD-2751]. Individual subunits are depicted in various
colors. The hetero-trimeric HIV envelope glycoprotein in A can be divided into the gp120 trimer (yellow, orange, and red)
and gp41 trimer (pink, blue, and cyan). Bound Fab fragments (green) are indicated by black arrows. Heptameric anthrax
protective antigen pore (yellow, orange, light red, dark red, magenta, cyan, and green) in (B), pentameric TcdA1 (yellow,
orange, red, green and blue) in (C), and tetrameric RyR1 (yellow, orange, red, and green) in D.
94
Baculovirus biology
• Extracellular enveloped baculovirus virions
can be found in two forms: OV (occluded
virus) and BV (budded virus). The
nucleocapsid is about 21 nm x 260 nm.
• Circular dsDNA genome , 80-180 kb in
length, encoding for 100 to 180 proteins.
BACULOVIRUS INFECTION CYCLE
• Attachment of the viral glycoproteins to host receptors mediates endocytosis of the virus into the host
cell.
• Fusion with the plasma membrane.
• The DNA genome is released into the host nucleus.
• Immediate early phase: host RNA polymerase transcribes viral genes involved in the regulation of the
replication cascade, prevention of host responses and viral DNA synthesis.
• Late phase: The virally encoded RNA polymerase expresses late genes.
• Replication of the genome by rolling circle in nuclear viral factories.
• Nucleocapsids are formed which can either bud out through the cellular membrane and disseminate the
infection or be occluded for horizontal transmission.
• Occlusion phase: the virus becomes occluded in the protein polyhedrin and the polyhedral envelope
(calyx) is produced.
• Lysis of the cell releases the occluded virus.
95
Engineering and evolution of AAV vectors by DNA shuffling
• Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for
therapeutic human gene transfer due to a unique combination of beneficial properties.
• These include the apathogenicity of the underlying wildtype viruses and the highly advanced
methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors.
• A further particular advantage of the AAV system over other viruses is the availability of a wealth of
naturally occurring serotypes which differ in essential properties yet can all be easily engineered as
vectors using a common protocol.
• Development of strategies to use these natural viruses as templates for the creation of synthetic vectors
which either combine the assets of multiple input serotypes, or which enhance the properties of a single
isolate.
• The respective technologies to achieve these goals are either DNA family shuffling, i.e. fragmentation of
various AAV capsid genes followed by their re-assembly based on partial homologies (typically >80% for
most AAV serotypes), or peptide display, i.e. insertion of usually seven amino acids into an exposed loop
of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type.
• For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are
up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of
a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive
peptide within the same viral backbone (peptide display approach).
• The subsequent final step is iterative selection of such a library on target cells in order to enrich for
individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably
combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for
instance elimination of all capsids reacting with anti-AAV antibodies.
• This combination increases chances that synthetic capsids surviving the selection match the needs of the
given application in a manner that would probably not have been found in any naturally occurring AAV
isolate.
96
The MultiBac: a factory for synthetic virus-like particles
• A plasmid module comprising expression
cassettes for the capsid-forming influenza
H1N1 M1 protein (colored in grey) and a
fluorescent protein marker, mCherry
(colored in red), was introduced into the
MultiBac baculoviral genome by Cre
recombinase enzyme mediated plasmid
fusion into the LoxP site (circle filled in red,
gradient)
• Co-expression of HA, NA and M1 yields
synthetic influenza virus-like particles
(VLPs) resembling live influenza virus
97
Design of multi-component materials
The computational design models (top) and X-ray
crystal structures (bottom) are shown at left for a,
T32-28, b, T33-15, c, T33-21, and d, T33-28. Views
of each material are shown to scale along the 2-fold
and 3-fold tetrahedral symmetry axes (scale bar: 15
nm). The r.m.s.d. values given are those between the
backbone atoms in all 24 chains of the design models
and crystal structures. For T33-21, r.m.s.d. values are
shown for both crystal forms (images are shown for
the higher resolution crystal form with backbone
r.m.s.d. 2.0 Å), while the r.m.s.d. range for T33-28
derives from the four copies of the fully assembled
material in the crystallographic asymmetric unit. At
right, overlays of the designed interfaces in the
design models (white) and crystal structures (grey,
orange, green, and blue) are shown. Due to the
limited resolution of the T32-28 structure, the amino
acid side chains were not modeled beyond the beta
carbon. For the interface overlays, the crystal
structures were aligned to the design models using
the backbone atoms of two subunits, one of each
component.
98
Genetic circuits for signal processing
Genetic circuits are commonly built by synthetic biologists to enable cells to take
some input and compute what to do. Beyond a cell being activated by a single
input, cells could be engineered to respond to multiple signals and to make
decisions based on both its neighbouring cells and environment.
One published example of mammalian genetic circuits is ‘Boolean logic and
arithmetic through DNA excision’ (BLADE) and it was able to build 113 different
circuits in embryonic kidney and Jurkat T cells. The BLADE system
uses recombinases to create logic gates but can also interface with CRISPR–
Cas9 to regulate host cell genes. Large circuits like the ones demonstrated with
BLADE could be combined with synthetic receptors so cells can make
decisions based on what combination of signals they see.
99
Receptor engineering
New types of receptors can be engineered to detect different types of molecules
and turn on some cellular output function. The more receptors that can be
readily engineered the more molecules that can be used as inputs to a
therapeutic cell. It has been shown that synthetic notch receptors can be made
for use with different recognition domains as well as different transcription
factors as activation domains. That means that a researcher can decide both
what the receptor responds to and what it turns on after it binds its target.
Typical CAR-Ts only change the input of the receptor, but this kind of receptor
can swap out either the input or output or both. Having several independent
receptors would allow much more complex programming of therapeutic cells to
respond to signals within the human body.
100
Safety kill switch
Since toxicity can be an issue, it would be good to have an off switch in the event
of a problem. One form of toxicity that can happen with CAR-T is called
“Cytokine Release Syndrome” or “Cytokine Storm” for especially severe cases.
This can happen when the immune system has such a strong response that
many inflammatory cytokines are released triggering mild to severe symptoms
including fever, headache, rash, rapid heartbeat, low blood pressure, and
trouble breathing.
Synthetic biologists have already worked on a number of “kill switches” in
different organisms. A kill switch could be applied to CAR-T by having a druginducible
kill-switch in the the T-cells that would give the medical team a way to
kill off the modified cells as soon as a bad side effect starts to show up.
Researchers and companies are also seeking multiple ways to either switch off
or kill the T-cells when they’re not needed. These variations on a kill switch can
help to make sure CAR-T cell therapy has extra safety measures in place.
There are many types of cancer and many ways in which a cancer can avoid
successful treatment. Hopefully mammalian synthetic biology can add
something to the immunotherapy toolkit by improving CAR-T or other
approaches to enlist the immune against cancer.
101
(a) Recombinases can perform
simple BUF logic
operations, either by
tyrosine-recombinasemediated
excision (left) or
serine-integrase-mediated
inversion (right).
(b) Recombinases are tested
for their recombination
efficiency and orthogonality
on all BUF logic reporters.
(c) A 6-input AND-gate that
produces GFP when all
inputs are present. MFI,
mean fluorescence intensity
from n = 3 transfected cell
cultures; a.u., arbitrary units.
Error bars, s.e.m.
Implementation of multi-input gates in mammalian cells
Weinberg et al., Nature Biotechnology 35: 453-462 (2017)
102
(a)2-input BLADE template on one plasmid with a single
transcriptional unit. This template contains four distinct regions of
DNA (addresses) downstream of a promoter. Each address
corresponds to an output function and is accessed or deleted via
site-specific DNA recombination. Each address can be
programmed from different configurations ranging from zeroinputs
to Boolean functions. The first address (Z00), which is the
closest to the promoter, corresponds to a state where no
recombinase is expressed (A = 0, B = 0). If the Z00 address
contains a protein coding sequence, then that gene will be
expressed. Gene expression from the other addresses
downstream of Z00 will be blocked by the presence of the Z00
protein coding region. In the presence of recombinase A, which
corresponds to state (A = 1, B = 0), addresses Z00 and Z01 will
be removed, thus moving address Z10 directly downstream of
the promoter and allowing gene expression of address Z10 only
to occur. Similarly, when only recombinase B is present (A = 0, B
= 1), addresses Z00 and Z10 are excised, allowing Z01 to be
moved directly downstream of the promoter. Finally, when both
recombinases are expressed (A = 1, B = 1), addresses Z00, Z01,
Z10 are all excised, thus placing Z11 downstream of the
promoter unobstructed by the other addresses.
(a)Integrated 2-input BLADE decoder with tagBFP, EGFP,
iRFP720, and mRuby2 as addresses Z00, Z10, Z01, and Z11
respectively. Plasmids constitutively expressing Cre and/or Flp
are then stably integrated. Three days of doxycycline (DOX)
treatment is used to permit the rtTA-VP48 protein to bind to the
tetracycline response elements promoter (pTRE) to activate
gene expression. Mean fluorescence intensity (MFI) is plotted of
either n = 1 or n = 2 independent integrations. a.u., arbitrary
units.
Four distinct output functions based on two inputs
103
Baculovirus-mediated production of AAV vectors
Gene transfer and gene therapy are powerful approaches for many biological research
applications and promising avenues for the treatment of many genetic or cancer diseases.
The most efficient gene transfer tools are currently derived from viruses. Among them, the
recombinant adeno-associated viruses (AAVs) are vectors of choice for many
fundamental and therapeutic applications. The increasing number of clinical trials involving
AAVs demonstrates the need to implement production and purification processes to meet
the quantitative and qualitative demands of regulatory agencies for the use of these vectors
in clinical trials. In this context, the rise of production levels on an industrial scale appeared
essential. The introduction, in 2002, of an AAV process using a baculovirus expression
vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in
expanding the AAV production in insect cells has been to switch the process to bioreactor
systems, which are the ideal equipment for scaling up. We describe here a method for
producing AAV vectors using the BEVS which can be easily used by research laboratories
wishing to overcome the difficulties associated with the scaling up of production levels. The
method provides sufficient quantities of AAV vectors to initiate preclinical projects in large
animal models or for research projects where a single batch of vectors will consolidate the
repeatability and reproducibility of in vitro and especially in vivo experimental approaches.
Methods Mol. Biol. 2019; 1937: 91-99. doi: 10.1007/978-1-4939-9065-8_5
104
Baculovirus-mediated multigene delivery in cells
• Multigene delivery and subsequent cellular expression is emerging as a key technology
required in diverse research fields including synthetic and structural biology, cellular
reprogramming and functional pharmaceutical screening.
• Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA
cargo capacity, thus impeding unrestricted multigene expression.
• The MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector
system expediting highly efficient transient multigene expression from a variety of
promoters.
• MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing
primary neurons and induced-pluripotent stem cells (iPS).
• The MultiPrime can be used for reprogramming, and for genome editing and
engineering by CRISPR/Cas9.
• Implementation of dual-host-specific cassettes enabling multiprotein expression in
insect and mammalian cells using a single reagent.
• The MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a
wide range of applications in primary and established mammalian cells.
105
Applications of MultiBac system
The MultiBac system is depicted schematically in the centre. It consists of an engineered baculoviral
genome which exists as a BAC in specialized bacterial cells. Foreign DNA can be introduced into
this BAC using a set of helper plasmids comprising the multigene circuitry of choice, by a variety of
means including Tn7 mediated transposition and/or site-directed insertion by Cre recombinase.
Originally, we designed the MultiBac system for expressing heterologous multiprotein complexes in
insect cells. A selection of recent high-impact structures of samples produced using MultiBac is
shown on the left, marked by their Protein Data Bank identifier (PDB-ID). 6GEJ, SWR1-nucleosome
complex; 6F3T, human TFIID subcomplex TAF5–TAF6–TAF9; 6HLP, human neurokinin 1 receptor
GPCR [36]; 6R8F, human BRISC–SHMT2 complex; 6QJ4, Ycs4–Brn1/Smc4hd–Brn1C complex;
6HCR, ADDomer virus-like particle (VLP). MultiBac is successfully used for a range of applications
beyond producing protein complexes for structural and mechanistic studies. For example,
customized MultiBac baculoviral genomes were prepared to express virus-like particles (VLPs),
which are promising vaccine candidates as they resemble live viruses but do not contain genetic
material and are thus safe. A different example is MultiBacTAG, a customized MultiBac baculovirus
capable of genetic code expansion. MultiBacTag exploits an orthogonal tRNA/tRNA synthetase pair
to insert artificial amino acids (ncAA) into polypeptide chains at defined sites by means of AMBER
codon (UAG) suppression. By altering the tropism of the baculovirion, MultiBac can be turned into a
powerful high-capacity DNA delivery device into mammalian cells, tissues and even organisms
(right), to faithfully deliver multicomponent DNA circuitry transiently or stably by genomic insertion
using CRISPR/Cas technology. For a more detailed conspectus of MultiBac applications see recent
review articles by our group.
106
Baculovirus-mediated production of AAV vectors
Gene transfer and gene therapy are powerful approaches for many biological research
applications and promising avenues for the treatment of many genetic or cancer diseases.
The most efficient gene transfer tools are currently derived from viruses. Among them, the
recombinant adeno-associated viruses (AAVs) are vectors of choice for many
fundamental and therapeutic applications. The increasing number of clinical trials involving
AAVs demonstrates the need to implement production and purification processes to meet
the quantitative and qualitative demands of regulatory agencies for the use of these vectors
in clinical trials. In this context, the rise of production levels on an industrial scale appeared
essential. The introduction, in 2002, of an AAV process using a baculovirus expression
vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in
expanding the AAV production in insect cells has been to switch the process to bioreactor
systems, which are the ideal equipment for scaling up. We describe here a method for
producing AAV vectors using the BEVS which can be easily used by research laboratories
wishing to overcome the difficulties associated with the scaling up of production levels. The
method provides sufficient quantities of AAV vectors to initiate preclinical projects in large
animal models or for research projects where a single batch of vectors will consolidate the
repeatability and reproducibility of in vitro and especially in vivo experimental approaches.
Methods Mol. Biol. 2019; 1937: 91-99. doi: 10.1007/978-1-4939-9065-8_5
107
Oncolytic viruses
Seymour and Fisher
British Journal of Cancer (2016) 114,
357-361 doi:10.1038/bjc.2015.481
108
Historical overview of gene therapy
Abbreviations: HSCT: hematopoietic stem cell transplantation; HSC: Hematopoietic stem cell; SCID:
severe combined immunodeficiency; NHP: nonhuman primate; ZFN: zinc finger nuclease; TALEN:
transcription activator–like effector nuclease; CRISPR/Cas9: clustered regularly interspaced short
palindromic repeat (CRISPR)–CRISPR-associated 9 (Cas9) nucleases.
Dunbar et al.,Science 359: eaan4672 (2018), DOI: 10.1126/science.aan4672
109
Historical overview of AAV gene therapy for hemophilia
Abbreviations: AAV: adeno-associated viral vector; FVIII: factor VIII; FIX: factor IX; Mfg: Manufacturing.
Dunbar et al.,Science 359: eaan4672 (2018), DOI: 10.1126/science.aan4672
110
Oncolytic viruses
• The activity of OVs is very much a reflection of the underlying biology of the viruses from
which they are derived and the host-virus interactions that have evolved in the battle
between pathogenesis and immunity
• Typically, OVs fall into 2 classes:
(i) viruses that naturally replicate preferentially in cancer cells and are nonpathogenic
in humans often due to elevated sensitivity to innate antiviral signalling
or dependence on oncogenic signalling pathways (parvoviruses, poxviruses,
paramyxoviruses, reoviruses, picornaviruses)
(ii) viruses that are genetically-engineered with mutations/deletions in genes
required for replication in normal, but not cancer cells (adenoviruses,
herpesviruses, rhabdoviruses)
111
Oncolytic viruses offer several important advantages
Seymour and Fisher
British Journal of Cancer (2016) 114,
357-361 doi:10.1038/bjc.2015.481
• Amplification of the active agent (infectious virus particles) within the tumour.
This avoids unnecessary exposure to normal tissues experienced during
delivery of traditional stoichiometric chemotherapy and maximises the
therapeutic index
• The active cell-killing mechanisms, often independent of programmed death
mechanisms, should decrease the emergence of acquired drug resistance
• Lytic death of cancer cells provides a pro-inflammatory microenvironment
and the potential for induction of an anticancer vaccine response
• Tumour-selective expression and secretion of encoded anticancer biologics,
providing a new realm of potent and cost-effective-targeted therapeutics
112
Emerging issues in AAV-mediated in vivo gene therapy
• In recent years, the number of clinical
trials in which adeno-associated virus
(AAV) vectors have been used for
in vivo gene transfer has steadily
increased
• The excellent safety profile, together
with the high efficiency of transduction
of a broad range of target tissues, has
established AAV vectors as the platform
of choice for in vivo gene therapy
• Successful application of the AAV
technology has also been achieved in
the clinic for a variety of conditions,
including coagulation disorders,
inherited blindness, and
neurodegenerative diseases, among
others
• Clinical translation of novel and effective
“therapeutic products” is, however, a
long process that involves several
cycles of iterations from bench to
bedside that are required to address
issues encountered during drug
development
113
https://www.autodeskresearch.com/
A The Bio/Nano/Programmable Matter group at Autodesk Research is working
with biology at the nanoscale level, to programatically define new matter. This is
called synthetic biology and they envision this science being applied to designing
products, buildings and cities.
114
Combined oncolytic virus and checkpoint blockade therapy
1: The delivery of oncolytic virus leads to specific infection and replication within the tumor. 2: Some of the infected tumor cells
undergo cell lysis and release antigen. Antigen-presenting cells, such as dendritic cells, are activated and migrate to lymph
nodes to present antigen to T cells, where the administration of anti–CTLA-4 mAb may increase the amplitude of T-cell
activation. 3: Concurrently, innate cells, including natural killer (NK) and natural killer T (NKT) cells, are recruited to the
inflammatory tumor bed that may further contribute to antitumor immune responses, through secretion of cytokines such as
IFNγ. 4: The activated T cells traffic to the tumor site and react against cancer cells through recognition of MHC/peptide
complexes 5: Administration of CTLA-4 antibody may further contribute to increasing T-cell activity at the tumor site. MDSC,
myeloid-derived suppressor cell; TCR, T-cell receptor.
115
Structures of designed two-component protein nanomaterials
The computational design models (top) and X-ray crystal
structures (bottom) are shown at left for a, T32-28, b, T33-15, c,
T33-21, and d, T33-28. Views of each material are shown to
scale along the 2-fold and 3-fold tetrahedral symmetry axes
(scale bar: 15 nm). The r.m.s.d. values given are those between
the backbone atoms in all 24 chains of the design models and
crystal structures. For T33-21, r.m.s.d. values are shown for
both crystal forms (images are shown for the higher resolution
crystal form with backbone r.m.s.d. 2.0 Å), while the r.m.s.d.
range for T33-28 derives from the four copies of the fully
assembled material in the crystallographic asymmetric unit. At
right, overlays of the designed interfaces in the design models
(white) and crystal structures (grey, orange, green, and blue) are
shown. Due to the limited resolution of the T32-28 structure, the
amino acid side chains were not modeled beyond the beta
carbon. For the interface overlays, the crystal structures were
aligned to the design models using the backbone atoms of two
subunits, one of each component.
116
Synthetic large protein nanocages to improve drug delivery
Computational models of the 10 successful designs are shown via molecular surface representations
(design names are shown above each model). Each design comprises a pairwise combination of
pentameric (grey), trimeric (blue), or dimeric (orange) building blocks aligned along icosahedral fivefold,
threefold, and twofold symmetry axes, respectively. All models are shown to scale relative to the 30
nanometer scale bar.
117
Examples of natural and non-natural protein compartments
(A), Bacteriophage P22, (PDB: 2XYZ); (B), Ferritin, (PDB: 6A4U); (C), Encapsulins, (PDB:
4PT2); (D), Artificial protein icosahedral, (PDB: 5KP9); (E), Artificial protein dodecahedron,
(PDB: 5IM5); (F), Artificial 12-subunit protein cage, (PDB: 3VDX).
118
Viral capsid-based nanocontainers as nanoreactors
119
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz