Sonogenetic Locale Specific Activation of Universal Vectors for Xenobiotics - iGEM project proposal

N E I T S (U R), Vol. I, No. 2, 2-6, 2016
A project proposal – iGEM 2016
Sonogenetic Locale Specific Activation of
Universal Vectors for Xenobiotics.
Nejc Draganjec1*, Roman Jerala2
Abstract
The final goal of the project is to develop “BioBrick” for liposome produced by means of synthetic biology, that
has a construct for disintegration embedded in its membrane. Xenobiotic packaged in a liposome is not part
of pharmacodynamics since it is biologically unavailable. Which makes liposomes interesting candidates for
universal drug delivery vectors. In our case, liposome disintegration is initiated by non-invasive sonic signal
and carried out by a construct of a sensor and an active part embedded in a membrane. Sensor part of a
construct is mechanoreceptor/mechanotransducer which activates protein representing the active part of a
construct. After activation, active part carries out the dissolution of a compartmentalization function by means
of total disintegration of vector or only membrane perforation. After an opening of a vector, previously packed
xenobiotic becomes locally available with a high concentration in locale and thus high effect and low systemic
concentration and thus smaller chance of side effect. This approach is very specific for both, time and space
factors and at the same time has a very broad area of potential biomedical applications. Vector would be, in
a hypothetical scenario of practical use in oncology, first packed with chemotherapeutics/biological drugs,
administered intravenously and then medical staff would have an option of drug activation in specific locations.
Activation is very precise and at the same time offers an option of easy switching among many different
targets, for example between dominant tumor to many potential metastasis. Since location of activation is
not tied to biomarker, but rather takes advantage of other rapidly developing medical technologies, vector
remains universally and directly applicable for any patient and for a broad spectrum of pathologies in fields of
oncology (chemotherapeutics/biological drugs and other payloads, like local immune response enhancers),
autoimmune diseases (local immune suppressors, diabetes), parasitology (malaria drugs and plasmodium
sporozoite), local pathologies (ulcer, trauma healing) . . .
Keywords
sonogenetics — pharmacodynamics — oncology — drug delivery — xenobiotics — autoimmune diseases —
igem
1Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
2Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia
*Corresponding author: nejc@draganjec.me
Contents
Introduction and motivation 2
Methods and project pipeline 3
iGEM and proof of concept . . . . . . . . . . . . . . . . . 3
Practical application and publication . . . . . . . . . . 4
References 4
Introduction and motivation
Already established approaches with pharmaceuticals [1,
2], optogenetics [3, 4] and sonogenetics [5, 6, 7, 8, 9, 10]
all have their own advantages for basic research and indus-
try and they compliment each other. But no method pub-
lished up until now has managed to avoid general applicative
problems of synthetic biology and genetic engineering in
medicine:
• In West, there is still a taboo on the direct engineer-
ing of the human genetic pool. But there is general
acceptance of products made by synthetic biology,
like almost all modern insulin supply [11, 12, 13].
• Since we already have an effective treatment, for an
instance injection of insulin, taking risks with new
methods of GE, cell therapy, implantation ...is un-
founded for [4].
• Very often solution offered is not actually a solution
for pathology but rather for a consequence of it. In
such case, a patient is offered an impossible choice
between being dependent on old vs. being dependent
on new technology. For example, switch from de-
pendency on insulin injection to dependency of an
external signal that activates insulin production in
micro-encapsulated implanted cells. [8, 9, 4].
For organizations, media attention is often a very real
part of the decision to participate in iGEM. Which makes
choosing the right topic at least as important as having the
right team and support environment [14, 15]. Media cov-
erage, which follows egocentric and topical attention of
public, is focused mostly on applicative value of research

Sonogenetic Locale Specific Activation of Universal Vectors for Xenobiotics. — 3/5
Figure 1. a) VesiColi BioBricks. b) Luciferase as a payload for liposome. c) Sequences of different sensor and active parts
of membrane construct. d) Transformation of a host organism in case of “in vivo” liposome production. e) Liposome
production in a bioreactor. f) Extraction of the product. g) There are many alternatives if it turns out that quality of
VesiColi BioBricks is not sufficient. We can use some other well-established liposome producing host, or turn away from
“in vivo” liposome production altogether and go with, for example, hydration of thin layer method. This is critical point and
a decision made at this step affects almost all other steps too. h) If we go with “in vivo” and we have time, resources and
interest, we can check the success of transformation at this point. Most of commercially available kits allow for easy
checkpoints. i) Product testing. Testing for payload, in the previous example for luciferase, is easily done by maceration
and mixing with enzyme substrate (luciferin) and then comparing the signal with control without maceration. Testing of
membrane construct is dependent on its components, but we could use immunolabeling and microscopy, patch-clamp for
ion channels, hybrid systems ...
from fields that catch public attention in certain period the
most (usually fields like oncology, infectology, metabolic
disorders, gerontology ...) [16, 17, 18]. So it is in no
surprise that anecdotal experiences scientist have with jour-
nalist’s questions like “ ...how does this cure cancer ...”
translate in only 0,001–0,005 % media coverage of scien-
tific research outside of medicine and health general topics
[18].
The fact that a lot of pharmaceuticals that are commonly
administered only works as expected for 25–60 % of pa-
tients [19] and that this resulted in 142.000 deaths only in
the year 2013 [20] was acknowledged as one of the biggest
current issues in medicine. Many methods were already
researched and proposed to improve on pharmacokinetics
and to assure more targeted delivery [21, 22, 23]. An issue
with current approaches is that specificity negates flexibil-
ity which makes medical treatment time consuming and
costly. This is furthermore complicated by pathologies that
combine homogeneity between pathology and healthy soma
and heterogeneity inside pathology tissue. Perfect examples
are tumors where systemic therapy, because of similarity
with healthy soma, carries devastating side effects. And
at the same time, cancerous cells go through cell clonal
evolution and extensive differentiation which makes precise
and dependable specific targeting very difficult [24].
Methods and project pipeline
As experienced mentors can surely testify, there is never an
excess of time for a big project like iGEM. That is why I
propose we split path to a final product in 2 stages – proof
of concept for iGEM and applicative test for a publication.
iGEM and proof of concept (Figure 1 and 2)
In the spirit of iGEM, where cooperation is the norm, I
think it makes sense if we start with a good idea developed
Figure 2. a) Isolated vectors are transferred to medium
with an enzyme substrate. Luciferin for example that was
given before. b) We sonicate samples and measure the light
signal. c) Between different membrane constructs we
measure and compare stability (unspecific permeability for
payload or even full disintegration of liposome), dynamics
(dissolution of the compartmentalization function after
activation) and interval of effective signal
amplitude/frequency (energy must not be too high so that
tissue “in vivo” is not damaged and not too low so that we
don’t have unspecific activations and poor stability).
by VesiColi iGEM NTNU 2013 team (bronze price) and
upgrade it to excellent. In case “bioBricks” from VesiColi
team are not of sufficient quality, we still have many other
methods of liposome production on our disposal (other

Figure 3. a) We prepare 3 combinations of vector and cell line – the first vector with membrane construct and payload,
second with payload but without construct and third as a negative control without construct and payload. b) We start with
cell culture and monitor stability and potential toxicity of vector. c) We sonicate cultures and analyze differences between
active, passive and negative control vectors. The difference between active and passive gives the efficiency of membrane
construct and negative control represents baseline normalization and test for potential general toxicity of liposome in
culture. d) If we stay in the field of oncology, we can continue tests on some of many oncology model lines of rodents. But
since this method of delivery is universal, we also have the option to switch payload and focus on a mirage of other
possible pathologies. For example, we could deliver local immune suppressors to locale of pancreas and follow the
prognosis of diabetes 1 in diabetic rodent line.
liposome producing hosts, hydration of thin layer ...).
Our main contribution to synthetic biology would be
“bioBrick” construct for controlled opening of the liposome
by means of a sonic signal. For mechanoreceptor, we have
many options, but I would recommend we look at MscL,
MscS and MCA protein families since they do not have a
homologue in animals. The absence of homology lowers
chance of unspecific effects in the final practical application
as a biomedical tool. Native presence in liposome producing
host (or perhaps just membrane construct if we decide on
other liposome production method) also makes synthesis
part of the project easier since we only have to modify and
not design cell pathways “de novo”. The active part of a
construct is porin (example [25]) or split enzyme which
gets activated after sensor part of constructs receives sonic
signal. Activation could be direct by on enzyme attached
ligand/receptor (as in [26]) or indirect through cell signaling
cascade. Main work in this segment of the project would
be building and testing different combinations of sensor
and active part of construct and testing which combination
offers the right combination of precision, dynamics and
reliability.
Furthermore, I propose, we make sure that in testing
period liposomes carry a payload which offers an easy as-
sessment of approach viability in next steps. As an example,
we could pack liposomes with luciferase during production.
After successful transformation of the host organism (in
a case of “in vivo” production) for test payload and mem-
brane construct we can start with production in a bioreactor.
Isolation and purification of product out of reactor media is
dependent on steps before, but as an example, we could po-
tentially use some well-established detergent method ([27]).
Isolated vectors can then be transferred to test medium
with added substrate for the enzyme of choice in a pay-
load. In the beforehand example that would be luciferin.
Then we sonicate test dishes and we record light signal as
a result of the disintegration of compartmentalization and
mixing of enzyme/substrate with optical methods (for ex-
ample microscopy). In case statistically significant signal in
the locale of sonic signal focus we have proof of working
concept. We can also check for time-dependent stability of
vectors by the same principle but without sonic signal.
Practical application and publication (Figure 3)
We follow up the basic proof of concept with a test of per-
formance in a more complex system of cell culture. The
best platform would be one of immortal human cell lines to
which we add a vector with payload of appropriate cytotox-
icity. After cells in culture adhere in place we can sonicate
and then we compare the formation of plaques between test
and control dishes. After success in cell cultures, we can
continue tests on some of many oncology model lines of
rodents.
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