N E I T S (U R), Vol. I, No. 2, 2-6, 2016A project proposal – iGEM 2016

Sonogenetic Locale Specific Activation ofUniversal Vectors for Xenobiotics.Nejc Draganjec1*, Roman Jerala2

AbstractThe final goal of the project is to develop “BioBrick” for liposome produced by means of synthetic biology, thathas a construct for disintegration embedded in its membrane. Xenobiotic packaged in a liposome is not partof pharmacodynamics since it is biologically unavailable. Which makes liposomes interesting candidates foruniversal drug delivery vectors. In our case, liposome disintegration is initiated by non-invasive sonic signaland carried out by a construct of a sensor and an active part embedded in a membrane. Sensor part of aconstruct is mechanoreceptor/mechanotransducer which activates protein representing the active part of aconstruct. After activation, active part carries out the dissolution of a compartmentalization function by meansof total disintegration of vector or only membrane perforation. After an opening of a vector, previously packedxenobiotic becomes locally available with a high concentration in locale and thus high effect and low systemicconcentration and thus smaller chance of side effect. This approach is very specific for both, time and spacefactors and at the same time has a very broad area of potential biomedical applications. Vector would be, ina 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 differenttargets, for example between dominant tumor to many potential metastasis. Since location of activation isnot tied to biomarker, but rather takes advantage of other rapidly developing medical technologies, vectorremains universally and directly applicable for any patient and for a broad spectrum of pathologies in fields ofoncology (chemotherapeutics/biological drugs and other payloads, like local immune response enhancers),autoimmune diseases (local immune suppressors, diabetes), parasitology (malaria drugs and plasmodiumsporozoite), local pathologies (ulcer, trauma healing) . . .

Keywordssonogenetics — pharmacodynamics — oncology — drug delivery — xenobiotics — autoimmune diseases —igem

1Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia2Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia*Corresponding author: nejc@draganjec.me

Contents

Introduction and motivation 2

Methods and project pipeline 3iGEM and proof of concept . . . . . . . . . . . . . . . . . 3Practical application and publication . . . . . . . . . . 4

References 4

Introduction and motivationAlready 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 applicativeproblems of synthetic biology and genetic engineering inmedicine:

• 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 aninstance injection of insulin, taking risks with newmethods of GE, cell therapy, implantation . . . is un-founded for [4].

• Very often solution offered is not actually a solutionfor pathology but rather for a consequence of it. Insuch case, a patient is offered an impossible choicebetween being dependent on old vs. being dependenton new technology. For example, switch from de-pendency on insulin injection to dependency of anexternal signal that activates insulin production inmicro-encapsulated implanted cells. [8, 9, 4].

For organizations, media attention is often a very realpart of the decision to participate in iGEM. Which makeschoosing the right topic at least as important as having theright team and support environment [14, 15]. Media cov-erage, which follows egocentric and topical attention ofpublic, 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 partsof membrane construct. d) Transformation of a host organism in case of “in vivo” liposome production. e) Liposomeproduction in a bioreactor. f) Extraction of the product. g) There are many alternatives if it turns out that quality ofVesiColi 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 anda decision made at this step affects almost all other steps too. h) If we go with “in vivo” and we have time, resources andinterest, we can check the success of transformation at this point. Most of commercially available kits allow for easycheckpoints. i) Product testing. Testing for payload, in the previous example for luciferase, is easily done by macerationand mixing with enzyme substrate (luciferin) and then comparing the signal with control without maceration. Testing ofmembrane construct is dependent on its components, but we could use immunolabeling and microscopy, patch-clamp forion channels, hybrid systems . . .

from fields that catch public attention in certain period themost (usually fields like oncology, infectology, metabolicdisorders, gerontology . . . ) [16, 17, 18]. So it is in nosurprise 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 commonlyadministered only works as expected for 25–60 % of pa-tients [19] and that this resulted in 142.000 deaths only inthe year 2013 [20] was acknowledged as one of the biggestcurrent issues in medicine. Many methods were alreadyresearched and proposed to improve on pharmacokineticsand to assure more targeted delivery [21, 22, 23]. An issuewith current approaches is that specificity negates flexibil-ity which makes medical treatment time consuming andcostly. This is furthermore complicated by pathologies thatcombine homogeneity between pathology and healthy somaand heterogeneity inside pathology tissue. Perfect examplesare tumors where systemic therapy, because of similaritywith healthy soma, carries devastating side effects. Andat the same time, cancerous cells go through cell clonalevolution and extensive differentiation which makes preciseand dependable specific targeting very difficult [24].

Methods and project pipelineAs experienced mentors can surely testify, there is never anexcess of time for a big project like iGEM. That is why Ipropose we split path to a final product in 2 stages – proofof 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, Ithink it makes sense if we start with a good idea developed

Figure 2. a) Isolated vectors are transferred to mediumwith an enzyme substrate. Luciferin for example that wasgiven before. b) We sonicate samples and measure the lightsignal. c) Between different membrane constructs wemeasure and compare stability (unspecific permeability forpayload or even full disintegration of liposome), dynamics(dissolution of the compartmentalization function afteractivation) and interval of effective signalamplitude/frequency (energy must not be too high so thattissue “in vivo” is not damaged and not too low so that wedon’t have unspecific activations and poor stability).

by VesiColi iGEM NTNU 2013 team (bronze price) andupgrade it to excellent. In case “bioBricks” from VesiColiteam are not of sufficient quality, we still have many othermethods of liposome production on our disposal (other

Sonogenetic Locale Specific Activation of Universal Vectors for Xenobiotics. — 4/5

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 withcell culture and monitor stability and potential toxicity of vector. c) We sonicate cultures and analyze differences betweenactive, passive and negative control vectors. The difference between active and passive gives the efficiency of membraneconstruct and negative control represents baseline normalization and test for potential general toxicity of liposome inculture. d) If we stay in the field of oncology, we can continue tests on some of many oncology model lines of rodents. Butsince this method of delivery is universal, we also have the option to switch payload and focus on a mirage of otherpossible pathologies. For example, we could deliver local immune suppressors to locale of pancreas and follow theprognosis 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 liposomeby means of a sonic signal. For mechanoreceptor, we havemany options, but I would recommend we look at MscL,MscS and MCA protein families since they do not have ahomologue in animals. The absence of homology lowerschance of unspecific effects in the final practical applicationas a biomedical tool. Native presence in liposome producinghost (or perhaps just membrane construct if we decide onother liposome production method) also makes synthesispart of the project easier since we only have to modify andnot design cell pathways “de novo”. The active part of aconstruct is porin (example [25]) or split enzyme whichgets activated after sensor part of constructs receives sonicsignal. Activation could be direct by on enzyme attachedligand/receptor (as in [26]) or indirect through cell signalingcascade. Main work in this segment of the project wouldbe building and testing different combinations of sensorand active part of construct and testing which combinationoffers the right combination of precision, dynamics andreliability.

Furthermore, I propose, we make sure that in testingperiod 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 (ina 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 isdependent 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 mediumwith 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 asa result of the disintegration of compartmentalization andmixing of enzyme/substrate with optical methods (for ex-ample microscopy). In case statistically significant signal inthe locale of sonic signal focus we have proof of workingconcept. We can also check for time-dependent stability ofvectors 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. Thebest platform would be one of immortal human cell lines towhich we add a vector with payload of appropriate cytotox-icity. After cells in culture adhere in place we can sonicateand then we compare the formation of plaques between testand control dishes. After success in cell cultures, we cancontinue tests on some of many oncology model lines ofrodents.

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