Contribution

In our quest to produce a versatile system for the production of designer exosomes (1), we faced a vast array of adversities and setbacks, which forced us to rethink certain strategies and eventually, propose some improvements. In this wiki, we register some of our boldest innovations including a direct assembly strategy for a lentiviral transfer plasmid construction and an original labelling strategy for shRNAs. We hope that our contribution will be useful for upcoming iGEM teams.

Abstract

Extracellular vesicles (EVs) are a heterogeneous group of small, lipid-based nanoparticles that play a crucial role as mediators of many physiological and pathophysiological processes, mainly by the delivery of RNAs.
As opposed to the widely known artificial liposomes, EVs are coated with surface proteins that provide specific tissue targeting. This interesting feature, alongside their biocompatibility, bioavailability, and ability to cross the blood-brain barrier, has boosted EVs as potential RNA drug carriers. These are the reasons why EVs are starting to be used in therapy. However, the capacity to create designer EVs is still lacking. Unfortunately, due to the low efficiency and high costs for their obtention, there is no consensus in the scientific community about the collection and purification techniques. Here we provide a solution for these two issues. We present a molecular biology kit to use in a biological system to produce designer vesicles. At the same time, we have included a tag system to improve and simplify the collection and purification techniques, making it more feasible for laboratory groups. Moreover, we have taken a step further: we propose to load the EVs with the c-myc shRNA to use them as an innovative and side-effect-free therapy for Burkitt's lymphoma.

OPTIMIZATION OF THE GOLDEN GATE PROTOCOL FOR ASSEMBLIES

Design and assembly of Level 1 modules, as first step

Modular Cloning (MoClo) is an assembly method, based on Golden Gate Assembly, that allows the creation of any eukaryotic multigene construct (2)(3)(4). The conventional MoClo system comprises 3 sets of cloning vectors [Fig.1]

Level 0 modules: Basic genetic elements.

Level 1 modules: Assembled transcription unit.

Level 2 modules: Multigene construct.

Scheme of golden gate cloning
Fig.1. Overview of the Modular Cloning system (based on Golden Gate). “Level 0 modules” include basic genetic elements cloned in a set of level 0 vectors (based on pUC19 backbone). The creation of “level 0 modules” involves an amplification step PCR-based and a cloning process via Golden Gate. Compatible sets of “level 0 modules” are assembled via a second Golden Gate reaction (into level 1 vectors), to create the “level 1 module”, consisting of individual assembled transcription units. Eventually, multiple “level 1 modules” can be directionally cloned into a level 2 destination vector, creating the “level 2 module”.

As an alternative to this hierarchical process, we designed our constructs as if they were “level 1 modules”. Instead of generating a library with all the single basic genetic elements (such as the CMV promoter or the SV40 terminator) to create “level 0 modules” in level 0 vectors, we directly designed individual transcription units in a level 1 vector. First of all, we designed, optimized and domesticated our constructs. Then, we divided each construct (see Parts, Composite Parts) into different parts, and flanked each of them with recognition sites of Type IIS restriction enzymes (BsmBI, BbsI, AarI). By these means, each part of the construct can be directionally assembled into a level 1 vector ("pJUMP28-1A KanR Type IIS level 1 vector. Origin pUC". BBa_J428353) on a single reaction (see Protocols).

Assembly reaction

However, what is thought in theoretical matters doesn’t always apply as easily in real life. That’s why we tried out different assembly protocols and checked whether our constructs and primers were correct. For this issue, we highly recommend consulting New Englands BioLabs tools (5)(6)(7). In our case, the protocols provided were not enough to achieve a successful assembly, but they were very useful to contrast information. Thanks to these resources and further research done by the team, we found the following conditions to work best in each case:

·1 fragment: 25 minutes 37ºC, 25 minutes 16ºC

·2 fragments: (2 minutes 37ºC, 1 minute 16ºC)x30, 10 minutes 16ºC, 10 minutes 65ºC

LENTIVIRAL TRANSFER PLASMID BASED ON pJUMP BACKBONE PLASMID

The second-generation system of lentiviral vectors splits essential components of the lentiviral system across 3 plasmids that are delivered separately for safety.(8) Transfer plasmid: Encodes for the transgene. It also contains cis-acting elements such as the 5’ and 3’ LTRs essential for promoting RNA polymerase II to begin transcription of viral mRNA, and the y sequence (which signals genome packaging). Packaging plasmid: Provided in trans. Encodes the essential trans-acting genes (gag, pol rev, tat) that are required for entry and integration of the viral genome. Envelope plasmid: Contains genes encoding for envelope proteins.

If we followed the conventional process, we would have to clone our transgenes (which are the different genomic constructs encoding for the exosomes boosting and the shRNA cargo loading) in a transfer plasmid such as pLVTHM by classic restriction cloning, which is sometimes difficult and time-consuming.

In that context, an adventurous idea occurred to us: could we improve our basic Golden Gate backbone to become a transfer plasmid itself? That way, no intermediate steps after the assembly should be required to use the construction in a lentiviral vector. Therefore, we conceived our favourite composite part, the so-called pJUMP-based lentiviral transfer plasmid (BBa_K4501022, see Fig.2 and Fig.3). By incorporating the required MSCV modules on the secondary restriction sites in the 3’ and 5’, we have been able to transform our basic backbone for the assembly of plasmids (pJUMP29-1A KanR TypeIIS Level 1 vector) into a transfer plasmid that can be used right away after the assembly.


Fig. 2. pJUMP information and restriction map.



Fig. 3. Workflow to produce recombinant lentivirus particles containing our desired constructs, on the basis of starting with our designed pJUMP lentiviral transfer. As a result, there is no need to clone the constructs from the pJUMP plasmid (Golden Gate result) to another transfer plasmid, because we have designed our “home-made” pJUMP plasmid to act as a transfer plasmid itself.

STABLE AND ELEGANT shRNA labelling

For our project, we designed our own shRNA against myc gene (BBa_K4501000) and a expression cassette to be produced directly by our cell line (BBa_K4501018, see Fig 4A). Additionally, we wanted to introduce a C/D-box sequence into the shRNA in order to be loaded directly into the exosomes by the L7Ae (ligand of C/D-box) present in the luminal domain of the exosome-enriched tetraspanin CD63. However, we saw that incorporating the C/D-box in the 3’ terminus of the shRNA altered the secondary structure of the shRNA (BBa_K4501001; BBa_K4501019), which is vital in order to be recognized as a double-stranded RNA by the silencing machinery of the cell in which shRNA and siRNA rely on.

To solve that issue, we envisioned and designed a novel approach to add a label to shRNAs. Since the C/D-box recognition site of L7Ae is a double-loop structure, we thought that substituting the traditionally used loop (TTCAAGAGA) for our C/D-box sequence may work, and thus our favorite basic part was born: the shRNA in-loop-C/D-box (BBa_K4501002; BBa_K4501020). Subsequent structural analysis revealed that not only the resultant structure created the stem-loop structure of a traditional shRNA, but that it was also more entropically stable! With this innovative solution, the labelling of shRNA with loop-structures becomes much more easier!


Fig. 4. Structure of our designed shRNA

EDUCATION CONTRIBUTION


In order to bring all this knowledge to future generations we have created a series of educational videos (link) that explain our project in a fun and simple manner. This way we bridge the educational gap and make Vesiprod accessible to all ages.


BUILDING UP A BRAND

Being the first iGEM team of the University is not easy. You need people to trust you in order to get the financial resources and support to enter the competition. That is why we have focused a lot of our efforts on creating a visual brand, getting noticed and making ourselves known. We have created a very personalized and visual aesthetic, we have established a color palette and typography that dominate all our products, so the following UB teams have half the work done. Furthermore, we have established strong relationships with many people from the university and other entities that will last over time so the following Barcelona_UB teams have it easier to find a supportive network.

Bibliography

  1. He.J., Ren.W., Wang.W., Han.W., Jiang.L., Zhang.D., Guo.M. (2022). Exosomal targeting and its potential clinical application. DOI: https://doi.org/10.1007
  2. Werner.S., Engler.C., Weber.E., Gruetzner.R., Marillonnet.S. (2012). Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo System. DOI:http://dx.doi.org/10.4161/bbug.3.1.18223
  3. Weber.E., Engler.C., Gruetzner.R., Werner.S., Marillonnet.S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. DOI:https://doi.org/10.1371/journal.pone.0016765
  4. Grützner.R., Marillonnet.S. (2020). Generation of MoClo Standard Parts Using Golden Gate Cloning. DOI: https://doi.org/10.1007/978-1-0716-0908-8_7
  5. NEB Golden Gate Assembly Tool: https://goldengate.neb.com/#!/
  6. NEB Technical Tips For Optimizing Golden Gate Assembly Reactions: https://international.neb.com/tools-and-resources/usage-guidelines/technical-tips-for-optimizing-golden-gate-assembly-reactions
  7. Golden Gate Assembly Protocol: https://international.neb.com/golden-gate/~/link.aspx?_id=2A5FC74C7C6143DAAAC058036122BD4E&_z=z
  8. Addgene. Lentiviral Guide: https://www.addgene.org/guides/lentivirus/
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