Team:Munich/Description

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Abstract

There is an increasing demand in biomedical research for techniques to monitor the dynamics of multiple genes over several time points. However, current methods such as gene reporters are limited to a few genes of interest or require sample destruction in the case of transcriptomic analysis. We thus engineered ALiVE as a diagnostics platform for the Analysis of Living cells via Vesicular Export. In particular, we hijacked the mechanisms of exosome secretion and viral budding to export specific transcripts from living cells repeatedly over time. Based on versatile BioBricks, we modified membrane proteins with affinity tags which are fused to bio-orthogonal RNA-adapters to enable convenient purification of the vesicles and thus the exported RNA. We also introduced sensitive luciferase reporters to quantify vesicle secretion efficiency and collateral transfection. ALiVE is a generalizable technology for minimally invasive diagnostics of gene expression dynamics in cellular model systems and holds great promise for monitoring cellular therapies in regenerative medicine.

ALiVE

a diagnostic tool for minimal-invasive longitudinal monitoring of cellular RNA

Achievements

Our goal for iGEM was to create a successful proof of concept for ALiVE, in several mammalian cells to highlight the strong potential impact of ALiVE for regenerative medicine.

ALiVE was so far established in HEK293T cells.

RNA Encodes Cellular Information

In order to retrieve cellular information without lysis, an exportable, information-encoding biomolecule is required. RNA fulfills these requirements as its 4 bases can hold a lot of information even with short lengths (4ⁿ). For our proof-of-principle study we exported mRNA that was coding for a Luciferase and also contained a handle to bind the RNA adapter. Although we mainly used qPCR to quantify the secreted mRNAs, this single channel can be easily expanded to many channels by introducing silent mutations that can be read by sequencing using the same primers used for qPCR.

Exported mRNA monitors the current gene expression.

RNA Enrichment on Binding Proteins

Specific RNA sequences are known to form RNA secondary structures. Their high affinity interaction with RNA-binding proteins (RBPs) can be exploited to direct RNA from the cytosol to cellular membranes via RBPs fused to membrane proteins.

The 50S ribosomal protein L7Ae from Archaeoglobus fulgidus is a well characterized RNA-binding protein, that interacts with a secondary RNA motif called C/D-box. This system has already been used in exosomes to deliver therapeutic mRNAs from one cell type to another (Kojima et al. 2018).

The MS2 bacteriophage coat protein (MCP) is a RBP which interacts with a stem-loop (MS2) motif from the phage genome and has been shown to be able to target RNA to VLPs (Prel et al. 2015).

Vesicle Loading

RNA is an unstable molecule with a half-life of 30 min to 10 h depending on its protective characteristics. Therefore, we engineered protective cargo vesicles for exported RNA, formed continuously over time.

average size: 30 - 100 nm

endogenous

minimal cell stress

As a first option, we hijacked the endogenous exosomal pathway implicated in intercellular communication and immune regulation (Batista and Melo 2019). By fusing RNA-binding proteins to the exosomal marker CD63, cytosolic RNA of interest will be enriched in exosomes and consequently exocytosed. We further engineered CD63 with an internal 6xHis-tag that faces the outside of the vesicle to purify exosomes without the need for expensive kits. Sensitive luciferase reporters allow the quantification of vesicle secretion efficiency and collateral transfection.

average size: 100 - 150 nm

orthogonal export mechanism

higher RNA loading capacity

As a second option, we included an orthogonal export mechanism using virus-like particles (VLPs). These can be secreted by human cells when expressing the HIV’s group antigen (Gag) protein (Eyckerman et al. 2016; Titeca et al. 2017). Gag itself is harmless as it exclusively forms the viral matrix, capsid, and nucleocapsid (Langer et al. 2017). To enable RNA loading, we fused RNA-binding proteins to the Gag monomers, leading to higher amounts of RNA-binding proteins per vesicle in comparison to the exosomes.

Both vesicles have strengths and weaknesses. We created a model to calculate the more suitable export system for a desired application.

Modular Vesicle Composition

To introduce modularity into the system, interacting protein domains were fused to CD63 and Gag such that vesicle loading could be tuned at will. Two sets of alpha-helical heterodimerizing peptides were tested for this purpose.

The first one consists of the 32 aa long coiled-coil domains P9SN and P10SN, which bind to each other in parallel orientation (N-terminus to N-terminus; from Ljubetič et al. 2017).

The second set is composed of the double-helical domains DHD154a and DHD154b from Chen et al. 2019. Each domain consists of two alpha-helical domains linked by a turn, which upon dimerization form a four-helix bundle.

Vesicle Purification

In order to isolate the exosomes, we fused a 6xHis-Tag to an external loop of the exosomal marker CD63. To our knowledge, Ni-NTA affinity chromatography has not been previously used to purify exosomes, it has only been applied to other His-tagged membrane structures (Alves et al 2017). See our best Basic Part here

VLP isolation is currently based on a recently published heparin affinity purification method that allows getting rid of other similarly sized particles such as exosomes (Reiter et al 2019).

Quality Control

We have equipped our vesicles with a luminescence-based tag: Promega's 11 amino acids long High Bit (HiBiT) peptide (Dixon et al. 2016; Promega®). This short sequence is able to reconstitute a functional NanoLuc luciferase enzyme when bound to the complementing polypeptide Large BiT (LgBiT), to which it has a high affinity (Kd ~1nM). The HiBiT tag is fused to the C-terminus of both CD63 and Gag, such that it is only present inside the exosomes or VLPs respectively.

RNA Analysis

The RNA exported in the vesicles can be isolated by PEG-based precipitation and phenol-chloroform extraction for exosomes or a viral RNA extraction protocol for VLPs. Subsequently, isolated RNA is reverse transcribed into cDNA for downstream analysis.

qPCR

The vesicular RNA content can be precisely quantified by quantitative PCR. The use of Exon-Exon-junction primers eliminates false positive signals and trace plasmid contamination because most eukaryotic promotors contain intronic sequences. The samples can be normalized to cellular housekeeping genes like GAPDH.

RNA-Seq

Thanks to the stable performance of ALiVE, sufficient amounts of RNA would have been achievable in our cell systems to run transcriptomic analyses which could, however, not been carried out within iGEM due to financial constraints.