miRNA strategy

The purpose behind the overexpression of a microRNA instead of a protein for our gene therapy model is to target multiple pathways and phenomena in the core of Alzheimer’s pathophysiology. With this principle in mind, we chose the hsa-mir-195-5p because of its targeting of virtually all of AD’s pathological roots. In contrast to other microRNAs that target neuronal inflammation and apoptosis (miR-34a), Αβ amyloid plaque formation, and Tau aggregation (miR-132) separately, mir-195-5p pushes back against all three of these fronts simultaneously, essentially fighting AD to its core.

Transfer Plasmid Design Strategy

Our transfer gene plasmid (pAAV-EF1a-mir195-eGFP-SV40pA) (BBa_K5471020 ) was carefully designed to accommodate the purposes of our experiment. This plasmid is composed of the original pUC57 vector provided by Genscript, and Cassette 2 cloned into position 2710. The reasoning behind its design is for it to be usable as a transfer gene plasmid, which, along with pRepCap and pHelper, will perform the triple transfection of HEK293T cells and produce rAAV2 viral particles with the desired properties. These properties include the simultaneous expression of mir-195 and eGFP, accomplished by Cassette 2. Additionally, due to the properties of pUC57, this plasmid can independently auto-replicate inside any cell and be selected later with the use of the antibiotic Ampicillin.

Cassette 2 (ITR-EF1a-mir195-eGFP-SV40pA-ITR)(BBa_K5471013) is an autonomous device for the simultaneous expression of microRNA 195 and the eGFP reporter gene protein (Cassette 1 ), specifically placed between a pair of ITR (inverted terminal repeats) sequences for the AAV2 serotype (BBa_K5471004). The ITR pair flanking makes the cassette ideal for rAAV serotype 2 production because they enable the cassette they flank to constitute the entire viral DNA (including the pair of ITR sequences) of the produced rAAV2 particles.

Cassette 1 (EF1a-mir195-eGFP-SV40pA)(BBa_K5471012) is an autonomous device for the simultaneous expression of microRNA 195 and the eGFP reporter gene protein.

This cassette is comprised of (from the 5' end):

1. EF1a mir195 infused promoter, which is a new composite part (BBa_K5471011, BBa_K5471009, BBa_K5471005)

2. a Kozak sequence (BBa_K5471006)

3. eGFP (BBa_K5471007)

4. SV40 polyadenylation signaling sequence (BBa_K5471008) for the termination of transcription.

EF1a Core Promoter: The human eukaryotic translation elongation factor 1 alpha (EF1a) promoter boasts strong gene expression in most cell lines (including HEK293T), while being more resilient and stable due to its mammalian origin, compared to a viral-origin promoter that might fail to express a gene or be silenced over time.

EF1a Intron: The human eukaryotic translation elongation factor 1 alpha (EF1a) first intron is added downstream of the EF1a core promoter to boost the gene expression power of the promoter. This intron also provides an ideal location to insert our “Gene of Interest.”

The EF1a mir195 infused promoter is a new composite part responsible for driving the intragenic expression of mir195 inside the EF1a intron while also ensuring the strong transcription of a (eGFP) protein gene. This new composite part provides the ability to efficiently express microRNA 195 and simultaneously express a reporter protein that confirms the expression of the microRNA.

This design simulates the intragenic expression of most microRNAs by imitating the genomic locus where the hsa-mir-195 gene (87bp) is located.

The genomic locus chr17:7017465:7017851 (387bp) can be treated as the “gene of interest” that is inserted into an already existing cassette or plasmid vector. In our case, the 150bp extra sequences flanking the hsa-mir-195 gene on either side are identical to the ones that naturally flank the gene in the human genome (150 + 150 + 87 = 387).

The goal of this faithful imitation of the human genomic locus is to drive the natural transcription and modification processes so that the mature hsa-mir-195-5p is produced inside the treated cells. This method promises a stronger expression of the mature microRNA, resulting in greater amounts compared to other methods that insert the mature miRNA directly or use shRNA mimics.

Specifically, the EF1a promoter used is borrowed from the plasmid (scAAV GFP). Since we aim to insert a 387bp sequence, it was crucial to preserve the parts of the intronic sequence near its 5’ and 3’ ends. With this in mind, we decided to insert our “gene of interest” at position 334 of the original EF1a promoter. The combined length of this new part is 1178 bp, which is longer than the original EF1a promoter, but similar in size to many commonly used EF1a promoters. At the same time, the sequences of the EF1a intron near the 5’ and 3’ ends remained untouched, allowing the natural excision of the intron via cellular mechanisms.

eGFP.The gene downstream of the new part could be any protein, but we deemed a reporter protein gene more useful for our experiments. We chose eGFP due to its relatively small size and strong fluorescence. This way, if fluorescence is detected, it strongly indicates that the microRNA is also being produced.

The Kozak sequence (vertebrate consensus, a DNA/RNA motif for strong initiation of translation, 1987) was placed directly upstream of the eGFP coding region to achieve stronger and more stable initiation of translation.

The SV40 polyA signal, or simian virus polyadenylation signal, is responsible for signaling the polyadenylation of the 3' end of an mRNA transcript. Its role is to shield the mRNA from enzymatic digestion and degradation inside the cell and to signal the termination of the translation process. It was chosen for its small size (63bp), stability, and reliability.

Cell Culture

For the execution of our experiments, we selected adherent HEK293T cells, a widely used derivative of the HEK293 line (Human Embryonic Kidney cells) that has been transfected with the SV40 large T antigen. This modification enhances their suitability for transfection protocols and gene therapy research. The combination of this genetic alteration with the cell's high reproducibility and ease of handling makes them an ideal choice for our initial experimental workflow. This cell line was optimal for our experiment for the reasons described above, in addition to the fact that this cell line was more accessible and familiar to our lab and their use would not compromize the safety of our experiments, compared to the use iPSCs.

To ensure optimal preparation and cell viability, we adhered to specific protocols for freezing, thawing, splitting, and seeding, all of which are detailed extensively in our experiment archive, as documented in the lab notebook.

Transfection

The construction of rAAVs began with the transient transfection of HEK293T cells in culture using the three essential plasmids: Transfer plasmid, Adenoviral Helper plasmid, and Packaging plasmid. Three days later, we evaluated our results by detecting eGFP, integrated within the transgene plasmid, under a fluorescence microscope.

Exosome Isolation

68 hours post-transfection, instead of proceeding with the harvest of our vectors, we opted to isolate exosomes from the culture supernatant, aiming to capture the virus contained within them. To achieve this, we used exosome isolation reagent, successfully obtaining vexosomes, which were subsequently diluted in PBS buffer. This isolation method was preferred to other methods (ultracentrifugation), due to its high specificity and purity.

Measurement of Vexosome Load

After designing the exosome isolation procedure, we also aimed to measure the quantity of rAAV encapsulated in the exosomes to assess the viral load, offering vital information for determining the ideal dosage required for the transduction process. Furthermore, this information is crucial for evaluating the overall effectiveness of vexosome production, ensuring that we achieve the desired efficiency and effectiveness in our experimental results. To meet our objective, we selected qPCR as the method for precisely quantifying the vector load.

Testing Model

Preparing our testing model presented several challenges. After rejecting the final testing of our product in Alzheimer's 3D cell culture due to the complexity of handling and the elevated costs associated with the procedure, we turned to tau-expressing HEK293 cells. Normally, HEK293 cells do not express tau proteins, which primarily stabilize microtubules in neuronal cells. However, a specific modification of their expression system allows HEK293 cells to mimic the tau expression capacity of neuronal cells.

Unfortunately, due to technical and delivery issues, we were unable to acquire these modified cells. As a result, we opted to utilize the HEK293T cells already available and assess the success of our therapy in relation to the functionality of phosphatase PP2A, which is strongly and directly linked to the dephosphorylation of Tau proteins. Specifically, we decided to measure the effect of microRNA-195 on the methylation of the amino acid Leucine 309 in the catalytic subunit. As already mentioned, this methylation is reduced in AD, and our goal is to upregulate the methylation and, thus, the catalytic activity of PP2A.

To better simulate the pathophysiology of Alzheimer's disease, we also incubated our cell culture with Calyculin A, a chemical compound known for its inhibitory effect on PP1 and PP2A phosphatases. The diminished activity of PP2A in Alzheimer's cells is associated with the increased phosphorylation of tau proteins via qPCR and Flow Cytometry phosphorylation of tau proteins.

Exosome modification

The next step in our experiment involves the incorporation of the RVG29 peptide onto the exosome membrane through a click chemistry reaction, in order to achieve specific targeting of neural cells. However, the absence of neural cell culture, combined with the pressing timeline in which the experiments were conducted, led us to decide against testing this modification experimentally. Instead, we opted to simulate it using Agent-Based Modeling (ABM), in Netlogo.
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Treatment/Transduction

Our exosomes were administered to HEK293T cells, that had been treated with Calyculin A for 30 minutes, and incubated for a duration of 72 hours, with the aim to facilitate the transduction of these cells with the recombinant adeno-associated virus (rAAV) that we had produced. The transduction was assesed with the methods of qPCR and Flow Cytometry.