iGEM 2025 — Team patras-med

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RNAi engineering

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* [miR-E-based shRNA design strategy](#strategy2)
* [Why miRE-based shRNA…](#miRE1)
* [Our plasmids](#plasmids1)
* [Cell cultures](#cell1)
* [Lentiviral production and delivery](#lenti1)
* [Choice of biomarker: LepRb receptor – 1st layer of safety](#biomarker1)
* [Lentiviral transduction and experimental validation](#lenti3)
* [References](#refContent)

## miR-E-based shRNA design strategy

To target HDAC6, we designed a short hairpin RNA (shRNA) embedded within the **optimized miR-E
backbone**2, following the design principles outlined in *“Guidelines for the Optimal
Design of miRNA-Based shRNAs”* and *“An Optimized microRNA Backbone for Effective Single-Copy
RNAi”*.

The **target sequence** (CTCACTGATCAGGCCATATTT) was selected using the **Broad Institute's
GPP Web Portal**,
based on its strong predicted efficacy, receiving a
Sequence-Dependent RNAi (SDR) score of 100%, with an
intrinsic score of 15.000
and an
adjusted score of 21.000, quantitative metrics reflecting on-target potency and design quality.

As **miRNA-based shRNAs** typically **require a 22-nt guide strand**  for
**optimal Dicer processing**, we performed a BLAST alignment against the HDAC6 mRNA using NCBI
tools to identify the upstream nucleotide, which was a cytosine. This cytosine was added to the 5′ end of the
guide to complete the final 22-nt antisense strand (AAATATGGCCTGATCAGTGAGG), which was then embedded into the 3′
arm of the miR-E scaffold.

The entire hairpin structure was manually constructed to conform to the optimized miR-E design, and crucial
structural corrections were made by Dr. Xavier Bofill De Ros, co-author of the shRNA design guidelines,
including the removal of an extra nucleotide in the loop and the addition of a missing base at the lower stem.
**These adjustments allowed the construct to fold identically to validated miR-E backbones**.

Final validation was performed using **RNAstructure**, **RNAfold**, and
**SplashRNA** to confirm correct folding and targeting efficacy.

To enable the insertion of our sequence into the transfer plasmid, we designed custom flanking sites. This
allowed us to generate the final nucleotide sequence as follows:

5’TGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCGACTTCTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG
ACTCACTGATCAGGCCATATTTTAGTGAAGCCACAGATGTAAAATATGGCCTGATCAGTGAGG
TGCCTACTGCCTCGGACTTCAAGGGGCTAGAATTCGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTT
3’

## Why miRE-based shRNA…

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/mirebasedshrna-why-1.webp)

## Curious how miR-E achieves all this? Dive deeper into the science

▼

#### Accurate guide strand selection

**Precise cleavage by Dicer** ensures that the correct strand, the guide strand, is incorporated
into the RNA-induced silencing complex (RISC), **minimizing off-target** effects. Deep sequencing
shows that miR-E constructs are processed accurately over 82% of the time, compared to just 25–70% with
conventional shRNA designs. This high fidelity enhances both the specificity and reliability of gene
silencing1.

#### Lower expression, reduced cytotoxicity

Although miR-E shRNAs are expressed at lower levels than traditional pol III–driven constructs (such as those
using U6 or H1 promoters), their improved processing leads to comparable or superior gene knockdown. The lower
expression level helps to avoid off-target effects, immune activation, and cellular stress, making the system
**especially advantageous for in vivo experiments and therapeutic applications**, where
minimizing toxicity is critical1,2.

#### Tissue-specific and inducible expression

A key limitation of traditional shRNA systems is their use of constitutive RNA polymerase III promoters, which
cannot be easily regulated. In contrast, **miR-E constructs are compatible with RNA polymerase
II** (pol II) **promoters**, which allow for spatial and temporal control of expression.
Researchers can drive shRNA expression in specific tissues, at certain developmental stages, or in response to
drugs. This makes the system **highly versatile for experimental setups** as well as
translational applications, that aim to move from basic science toward clinical or real-world
use1-3.

#### Multiplexed gene regulation

The miR-E system also supports the expression of multiple shRNAs from a single transcript, enabling the
**simultaneous targeting of several genes**. Thanks to optimized processing, each shRNA is
efficiently released and functional. This makes miR-E constructs ideal for coordinated regulation of gene
networks, synthetic biology circuits, and combinatorial strategies, approaches where multiple targets are
modulated together to better study complex pathways or enhance therapeutic outcomes1.

## Our plasmids

Due to limited expertise in neuronal systems, we opted to work with non-neuronal cells, which in turn influenced
our plasmid design strategy.

#### Therapeutic plasmid (K562 experiments)

This plasmid was **primarily designed** to evaluate the efficiency of our miRE-based shRNA in
knocking down HDAC6, and **secondarily** to demonstrate the correlation and rescue of the insulin
signaling pathway upon knockdown in K562 PA-induced insulin-resistant cells.

**In K562 cells**, the use of condition-specific promoters (AgRP and FoxO1), as originally planned,
was not feasible. As a result, we altered our therapeutic design, demonstrating the effectiveness of the shRNA
molecule independently of the AND
gate system [(see project description)](https://2025.igem.wiki/patras-med/description).

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/vb250626-1642qkm-keynotes-2.webp)

## Want the full breakdown of each element?

▼

#### EF1α core promoter

The human EF1α promoter is widely used for driving strong and stable gene expression across various
mammalian cell lines, including HEK293T and K562. Due to its mammalian origin, it is less prone to
epigenetic silencing, making it ideal for sustained expression. As a Pol II promoter, it is also compatible
with miRNA-based shRNA (miRE) expression.

#### Kozak sequence

Positioned just upstream of the start codon, the Kozak sequence improves translation efficiency by ensuring
that the ribosome recognizes the correct initiation site. Its presence is crucial for consistent expression
of the EGFP reporter.

#### EGFP

EGFP was placed upstream of the miRE-based shRNA to act as a visual reporter. Because both are transcribed
from the same promoter, the presence of EGFP fluorescence indirectly confirmed successful transcription of
the shRNA as well. We selected EGFP for its compact size, strong signal, and widespread use in mammalian
cell imaging.

#### WPRE

The Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element improves gene expression by enhancing
mRNA stability and export from the nucleus. It does not interfere with the promoter itself but acts at the
post-transcriptional level. WPRE is a standard element in lentiviral vectors and gene therapy applications.

#### hPGK promoter

The human phosphoglycerate kinase (hPGK) promoter drives constitutive, moderate expression of the puromycin
resistance gene. Its endogenous origin makes it less susceptible to silencing, ensuring reliable selection
in both HEK293T and K562 cells over time.

#### Puromycin

Puromycin is a fast-acting antibiotic that selects cells expressing the pac gene, which encodes puromycin
N-acetyltransferase. It causes premature termination of protein synthesis, allowing us to efficiently
eliminate non-transduced cells. Given the time constraints of our project, puromycin was ideal for rapid
selection.

#### Logic gate & fail-safe plasmids (HEK293T experiments)

#### Proof-of-concept circuits

Before finalizing our AND gate strategy, we tested two candidate proof-of-concept (POC) circuits to model how
independent inputs (like AgRP and FoxO1) could combine into a logical output.

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/option-a-1.webp)

## Curious about how each system worked in detail?

▼

#### Option A – Visual dual-input system (Selected for POC)

Upon doxycycline addition, rtTA was activated, leading to expression of TagBFP2 (blue). Cumate addition
inactivates CymR, allowing expression of
mCherry (red). When both inducers are present, cells co-express TagBFP2 and mCherry, resulting in purple
fluorescence—a fully visual proxy for AND logic. Gal4 and VP16
were excluded, as their function as split activators is already well validated in the literature. Instead,
we focused on a simplified, modular, and visual design.

#### Option B – compact genetic AND gate (Not selected)

Upon cumate addition, CymR is released from the CuO operator, allowing rtTA to be expressed; however,
without doxycycline in the media, rtTA cannot activate TetO, so TagBFP2 is
not produced. When only doxycycline is added, rtTA is not expressed, and TagBFP2 remains off. Only when both
cumate and doxycycline are present does CymR release rtTA and doxycycline binds to rtTA, forming an active
complex that binds TetO and drives TagBFP2 expression.

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/option-2.webp)

While this system functions as a true genetic AND gate, it produces only a single output, offering no
internal control and making negative results difficult to interpret. It also requires molecular assays, such
as qPCR,
for validation, which were not feasible within our budget and timeline. Furthermore, it is more prone to
failure and less visually informative for a proof-of-concept study.

After considering both options, we proceeded with Option A and used it as the basis for our experimental design.
This choice set the direction for the plasmids we constructed and determined the overall workflow of the
project.

These two plasmids demonstrate our **AND gate logic system** and to validate the functionality of
our **fail-safe mechanism based on hsa-miR-7-5p**.

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/vb250714-1090-cvh-keynotes-1.webp)

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/vb250714-1135hje-keynotes-recovered.webp)

For the AND gate, we built on the validated **GAL4-VP16 system**, replacing its DNA-binding and
activation domains with two fluorescent proteins: mCherry and TagBFP2. To enhance biosafety and achieve precise
control, we employed inducible promoters-**Cumate and Doxycycline**-(instead of the AgRP and Foxo1,
[see
project description](https://2025.igem.wiki/patras-med/description)) which function independently of cell type.

Key point: Only when both inducers (cumate and doxycycline) were present, did cells co-express TagBFP2 and
mCherry, thereby recapitulating AND gate behavior.

Fail-safe: As HEK293T cells lack endogenous miR-7-5p, we introduced a synthetic mimic. In its presence,
cumate-induced mCherry expression was suppressed, demonstrating effective disruption of the AND gate output via
RNA interference.

## Want the full breakdown of how each element works?

▼

#### Tet-On System: TRE (TetOx7) and rtTA-M2

The TetOx7 sequence contains seven tetracycline operator (TetO) sites, forming TRE (Tetracycline Response
Element). This promoter is activated only in the presence of doxycycline, which binds to the rtTA-M2
transcriptional activator. The M2 version offers reduced leakiness and improved sensitivity, resulting in
tight and reliable inducible expression of TagBFP2.

#### Cumate system: 2xCuO and CymR

The cumate-inducible system functions similarly to the Tet-On system but does not rely on antibiotics. The
CuO operator sites bind the CymR repressor protein in the absence of cumate, blocking transcription. When
cumate is added, CymR undergoes a conformational change and dissociates from CuO, enabling transcription.
This system regulated mCherry expression in our design.

#### TagBFP2 & mCherry

We used TagBFP2 and mCherry as fluorescent reporters representing two separate inputs. TagBFP2 was expressed
only upon doxycycline induction (via TRE), while mCherry was controlled by the cumate system. Only when both
inducers were present did we observe simultaneous expression of both fluorophores, thereby demonstrating the
successful implementation of our synthetic AND gate.

#### YB\_TATA core promoter

The YB\_TATA promoter is a minimal core promoter (25 bp) known for low basal expression and high
inducibility, ideal for synthetic constructs with limited plasmid space. It ensures minimal leakiness and
strong activation when paired with upstream response elements, in our case TetO5.

#### Minimal CMV

This truncated version of the cytomegalovirus (CMV) promoter is used to drive CymR expression. It offers
strong, constitutive transcription across various mammalian cell types, ensuring consistent repressor
levels.

#### 4x hsa-miR-7-5p target sequences

To implement a fail-safe mechanism, we included four tandem miR-7-5p target sites downstream of mCherry.
This allows post-transcriptional silencing in the presence of a synthetic miR-7-5p mimic, even when
transcription is activated by cumate. The decision to use four sites is supported by literature, which
indicates that fewer sites are ineffective, while more do not significantly enhance repression6.

In a broader context, miR-7-5p is upregulated in AgRP neurons during fasting7. In our therapeutic
design, it targets VP16AD, preventing shRNA production under normal physiological conditions. This acts as a
third biosafety layer, ensuring activation only occurs during pathological states like insulin or leptin
resistance, thereby preventing unintended gene silencing outside the intended therapeutic context.

#### Blasticidin (Bsd)

Blasticidin is an antibiotic used in molecular biology for the selection of genetically modified cells
carrying the blasticidin resistance gene (bsd). It acts by inhibiting protein synthesis, enabling efficient
elimination of non-transfected cells in both prokaryotic and eukaryotic systems.

## Cell cultures

To align with our team’s expertise and available resources, we selected **HEK293T** and
**K562** cell lines, well-established models known for their high transduction efficiency, for
initial validation.

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/cell-line-2.webp)

## Want the full breakdown of how each element works?

▼

#### HEK293T cells

HEK293T is an **adherent cell line** derived from Human Embryonic Kidney (HEK293) cells and
engineered to express the SV40 large T antigen, which enhances both transfection and transduction
efficiency. These cells are widely used in molecular biology and gene therapy studies due to their robust
growth, ease of handling, and high reproducibility. They were selected for our proof-of-concept
experiments, primarily because our lab had prior experience culturing them, and they are readily
accessible.

While neuron-like cell lines such as SH-SY5Y would have been more biologically relevant to our therapeutic
target, our team lacked the expertise and resources to culture neuronal models. In contrast, HEK293T cells
are **extensively characterized in literature, allowing for reliable troubleshooting and
benchmarking of experimental results**. This helped ensure that any unexpected outcomes could be
attributed more confidently to our constructs rather than the cellular model itself.

To maintain consistency and cell health, we followed standard protocols for freezing, thawing, seeding,
and passaging, which are thoroughly documented in our lab notebook.

#### K562 cells

K562 is a **non-adherent, suspension cell line** derived from a patient in blast crisis of
chronic myelogenous leukemia (CML). It is a commonly used model in hematopoietic and molecular biology
research due to its stable growth, high lentiviral transduction efficiency, and ease of maintenance.

K562 cells were specifically chosen for the shRNA-mediated knockdown of HDAC6. Literature indicates that
K562 cells **express high endogenous levels of HDAC6 mRNA and moderate protein levels**,
providing a suitable model for assessing knockdown efficiency both at the transcript and protein level.
Their compatibility with lentiviral systems and biological relevance to our RNAi target made them an ideal
model for validating our miRE-based shRNA.

## Lentiviral production and delivery

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/lentiviral-production-1.webp)

#### Transfection

Lentivirus production began with transient co-transfection of HEK293T cells using the standard three-plasmid
system:

1. Envelope plasmid: *p23* (encodes VSV-G protein)
2. Packaging plasmid (p202): Encodes *Gag, Pol, Rev, Tat*
3. Transfer plasmid.

This protocol was performed using established lipid-based transfection reagents and optimized for high-yield
viral particle production.

#### Virus harvest

72 hours post-transfection, the viral supernatant was collected and filtered through a 0.45 μm PES membrane
to remove cell debris while retaining lentiviral particles.

Following harvest, we quantified the viral titer (method to be specified) to confirm successful virus
production before proceeding with cell transduction experiments.

#### Lentivirus surface modification

Our therapeutic design includes targeted delivery of lentiviral vectors to hypothalamic AgRP neurons via
**intranasal administration**. To enable cell-type specificity, we proposed modifying the viral
surface post-harvest using a click chemistry reaction8. This strategy involves conjugating a
leptin-derived peptide to the envelope, facilitating receptor-mediated endocytosis through interaction with
the LepRb receptor, which is highly expressed on AgRP neurons9,10.

Due to the absence of neuronal cultures and the lack of LepRb expression in our available cell lines,
combined with limited experimental time, we were unable to evaluate this step in vitro.
Furthermore, the click chemistry process under consideration is relatively straightforward, and modeling it
would not provide novel insights. Specifically, obtaining binding rate constants for this reaction
does not contribute meaningful information relevant to downstream analysis or impact the drug delivery
mechanism itself. Additionally, constructing differential equations to describe this system is
challenging due to uncertainties and lack of precise kinetic parameters. Therefore, investing effort into
modeling this aspect is unlikely to advance the project significantly.

## Choice of biomarker: LepRb receptor – 1st layer of safety

An essential requirement for gene therapy is minimizing off-target effects. **To achieve tissue
specificity, we needed a receptor highly expressed in target cells but absent in peripheral
tissues**. We conducted ROC-based expression analyses across multiple brain regions and highly
perfused organs (e.g. liver, heart, lungs, kidneys, skin) and consulted domain experts and our PIs.

Based on bioinformatics data, literature data and expert feedback, we selected the long isoform of the leptin
receptor (LepRb) as the most appropriate cell surface biomarker for our targeted delivery strategy. Its
restricted expression in AgRP neurons provides the foundation for our system’s first biosafety layer.

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/giannis.webp)

## Lentiviral transduction and experimental validation

Lentiviral **transduction** was performed in **K562** cells, while **HEK293T** cells were subjected to **transient co-transfection** of the lentiviral plasmids, without proceeding to viral particle production. For K562 cells, the viral supernatant was collected and used to infect the cells in the presence of polybrene, which enhances viral entry and infection efficiency.
After one week, **selection antibiotics** were applied to both HEK293T and K562 cells to enrich for successfully transduced populations, resulting in stable cell lines expressing the gene of interest.

**Note:** Our original plan included lentiviral packaging and transduction to generate stable cell lines for long-term testing. However, delays in the delivery of the transfer plasmids intended for the HEK293T cells compressed our experimental window. Given these time constraints, and since our goal was to characterize short-term inducible responses within a week rather than assess long-term integration over a month, we deliberately chose to perform transient plasmid transfections instead. While this strategy enabled rapid validation of circuit function, it comes with known limitations: transient expression is heterogeneous across cells, declines over time, and does not fully replicate stable genomic integration. Therefore, the data presented here should be interpreted as **proof-of-principle results**, with stable integration left for future work.

#### K562 experiments – HDAC6 knockdown

![Morphe Project](https://static.igem.wiki/teams/5998/experdesign/k562hardcore-2.webp)

In K562 cells, transduction efficiency was evaluated by monitoring EGFP expression via fluorescence
microscopy, confirming transcription from the same promoter driving the miRE-based shRNA. **To assess
knockdown efficiency**, we performed **mechanistic assays** involving:
**RT-qPCR** to quantify *HDAC6* mRNA levels and **flow cytometry** to
measure ***HDAC6*** protein expression, confirming successful knockdown at both the
transcript and protein levels.

Next, we performed a **functional assay** to investigate the relationship between
*HDAC6* and insulin signaling. Given that obese patients with insulin resistance often exhibit
elevated *HDAC6* levels, we hypothesized that HDAC6 knockdown could restore insulin sensitivity.

To test this, we carried out **immunofluorescence microscopy** (IF) to examine the subcellular
localization of **phosphorylated Foxo1** (pFoxo1), a downstream effector of the
**PI3K/AKT** pathway, as a readout of insulin pathway activation.

For this assay, K562 cells were treated with **palmitic acid (PA) overnight** to induce insulin
resistance, **serum-starved for 4 hours**, and subsequently stimulated with **insulin
for 15 minutes** to activate PI3K/AKT signaling. Control (non-transduced) and *HDAC6*
knockdown cells were processed in parallel.

We expect that comparative analysis of **pFoxO1 localization** and signal intensity will reveal that **HDAC6 knockdown**
restores insulin signaling, supporting our hypothesis that HDAC6 contributes to insulin resistance and that its reduction could enhance insulin sensitivity in obese patients.

#### HEK293T experiments – Logic gate and fail-safe validation

To experimentally validate our circuit architecture, we planned a series of transduction and induction assays in HEK293T cells. Following transient co-transfection of lentiviral plasmids and antibiotic selection, only successfully transduced cells were maintained to ensure stable construct integration.

To assess the functionality of our dual-inducible logic gate, cells were exposed to doxycycline and cumate individually and in combination11. This setup allows us to evaluate whether each inducer specifically activates its corresponding module and whether co-stimulation leads to concurrent activation (magenta colour), as predicted for our AND gate design.

To examine the robustness of our fail-safe mechanism, we designed an additional condition in which cells are co-transfected with a synthetic hsa-miR-7-5p mimic. This step aims to test whether the safety circuit can suppress downstream expression in the presence of the miRNA, even when the corresponding inducer is present-verifying post-transcriptional control at the design level.

## References

▼

1. An Optimized microRNA Backbone for Effective Single-Copy RNAi.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/24332856/)
2. Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial
   miRNA targeting luciferase and Apolipoprotein B100.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/22827812/)
3. Guidelines for the optimal design of miRNA-based shRNAs.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/27083402/)
4. Creating an miR30-Based shRNA Vector.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/23818675/)
5. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in
   Mammalian Cells.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/26883397/)
6. A mixed antagonistic/synergistic miRNA repression model enables accurate predictions of multi-input
   miRNA sensor activity.
   [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6014984/)
7. Fasting-induced miR-7a-5p in AgRP neurons regulates food intake.
   [PubMed](https://pubmed.ncbi.nlm.nih.gov/38942170/)
8. Lipid-based nano-carriers for the delivery of anti-obesity natural compounds: advances in targeted
   delivery and precision therapeutics.
   [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12057057/#Sec24)
9. Novel mechanisms involved in leptin sensitization in obesity.
   [ScienceDirect](https://www.sciencedirect.com/science/article/abs/pii/S0006295224001126)
10. Leptin and Obesity: Role and Clinical Implication.
    [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8167040/)
11. New dual inducible cellular model to investigate temporal control of oncogenic cooperating genes.
    [Nature](https://www.nature.com/articles/s41598-024-71227-3)

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* [Experiment 1: HDAC6 knockdown validation](#exper1)
* [Experiment 2: AND Gate & Fail-safe system validation](#exper2)
* [Experiment 3: HDAC6 KD and insulin pathway response](#exper3)

## Experiment 1: HDAC6 knockdown validation

To verify the functionality of our therapeutic RNA interference design, we conducted an initial validation of
HDAC6 silencing in K562 cells.

**Objective**: To confirm the efficiency and specificity of our miR-E-based
therapeutic construct in downregulating HDAC6 expression.

**Methods overview**: Lentiviral particles carrying the **EF-1α-driven miR-E
shRNA** targeting **HDAC6**, along with an **EGFP reporter** and
**puromycin resistance cassette**, were produced and used to transduce K562 cells. Transduction
efficiency was assessed through EGFP fluorescence.

![Morphe Project](https://static.igem.wiki/teams/5998/results/vb250626-1642gkm-addgenes.webp)

**Results**: Transduction was successful, as evidenced by strong EGFP fluorescence in
treated cells **(Fig. 1A).**

RT-qPCR analysis revealed a **4.02-fold (≈75.1%) reduction in two biological replicates (Fig.1B)**
in HDAC6 mRNA levels compared to untransduced controls, confirming robust transcriptional knockdown.

Flow cytometry analysis across two independent biological replicates further demonstrated a **30-35%
decrease** in HDAC6 protein levels, validating suppression at the protein level **(Fig.1E,
1F).**

To verify specificity, **α-tubulin** was used as a housekeeping marker. Tubulin expression
remained unchanged in both replicates, indicating that the knockdown selectively targeted HDAC6 without
affecting unrelated proteins **(Fig.1C, 1D).**

Since additional stock of transduced cells was available, we proceeded with **immunofluorescence staining
to visualize HDAC6 expression**. The analysis revealed a **clear reduction in HDAC6 signal
intensity** in cells treated with our construct compared to untransduced cells, further confirming
effective silencing **(Fig.1G).**

**Conclusion**: The miR-E shRNA construct achieved efficient and specific knockdown
of HDAC6 at both the mRNA and protein levels, as confirmed by qPCR, flow cytometry and immunofluorescence. This
experiment lays the groundwork for the subsequent validation of our therapeutic concept.

#### Figures

![Key Advantage 1](https://static.igem.wiki/teams/5998/giannis/eleni1.avif)
![Key Advantage 3](https://static.igem.wiki/teams/5998/giannis/eleni2.avif)

**Figure 1A.**
*EGFP fluorescence confirming successful transduction.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/3.webp)

**Figure 1B.** *RT-qPCR showing
HDAC6 mRNA reduction in K562 cells transduced with miR-E shRNA at 100 μL and 200 μL virus/mL.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/plots-a-tub.webp)

**Figure 1C.** *Flow cytometry
plots showing α-tubulin (AF647) expression. Results represent two independent experiments.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/4.webp)

**Figure 1D.** *Quantification
from two independent experiments showing the percentage of α-tubulin-positive cells. Knockdown of HDAC6 did
not alter the total α-tubulin protein levels.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/plot-hdac6neww.webp)

**Figure 1E.** *Flow cytometry
plots showing HDAC6 (PE) expression. Results represent two independent experiments.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/6.webp)

**Figure 1F.** *Quantification
from two independent experiments showing the percentage of HDAC6 - positive cells. Results show reduction of
HDAC6 protein upon knockdown (KD) in K562 cells.*

![Morphe Project](https://static.igem.wiki/teams/5998/results/eikonatelos.webp)

**Figure 1G.** *K562 cells
transduced with 200 µL lentiviral miR-E-based shRNA targeting HDAC6 show reduced HDAC6 immunofluorescence
compared to untransduced (UT) cells.*

## Experiment 2: AND Gate & Fail-safe system validation

**Objective:** To validate the dual-input AND gate logic and the integrated
fail-safe mechanism containing four tandem miR-7-5p target sites, demonstrating selective circuit activation and
miRNA-dependent repression under physiologically relevant conditions.

**Methods overview:** To simulate the layered logic of Morphe’s therapeutic system,
we built a **dual-inducible visual circuit** composed of two complementary plasmids:

* **Plasmid A:** Drives the reporters **TagBFP2** and
  **mCherry** under **doxycycline- and cumate-inducible promoters**, respectively.

  ![Morphe Project](https://static.igem.wiki/teams/5998/results/plasmid-1.webp)
* **Plasmid B:** Provides the constitutive expression of the **rtTA**
  and **CymR** regulators required for the two induction systems.

  ![Morphe Project](https://static.igem.wiki/teams/5998/results/plasmid-2.webp)

Together, these plasmids operate as a **biological logic processor**, expressing **blue
(TagBFP2)**, **red (mCherry)** or **both (purple)** depending on the
combination of inducers.

Because lentiviral packaging was time-limited, both plasmids were transfected in HEK293T cells with HBS
(HEPES-buffered saline) and CaCl₂. Each plasmid expressed a distinct antibiotic resistance gene (blasticidin and
puromycin), allowing dual selection (10 µg/mL blasticidin + 2 µg/mL puromycin) to enrich double-positive cells.

#### Induction Matrix

![Morphe Project](https://static.igem.wiki/teams/5998/results/matrix2.webp)

**Notes:** No combinations of mimic and siRNA or triple-inducer setups were included
in this experiment. Doxycycline was intentionally omitted from all mimic and siRNA conditions, as the mimic is
antibiotic sensitive. Each condition was tested in three technical replicates to ensure reproducibility and
reliable comparison across treatments.

**Results:** Confocal microscopy revealed distinct and reproducible fluorescence
patterns corresponding to inducer combinations.

**Control cells** showed little to no detectable fluorescence, confirming minimal background
activity. TagBFP2 was visible upon no induction indicating low basal leaky expression (**Fig.2A**,
upper left).

**Single induction** with doxycycline or cumate activated **TagBFP2 (blue)** or
**mCherry (red)**, respectively (**Fig.2A** second row).

**Dual induction** activated both reporters: the **purple signal** observed
represents the **merged overlap of blue and red fluorescence channels** in the confocal composite,
confirming correct AND-gate behavior (**Fig.2A**, down right 3rd row).

In **miR-7-5p mimic** conditions, **mCherry** fluorescence driven by the cumate
promoter was **completely absent**, while **TagBFP2** expression remained unaffected
(**Fig.2B**, 3rd and 4th row, 2nd column).

In contrast, **scramble siRNA-treated cells** retained **detectable red
fluorescence** (**Fig.2A**, 2nd row, 2nd column), though weaker than in the cumate-only
condition. This attenuation was attributed to **cellular stress** rather than incomplete
repression, since the cells underwent **CaCl₂ transfection, dual antibiotic selection**,
and **serum-free exposure in Opti-MEM** during transfection. To mitigate this, the **mimic
incubation period was extended to 48 h** (followed by 24 h induction) to allow recovery after medium
replacement.

Overall, the circuit was activated only under dual induction and effectively repressed in the presence of
miR-7-5p, validating both the precision of the AND gate and the reliability of the fail-safe mechanism.

**Conclusion:** The dual-inducible circuit successfully demonstrated AND gate
behavior, being activated only under simultaneous doxycycline and cumate induction. The integrated fail-safe
module responded precisely to miR-7-5p mimic treatment, completely suppressing mCherry expression while leaving
TagBFP2 unaffected. **These findings confirm the expected logic and specificity of the designed regulatory
system**, although extended quantitative studies will be required to evaluate long-term stability and
reliability.

TagBFP2 fluorescence was detectable without doxycycline induction **(Fig. 2A)** , indicating low basal expression
under control conditions. This likely reflects the TRE-YB\_TATA promoter architecture, cellular stress responses
and the use of regular FBS instead of Tet-free FBS, rather than genuine promoter leakiness.

**Limitations:** This experiment was performed under short-term transient
transfection, preventing long term evaluation. Follow-up assays are required to assess **signal stability,
reversibility, and durability** under sustained induction and eventual lentiviral integration.

#### Figures

![Morphe Project](https://static.igem.wiki/teams/5998/results/dox-cum.webp)

**Figure 2A.** *HEK293T cells
transfected with the dual-plasmid system (TRE\_YB-TATA\_TagBFP2 / rtTA\_CymR).*

Fluorescence images show uninduced cells, Dox induction, and combined Dox + cumate induction. Dox activates
TagBFP2, cumate induces mCherry, and co-induction leads to magenta/purple co-expression.

![Morphe Project](https://static.igem.wiki/teams/5998/results/mimic.webp)

**Figure 2B.** *HEK293T cells
transfected with the miRNA mimic upon cumate induction, compared with untreated cells and those transfected by
the scramble siRNA.*

## Experiment 3: HDAC6 KD and insulin pathway response

**Objective:** To evaluate whether **HDAC6 knockdown** could promote a
**metabolic rescue of insulin signaling** under obesity-mimicking, lipotoxic conditions induced by
**palmitic acid (PA)**.

**Method overview:** K562 cells were treated with **PA at 100 µM (data not
shown) and 200 µM** , followed by **serum starvation** and **insulin
stimulation**. Following treatment cells were fixed and stained for **phospho-FoxO1
(p-FoxO1)** to evaluate insulin-pathway activation by immunofluorescence.

![Morphe Project](https://static.igem.wiki/teams/5998/results/results-1.webp)

Each concentration was combined with **three viral transduction levels** (UT, 100 µL virus, 200 µL
virus), resulting in six total conditions:

* UT + 100 µM PA + starvation + insulin
* 100 µL virus + 100 µM PA + starvation + insulin
* 200 µL virus + 100 µM PA + starvation + insulin
* UT + 200 µM PA + starvation + insulin
* 100 µL virus + 200 µM PA + starvation + insulin
* 200 µL virus + 200 µM PA + starvation + insulin

Each condition was performed in **two technical replicates**.

**Results:** p-FoxO1 staining was **successfully detected** in all
groups, confirming antibody performance and reproducibility between replicates.

However, **no measurable change in p-FoxO1 signal intensity** was observed between
**HDAC6-knockdown** and **untransduced (UT)** cells at either palmitate concentration.

Similarly, **no localization shift** of p-FoxO1 was detected, indicating that HDAC6 silencing
**did not modify insulin-pathway activation** in this cellular context.

Confocal imaging was performed only for the **200 µL virus groups**, which showed the highest
transduction efficiency the **100 µL virus** conditions were not imaged but appeared similar under
fluorescence microscopy.

**Conclusion:** This assay showed **no detectable change in p-FoxO1 levels or
localization** following HDAC6 knockdown, suggesting that **insulin-pathway activation was
unaffected** in K562 cells under lipotoxic stress.

Nevertheless, the experiment confirmed the **technical feasibility** of integrating viral
transduction, palmitate treatment, and insulin stimulation in suspension cultures.
**K562 cells** are **leukemic and predominantly glycolytic (Warburg effect)**, meaning
they exhibit minimal **insulin receptor-mediated signalling**, which likely masks potential rescue
effects.

Future experiments will focus on **insulin-responsive models** (e.g. 3T3-L1 adipocytes, HepG2
hepatocytes) using quantitative **readouts** such as **p-Akt (Ser473)** to reveal any
true metabolic rescue.

**Limitations:** The use of **K562 cells**, which exhibit the
**Warburg effect** and limited insulin receptor activity, restricts the ability to model canonical
insulin-responsive pathways.

**Therefore, these findings primarily reflect technical feasibility rather than physiological recovery of
insulin sensitivity.**

Follow-up studies should incorporate **quantitative assays** (e.g. p-Akt or glucose uptake
measurements) in **metabolically active cell lines** to confirm HDAC6’s role in restoring insulin
responsiveness.

#### Figures

![Morphe Project](https://static.igem.wiki/teams/5998/results/7.webp)

**Figure 3A.** *K562 cells
pre-treated with palmitic acid (200uM), serum-starved, acutely stimulated with insulin (100nM). Cells are
stained with primary p-Foxo1 and secondary AF 647 antibodies.*

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