The siREN project introduces an innovative RNA interference (RNAi) therapeutic strategy for Chronic Lymphocytic Leukemia (CLL), a cancer of the blood and bone marrow marked by the buildup of abnormal B lymphocytes ¹.

Our strategy uses two precisely designed small interfering RNAs (siRNAs) to silence BCL2 and BTK, two genes essential for CLL cell survival and proliferation. By the targeted and selective degradation of their corresponding mRNAs, we aim to remove the molecular supports that keep leukemic cells alive and resistant to treatment ^{2 3}.

siRNAs provide unmatched specificity, recognizing only their intended targets and minimizing off-target effects. Furthermore, they act catalytically, degrading multiple transcripts even at low doses ⁵, and their modular design allows rapid development against virtually any disease-driving genes ⁶.

A critical component of our strategy is a targeted delivery system. We encapsulate these siRNAs within lipid nanoparticles functionalized to target ROR1, a receptor expressed in over 95% of CLL cells, but largely absent in healthy tissues ^{7 8}. This targeted system acts like a "Trojan horse"—selectively entering CLL cells via ROR1 and releasing the siRNAs into the cytoplasm, silencing BCL2 and BTK directly.

In summary, this dual-targeted, ROR1-guided approach offers high tumor specificity, low systemic toxicity, and the potential to overcome anti-leukemic therapy resistance by attacking the disease at its genetic roots.

One of the defining features of Chronic Lymphocytic Leukemia (CLL) is the malignant B cells’ dependence on the anti-apoptotic protein BCL-2 for survival. Overexpression of BCL2 occurs in approximately 95% of CLL cases compared to normal peripheral blood lymphocytes, and enables leukemic cells to evade programmed cell death (apoptosis). This dysregulation promotes uncontrolled proliferation through aberrant B-cell receptor (BCR)-mediated signaling, contributing to uncontrolled proliferation, disease persistence and therapy resistance ⁹.

High levels of BCL2 have been detected across numerous human lymphoid malignancies, including follicular lymphoma, multiple myeloma, and B-cell chronic lymphocytic leukemia, highlighting its central role in cancer development and resistance to treatment ^{10 11}. The deregulation of BCL2 family proteins allows cancer cells to resist apoptotic signals, leading to (chemo)therapy resistance and poor clinical outcomes.

Given its critical role in cell survival, disease progression, and treatment resistance, BCL2 represents a highly validated and therapeutically actionable target in CLL. Silencing BCL2 expression through RNA interference (RNAi) offers a precise and potent strategy to overcome resistance and improve treatment efficacy.

To test our hypothesis that BCL2 is overexpressed in chronic lymphocytic leukemia (CLL), we analyzed its expression in the MEC-1 cell line, which originates from CLL. RNA was extracted using two different protocols, and subsequent qPCR analysis using two distinct primer sets successfully amplified the BCL2 transcript in MEC-1 cells, confirming that the gene is actively expressed. The observed Cq values (~21–22) indicated moderate to high expression levels, which was consistent when compared to two other lymphoma cell lines, namely JMP-1 and SUP-M2. This expression profile aligns with BCL2’s known role as a key anti-apoptotic factor in malignant B cells.These findings validate MEC-1 cells as a model that robustly expresses BCL2, and provide a reliable system for evaluating BCL2-targeting siRNAs in subsequent proof-of-concept experiments. For detailed information, see our Results Page.

Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase that plays a central role in B-cell receptor (BCR) signaling, driving the proliferation, differentiation, and survival of both normal and malignant B cells. Proper regulation of BTK activity is essential for B-cell homeostasis, as aberrant activation of the BCR pathway is a hallmark of B-cell malignancies, including Chronic Lymphocytic Leukemia (CLL) ¹².

BTK is overexpressed and constitutively phosphorylated in CLL cells compared to normal B cells, leading to persistent BCR signaling that promotes leukemic growth and survival. This dysregulated signaling not only leads to autonomous cell survival but also enhances post-survival interactions with the tumor microenvironment (TME), creating a supportive niche that further protects CLL cells from apoptosis ¹³.

Targeting BTK has therefore emerged as a breakthrough therapeutic strategy in the treatment of CLL. BTK inhibitors (BTKis), such as ibrutinib, acalabrutinib, and zanubrutinib disrupt BCR signaling by inactivating BTK, leading to impaired leukemic cell proliferation and decreased microenvironmental support ¹⁴. The success of BTK inhibition in clinical settings validates BTK as a key oncogenic driver and therapeutic target.

Our siREN strategy offers a complementary and potentially, more precise approach: the direct silencing of BTK mRNA through RNA interference (RNAi). By eliminating the BTK-dependent signaling at its origin, we aim to achieve a profound and durable suppression of the BCR pathway, potentially enhancing efficacy while overcoming resistance mechanisms that can arise from long-term kinase inhibitor use.

Note: Experimental validation of BTK expression in MEC-1 cells is a planned next-step experiment, essential to confirm this model’s suitability for BTK-targeting experiments.

Delivering BCL2-targeting siRNAs into MEC-1 cells using INTERFERin as the delivery system is the critical first step for RNA interference. We hypothesized and tested whether INTERFERin could effictively deliver BCL-2 targeting siRNAs into MEC-1 cells across a concentration gradient (5–50 nM).

Since the effectiveness of RNA interference depends on the intracellular delivery of siRNA molecules, we aimed to determine whether MEC-1 cells are capable of internalizing siRNAs under varying conditions. To visualize and quantify the kinetics and efficiency of siRNA uptake, we transfected MEC-1 cells with a Cy3-labeled BCL2 siRNA and measured fluorescence intensity at multiple timepoints (6 h, 24 h, and 48 h).

Fluorescence measurements confirmed that cellular uptake occurred at both 20 nM and 50 nM concentrations, but with distinct kinetic profiles. At 20 nM, a distinct fluorescence peak was observed at 6 h, indicating rapid, efficient siRNA internalization through active endocytosis, followed by a gradual decrease and stabilization by 24–48 h, consistent with possible intracellular processing and engagement in gene silencing. In contrast, at 50 nM, fluorescence intensity continued to rise over time, suggesting saturation of the delivery pathway and possible vesicular accumulation of siRNA within endosomal vesicles, which may limit cytoplasmic availability.

These findings validate that Cy3-labeled siRNAs can be effectively delivered into MEC-1 cells using INTERFERin at higher concentrations into MEC-1 cells, with 20 nM representing an optimal concentration for efficient uptake and productive silencing rather than mere accumulation. For detailed information, see our Results Page (Figure 2-3).

Chronic lymphocytic leukemia (CLL) cells are known to rely heavily on BCL2 for their survival, as this anti-apoptotic protein prevents programmed cell death and allows malignant B cells to persist. The overexpression of BCL2 enables leukemic cells to evade apoptosis, sustaining their uncontrolled proliferation through continuous B-cell receptor (BCR)-mediated signaling. We hypothesized that by targeted silencing BCL2 expression with siRNA, we aim to disrupt this protective survival mechanism, thereby restoring the apoptotic pathway and selectively promoting the cell death in CLL MEC-1 cell model ⁹.

First Experiment: Contrary to our initial hypothesis, our initial experimental data revealed a distinct phenotypic outcome, where siRNA-treated MEC-1 cells did not exhibit clear signs of apoptosis, within the first 48h. Instead, cell growth and viability analyses indicated a potent yet transient cell cycle arrest, primarily observed at the 24-hour timepoint, particularly at 20 nM and 50 nM concentrations. Beyond 48 hours, the cells showed signs of recovery, and overall viability remained high, suggesting that BCL2 silencing led to temporary growth inhibition, initiating a cytostatic response, rather than immediately forcing cells to sustain apoptotic death. For detailed information, see our Results Page (Figure 4-5).

Based on the findings from our first experiment, we adjusted key parameters of our transfection protocol to enhance siRNA uptake and consistency. A second experiment was then conducted under these optimized conditions to confirm the improvement in delivery efficiency. For detailed information, see our Results Page.

Second Experiment: A follow-up experiment was conducted to further investigate the phenotypic effects of BCL2 silencing. Consistent with our first experiments’ results, transfection of BCL2-targeting siRNA did not confirm apoptotic effects. Instead, cell growth increased between 24h and 48h across all conditions, indicating that the cells remained viable and proliferative. The strongest effect was observed at 20 nM, where siRNA was efficiently internalized and processed without compromising viability. In contrast, at 50 nM, a mild growth inhibition was detected, likely due to cellular stress caused by excessive siRNA uptake.

These findings suggest that BCL2 silencing alone, under the tested conditions, is not sufficient to trigger apoptosis in MEC-1 cells. The primary outcome is a transient influence on cell cycle dynamics and stress responses in a concentration-dependent manner. For detailed information, see our Results Page.

Transfection of the CLL-derived MEC-1 cell line with BCL2-targeting siRNA will lead to a reduction in BCL2 mRNA levels compared to the control, through the catalytic action of the the RNA-induced silencing complex (RISC). RISC is known to specifically degrade complementary mRNA transcripts, thereby preventing their translation into protein ^{3 15}. Through this mechanism, BCL2 siRNA should directly silence the target mRNA, removing key molecular supports for cell proliferation and survival.

To assess whether BCL2-targeting siRNA effectively silenced gene expression in MEC-1 cells, BCL2 mRNA levels were measured at 6h and 24h post-transfection using two independent primer sets. The data revealed a dynamic transcriptional response. At 6h, a transient increase in BCL2 expression (~5-fold) was observed at lower concentrations (5–10 nM), suggestive of an early cellular stress response to the transfection process. By 24h, mRNA levels decreased across all siRNA conditions, with the most pronounced effects at 20 nM and 50 nM. Specifically, BCL2 mRNA levels were reduced by approximately 20% at 20 nM and by 40–50% at 50 nM relative to the controls. Control treatments (scrambled siRNA and INTERFERin alone) showed no significant changes after 24h, confirming that the reduction was siRNA-specific. These findings are consistent with the cellular uptake results, indicating effective siRNA internalization and activity at these concentrations. Although the results were not statistically significant, they establish a clear biological tendency that aligns directly with our other results, linking the cellular uptake, mRNA silencing, and overall cellular and functional response, strengthening our hypothesis for active, specific RNAi activity. For detailed information, see our Results Page (Figure 8-9).

To complete the efficacy validation of our siRNA-mediated silencing approach, we plan to perform a Western blot analysis to evaluate BCL2 protein levels. This critical next step will confirm whether the observed decrease in BCL2 mRNA levels translates to a reduced protein production, thereby verifying the effectiveness of the transfection. While this experiment could not be completed within the project deadline, it is essential for confirming that the observed downregulation of BCL2 mRNA translates into a corresponding fainter BCL2 band compared to the control, indicating lower protein abundance following siRNA treatment. Based on our mRNA results, we anticipate the most pronounced reduction at the 24h and 48h timepoints, particularly at the 20 nM and 50 nM concentrations where BCL2 mRNA silencing was most effective.