Chronic Lymphocytic Leukemia (CLL) is the most common type of Leukemia in adults in Western countries. It is a type of blood cancer that develops from the bone marrow where a type of white blood cells, lymphocytes, start to grow uncontrollably. Over time, these cells enter the bloodstream, leading to a decrease in healthy blood cells and subsequently affecting the immune system ¹. Regardless of the developments being made in the field in recent years, there are still patients facing difficulties with their therapeutic journey due to the side effects, treatment resistance, and high relapse rates ².

This is where we step in! As an iGEM team, we started our brainstorming right after the 2025 team formation in late January. We are a multidisciplinary team comprising 11 undergraduate students from 6 different departments. While our academic backgrounds may differ, we quickly discovered our common goal, making real world impact in the field of oncology. Hematological malignancies emerged as a key focus during our initial brainstorming sessions. However, it was through our discussions with Dr. Papaioannou and Dr. Georgiou (see our Integrated Human Practices Page) that we truly came to understand the significant, unmet needs of patients with Chronic Lymphocytic Leukemia (CLL). From that moment forward, the vision was clear and siREN was set in motion.

siREN is a novel therapeutic approach for the treatment and management of Chronic Lymphocytic Leukemia (CLL). By targeting the overexpressed BCL-2 and BTK proteins through siRNA-mediated gene silencing, we aim to inhibit the uncontrolled proliferation of leukemic cells and induce apoptosis. Our goal is to suppress the survival pathways driving disease progression, thereby restoring normal cell death mechanisms.

This gene silencing will be achieved by delivering siRNAs into cancerous cells via LNPs; doubly engineered to localize to secondary lymphatic tissues and to specifically target CLL cells. With an FDA-approved “gold standard” base lipid composition, we employ two strategies -for specific targeting of either spleen or lymph nodes- limiting accumulation in non-affected organs and tissues. These modified lipid nanocarriers are additionally equipped with Fab’ fragments designed to selectively interact with the surface of target cells and release the siRNA payload inside their cytoplasm: effectively silencing the genes responsible for CLL pathogenesis, while minimizing interaction with healthy cells.

The name siREN has a dual meaning, combining the name of our little soldiers, siRNAs and the mythological beings sirens. Sirens in Greek mythology are depicted as half-bird, half-woman creatures who used their enchanting voices to lure sailors to destruction ³. Our lipid nanoparticles take that role by locating the cancerous cells (the sailors) and using siRNAs (the enchanting voice) to annihilate them.

In the previous section, we explored what our project stands for, but we have yet to address a fundamental question: “What is the unmet need in CLL?”. To provide a clear answer to this question, we will outline key facts regarding the clinical landscape of the disease, focusing on the challenges patients face during their therapeutic journey.

Chronic Lymphocytic Leukemia constitutes 25-30% of all leukemia cases in Western countries ⁴, while, in contrast, it is rare among Asian and Arab populations ⁵. Globally, the number of patients annually diagnosed with this disease has been increasing. Just in the USA, it is estimated that there would be approximately 23,690 new cases in 2025 accounting for 1.2% of all new cancer cases ⁶. The average age of diagnosis ranges from 65-70 years, affecting mostly older adults while being rare for people under 40 ⁷. Furthermore, men are twice as likely to develop CLL as women ⁴.

A challenging aspect of Chronic Lymphocytic Leukemia is that most patients have no symptoms when they are first diagnosed. The majority of cases are incidentally identified during routine blood testing, which reveals an elevated white blood cell count ⁸. As the disease slowly progresses, it can cause a variety of symptoms. However, these are often vague and nonspecific ¹. Common symptoms include: enlarged lymph nodes, fatigue, fever, night sweats, and unintentional weight loss ⁴. As abnormal cells take over the blood and marrow, the resulting RBC and platelet underproduction can cause anemia and excessive bleeding. CLL can also undermine the immune system’s function, making patients more susceptible to infections, other cancers and autoimmune conditions ⁹. These symptoms are non-specific and not exclusive to CLL, which is why a suitable diagnosis is made based on laboratory test results.

In Chronic Lymphocytic Leukemia (CLL), as in most cancers, aberrant survival pathways are the driving force behind disease progression. Apoptosis, the programmed form of cell death, involves a sequence of biochemical events culminating in the activation of intracellular proteases known as caspases. The Bcl-2 protein family plays a pivotal regulatory role in this process. BCL2 was the first proto-oncogene identified to have anti-apoptotic properties, contributing to enhanced cell survival. Multiple independent studies have consistently demonstrated that overexpression of the anti-apoptotic BCL-2 protein is a hallmark of CLL ¹².

Bruton’s tyrosine kinase (BTK) plays a particularly critical role in the development and progression of chronic lymphocytic leukemia (CLL), making it an important therapeutic target. Silencing BTK expression using siRNAs offers a promising approach to limiting disease progression by inhibiting the uncontrolled proliferation of malignant B cells. Specifically, BTK inhibition has been shown to promote apoptosis and suppress the proliferation of CLL cells. The therapeutic relevance of BTK is further underscored by its consistent expression in CLL cells (minimizing the off-target effects) and its central role as a target in several existing treatment options ¹³.

Mechamism Explained

In CLL, the anti-apoptotic protein BCL‑2 is overexpressed, often due to genetic alterations such as the deletion of miRNA regulators on chromosome 13q14, enabling malignant B‑cells to evade mitochondrial-mediated apoptosis ¹⁴. By sequestering pro-apoptotic proteins like BIM, BAX, and BAK at the mitochondrial outer membrane (MOM), BCL‑2 blocks mitochondrial outer membrane permeabilization (MOMP), which prevents cytochrome c release and downstream caspase activation. This alteration in the balance of BCL-2 family proteins promotes cell survival and supports the clonal expansion characteristic of CLL ¹⁵. Functionally, CLL cells are highly “primed” for apoptosis, meaning they contain elevated pro-death signals held in check by BCL‑2, and are thus severely dependent on BCL‑2 for survival. BH3‑profiling studies across dozens of primary CLL samples demonstrate strong cytochrome c release in response to BCL‑2–specific BH3 peptides, underscoring this critical dependency ^{16 17}. Therapeutically, agents inhibiting BCL‑2 restore the apoptotic pathway by displacing pro-apoptotic effectors from BCL‑2 complexes, leading to rapid apoptosis in CLL cells independent of TP53 status ¹⁴.

In normal mature B-cells, the B-cell receptor (BCR) is composed of a membrane-bound immunoglobulin (Ig) receptor associated with the signaling subunits Igα (CD79A) and Igβ (CD79B). Under normal conditions, the BCR delivers two types of signals: a constitutive, antigen-independent survival signal mediated through PI3K, and a second signal triggered by antigen engagement. The latter promotes the recruitment and activation of intracellular signaling molecules such as LYN, SYK, Bruton’s tyrosine kinase (BTK), and PI3K, leading to downstream signaling cascades that result in cell activation and proliferation. Upon antigen binding, LYN and SYK kinases phosphorylate BTK. In CLL, BTK is often overexpressed, which enhances pro-survival signaling pathways and contributes to disease progression ¹⁸.

BTK functions as a critical downstream mediator in BCR signal transduction. During B-cell development, its activation leads to the stimulation of four key tyrosine kinase pathways: PLCγ2, MAPK, NF-κB, and the serine/threonine kinase AKT, all of which are essential for promoting B-cell survival and proliferation ¹⁹. When BTK activity is pharmacologically inhibited in CLL, the signaling interactions between leukemic cells and their supportive microenvironment are disrupted, thereby preventing the transmission of survival cues and promoting apoptosis in malignant lymphocytes ¹⁸.

Small interfering RNAs (siRNAs) are short (20–24 nucleotide) double-stranded RNA molecules that play a key role in the RNA interference (RNAi) pathway ²⁰. They are exogenously introduced or processed from longer double-stranded RNA (dsRNA) by the enzyme Dicer into fragments with characteristic 2‑nt 3′ overhangs. These siRNA duplexes are then loaded into the RNA-induced silencing complex (RISC), where one strand (the guide strand) is selected based on thermodynamic stability and retained, while the other (passenger strand) is cleaved and discarded ²¹.

Once assembled, the siRNA–RISC complex scans for messenger RNAs (mRNAs) that are fully complementary to the guide strand. Upon binding, Argonaute-2 (Ago2), a catalytic component of RISC, cleaves the target mRNA at a specific site, triggering its degradation and effectively silencing the gene’s expression ²². This mechanism is highly specific and efficient in repressing target genes, making siRNAs invaluable tools in research for gene knockdown and increasingly promising agents in therapeutic settings ²⁰.

Small interfering RNA (siRNA) targeting BCL2 efficiently downregulates the anti-apoptotic protein in primary CLL cells by binding to its mRNA and triggering its degradation. A transfection of BCL‑2–specific siRNA into CLL cells will lead to a significant, time-dependent decrease in BCL‑2 mRNA ²³. Importantly, targeting BCL‑2 by siRNA maximally affects CLL cells due to their high dependence on this protein for survival ²⁴. Cells pre-treated with BCL‑2 siRNA exhibit lower apoptotic thresholds, allowing chemotherapeutic drugs to more easily push them beyond the death threshold. This approach could affect cancerous cells and lead them to apoptosis, helping CLL patients deal with CLL ²⁵.

Targeting BTK with siRNA in CLL cells results in potent disruption of critical survival mechanisms. BTK knockdown leads to a decrease in downstream BCR signaling, manifested as reduced phosphorylation of PLCγ2 and AKT. As a result, the disrupted signal transduction weakens cell–stroma interactions necessary for leukemic cell survival, enhancing sensitivity to apoptotic stress. siRNA-based BTK silencing is a powerful strategy for dismantling multiple pro-survival pathways in CLL ^{26 27}.

The synergistic decrease of BCL-2 and BTK, in both proliferation and anti‑apoptotic defense, frequently induces robust apoptosis in CLL cells. Dual siRNA strategies targeting both BCL‑2 and BTK is vital in overcoming resistance inherent to monotherapies ²⁸.

An effective treatment with the minimum possible off-targets requires a specific delivery system to CLL cancer cells. Small interfering RNA (siRNA) encapsulated in modified lipid nanoparticles (LNPs) offers a promising strategy for targeted gene silencing in chronic lymphocytic leukemia cells by overcoming traditional delivery barriers ²⁹. With the help of stakeholders in the field of nanomedicine, such as Dr. Pippa, Dr. Karatasos, and Dr. Demetzos (see our Integrated Human Practices Page), we were able to outline a delivery strategy with an emphasis on precision and efficacy.

Following current RNAi “gold standards”, we have designed LNPs consisting of an ionizable lipid, cholesterol to improve cell entry and structure, a zwitterionic helper phospholipid, and a protective PEG coating to enhance stability and circulation time. The ionizable lipid provides the opportunity for efficient siRNA encapsulation by electrostatic interactions in its cationic form, while assuming a relatively neutral net charge in physiological pH; a fact crucial for avoiding toxicity or immunogenicity ³⁰. After endocytosis, the acidic environment of endosomes protonates the ionizable lipids, which then interact electrostatically with endosomal membrane lipids, destabilizing the membrane and creating transient pores that allow a fraction of the loaded siRNA to escape into the cytosol. The siRNAs are then able to effectively silence the pathogenic genes responsible for CLL pathogenesis ³¹.

Minimising the off-target effects is an extremely important issue, and one we have decided to tackle with two complementary strategies. First, we aimed to divert the localization of nanoparticles from the liver (as a notable disadvantage of intravenously injected ionizable LNPs is excessive liver homing) ³² to the secondary lymphatic tissues, where CLL cells accumulate and proliferate ³³. More specifically, our goal was to target the spleen and the lymph nodes, by creating two groups of modified LNPs that would be directed to the disease hotspots and largly spare healthy tissues. For our first group of LNPs, we incorporated the Selective ORgan Targeting (SORT) molecule 18PA along with the standard LNP components, which has been shown to enhance spleen-specific gene delivery in vivo following intravenous administration, and to be compatible with our ionizable lipid LNPs ³². We also designed a second group of LNPs for intramuscular administration: targeting the proliferative lymph node sites and the large amounts of CLL cells circulating in the lymphatic fluid ³⁴.

Secondly, the matter of non-specific uptake by non-malignant cell types could be largely overcome if an antibody fragment conjugated on LNPs from both groups targeted a specific “cancerous” protein on the surface of the CLL cells. For that reason, we designed Fab’ fragments that bind a target identified via literature review and validated through expert opinions: ROR1. Targeting ROR1 for siRNA delivery in chronic lymphocytic leukemia (CLL) represents a highly selective and promising therapeutic strategy, due to the unique expression pattern of this receptor, the reliability of its overexpression regardless of the patient’s disease manifestation, and its ability to mediate endocytosis of bound antibodies ³⁵. ROR1, an oncofetal receptor tyrosine kinase-like orphan receptor, is overexpressed in the vast majority of CLL cells but is largely absent in normal adult tissues, including healthy B lymphocytes. This cancer-specific expression enables the development of targeted delivery systems, such as antibody-based approaches, that use ROR1 as a molecular “address,” directing siRNA specifically into malignant CLL cells while avoiding uptake by healthy cells ^{36 37}.