On this page, you will find the proposed implementation of our siREN project. Firstly, we briefly refer to the actual issue it addresses and how we envision our project in the real world. Our project has the potential to improve the quality of life for many patients suffering from chronic lymphocytic leukemia (CLL) who are undergoing treatments that cause unpleasant and, in some cases, dangerous side effects. Through our innovative therapeutic proposal of silencing BTK and BCL2 via siRNA specifically delivered to CLL cells with Lipid Nanoparticles (LNPs), proliferation of malignant cells could be halted; leading to apoptosis and a way for CLL treatment. Nevertheless, it is a fact that bringing siREN to market is a difficult undertaking that requires careful, ethical, and responsible steps to achieve. As dreamers, we foresaw its future, and here is a glimpse of our vision.

In our research project on chronic lymphocytic leukemia, a form of cancer that has a plethora of treatments but whose side effects are numerous, significant, and dangerous, safety and ethical compliance have been our team's primary and most important goals from day one. We recognize that ethical considerations and patient safety must be a priority in our work. After all, this is the way to maintain public confidence in scientific endeavors and ensure the integrity of our research.

Understanding the importance of integrity, safety, and transparency in research, our team has acted throughout the course of its activities and the conduct of its experiments to ensure that our experiments were conducted fairly, with transparency, and with the utmost attention to safety protocols. It is our team's firm belief that this is the only way we can trust our results and utilize them in the future. This commitment to ethical practices and safety has been a non-negotiable position of our team, allowing us to consider and present our results as trustworthy.

Securing funding for our research has been crucial to the development of our project and has been instrumental in the progress of our team. However, we have made a collective decision that financial considerations will never override our ethical and safety standards, and that no external (financial) factors will influence or direct our team's work. By always prioritizing ethics and safety in every step of our work, we ensure that our research on CLL is conducted responsibly.

Chronic Lymphocytic Leukemia remains the most common adult leukemia in Western countries, accounting for 22–30% of all leukemia cases ¹, with an average crude incidence rate across Europe of 4.8 per 100,000 individuals ². The true incidence, however, is likely underestimated, as many patients remain asymptomatic. According to the Global Burden of Disease (GBD) 2019 study, the global burden of CLL has risen dramatically between 1990 and 2019, with more than 100,000 new cases and over 40,000 deaths reported globally in 2019. In Europe, the median age at diagnosis is 71 years for men and 74 years for women, with incidence approximately twice as high in males. Although CLL is considered indolent, it reduces survival among elderly populations. Epidemiological data reveal that incidence increases exponentially with age, reaching its peak among older adults ³.

Despite major advances with targeted therapies such as BTK and BCL2 inhibitors, CLL remains incurable. Patients frequently experience off-target effects from existing treatments, significantly affecting their quality of life. Moreover, resistance and relapse continue to pose substantial clinical challenges. These limitations underscore a clear unmet need within the CLL treatment landscape. There is an urgent demand for therapies that are both effective and selective, with minimal off-target effects, while also being scalable and cost-efficient in production.

The global market for CLL therapies was valued at approximately 5.4 billion USD in 2025 and is projected to reach nearly 7.5 billion USD by 2030 ⁴. This figure represents the Total Addressable Market (TAM), reflecting the overall global opportunity for all companies developing CLL treatments.

Within this broader context, the Serviceable Available Market (SAM) focuses on relapsed and refractory CLL patients. While current therapies such as BTK and BCL2 inhibitors have shown high initial efficacy, resistance often develops over time, leading to treatment failure ⁵. Τhe SAM for relapsed and refractory patients is estimated at 1.1–1.6 billion USD annually ⁶.

The Serviceable Obtainable Market (SOM) represents the portion that siREN could realistically capture during its initial clinical adoption phase. As novel therapies typically enter the market gradually through specialized oncology centers and partnerships with pharmaceutical companies, a reasonable early estimate would be 1–5% of the SAM during the first years of commercialization. This translates to an annual market potential of 10–80 million USD, depending on geographic focus and uptake rate. The SOM could expand considerably if siREN demonstrates safety, efficacy, and scalability, and if its platform extends to other malignancies expressing ROR1.

Our product is the proven design of correct siRNAs, targeting the oncogenic proteins BTK and BCL2, which play a decisive role in the onset and development of CLL. Therefore, its direct users will be pharmaceutical companies that have outsourced research into new therapeutic agents and will now undertake clinical trials and approval. For siREN, the ideal candidate users are companies operating in the field of pharmaceutical production, where they could support the production of siRNAs and, more generally, siREN as a final product, always obtaining the production rights and know-how from our company. They would then distribute it to the market through their sales and distribution networks to end consumers, who would be none other than people suffering from CLL.

The final product, which is the innovative siRNA therapy we have designed, will be promoted to clinics and government authorities for distribution to hospitals and, ideally, coverage by public insurance funds. The end users are, of course, as mentioned before, patients with chronic lymphocytic leukemia who will be administered the treatment following medical recommendations.

From the manufacturer and the production line to the end users, there are some crucial steps in the journey of the product flow. The design of our treatment –which consists of siRNAs in nanoparticle carriers– also includes the development of a plan for the management of the product, storage, and transport, mirroring real-world problems and maintaining the goal of ensuring the quality of our therapy, its effectiveness, and its accessibility to everyone around the world who may need siREN. The approach we present focuses on safety and quality assurance, without ever forgetting that many people may not live near big cities, and that this does not mean they should have to settle for substandard or inadequate health care. Our therapy remains both accessible and effective for patients worldwide.

The production of drugs containing siRNA encapsulated in lipid nanoparticles (LNPs) must be carried out in facilities that comply with Good Manufacturing Practice (GMP) principles and include documented production procedures, raw material control, and quality assurance systems ⁷. LNP formation technologies used industrially (e.g., microfluidic mixing or controlled ethanol/aqueous mixing) must be validated (process validation). Critical quality attributes (Critical Quality Attributes, CQA) must be defined and documented, and acceptable limits and appropriate analytical tests must be established for each CQA ⁸. The production process requires a documented cleaning/disinfection and contamination prevention strategy, often in controlled airflow environments (ISO/cleanrooms), as well as validation of intermediate stages and processes (process validation) [7].

The LNPs should be prepared by microfluidic mixing, as this enables both high inter-batch reproducibility, as well as the intra-batch uniformity of LNPs. To create LNPs, a dual syringe pump is used to transport the two solutions through the micromixer at a total flow rate of 12 mL/min. The particles are then transferred into dialysis overnight against PBS. During the LNP/siRNA formulation, loading of the negatively charged siRNA is performed under pH = 4. The acidic conditions ensure that all the lipid amino groups are fully protonated, allowing efficient electrostatic interactions and complexation between the siRNA and the lipid ⁹.

For distribution and logistics, storage and transport must comply with Good Distribution Practice (GDP) and a certified cold chain must be implemented, as the product requires conditions of 2–8°C, including validated shipping containers, temperature loggers, and deviation management procedures. Research on LNP models shows that refrigeration better preserves LNPs in solutions over a long period of time compared to freezing or storage at higher temperatures. It is thus important that products are transported in vehicles that can maintain a stable low temperature, as required for the transport of siREN, and that are certified for the transport of environmentally sensitive medicines ^{10 11}, ¹².

As with the approved product Patisiran (ONPATTRO), the final LNP product should be stored in a refrigerator, and regulatory decisions document recommended storage conditions (2–8°C) and warnings such as "do not freeze," as these have been documented by stability data . Laboratory studies on LNP samples have shown that stable refrigeration (e.g., 2°C–8°C) often better preserves physicochemical characteristics and biological activity compared to repeated freeze–thaw cycles without adequate protection ¹⁴. Freezing can cause ice crystal formation, freeze-concentration (concentration of dissolved substances), mechanical stresses, and changes in aggregation or morphology leading to disruption of the lipid structure and loss of siRNA encapsulation (unless a validated cryoprotection strategy or lyophilization has been used) ¹⁵. The effect of freezing depends on the lipid composition, the presence of PEG-lipids, the relative content of auxiliary lipids, and the presence of cryoprotectants (e.g., saccharides such as sucrose or trehalose).

Upon receipt at hospitals/clinics, the integrity of the cold chain must be confirmed (verification of temperature records), and products must be stored in supervised refrigerators in the unit's pharmacy or medicine storage areas, in accordance with the conditions required for the storage and preservation of the medicine for the specified period of time ¹⁰. Furthermore, at the end point (hospital/pharmacy), procedures must be followed for storage, handling during dose preparation, limits on permissible exposure to room temperature (30 min) during preparation/administration, and disposal instructions in case of cooling or other deviation from the instruction sheet ¹⁰, ¹², ¹⁶

Appropriate single dosage amount, as well as total number and interval of doses will be determined following appropriate clinical studies and adjusted depending on the profile of each individual recipient of therapy. The method of administration of our proposed pharmaceutical formulation will depend on the results of preliminary experimental validation of both our proposed molecular delivery systems. When administered intravenously (IV), the siRNA–LNP–anti-ROR1 formulation achieves rapid systemic exposure and direct access to circulating and lymphoid CLL cells but also carries a higher risk of acute infusion-related reactions, including complement activation, cytokine release, and hypersensitivity. IV dosing can also lead to transient hemodynamic changes (flushing, hypotension) and may increase the likelihood of systemic off-target exposure, necessitating slow infusion rates, premedication, and close monitoring during and after administration. In contrast, intramuscular (IM) administration provides slower absorption and potentially longer local depot release, which can reduce acute infusion reactions and improve tolerability but introduces new considerations, such as injection-site inflammation, pain, erythema, or localized immune activation. Relevant precautions should be taken in each case, and safety protocols must be followed by healthcare professionals administering the drug ^{33 34}.

Our investigational siRNA therapy for CLL that combines dual BCL2 and BTK knockdown, delivered via an LNP system with anti-ROR1 Fab’ targeting, may exhibit a complex safety profile. Given the lack of available experimental data, we considered and analysed the safety profiles of FDA-approved drugs targeting BTK and BCL2, FDA-approved siRNA therapies, including the drug that shares a similar LNP composition with our own, and of anti-ROR1 therapeutic antibodies in clinical trials. In this way, we could get a vague estimation of our product’s possible side effects by considering the side effects of agents similar with each of its individual components. Therefore, based on clinical data from comparable agents, the following categories of adverse effects must be considered in a worst-case scenario:

In a worst-case safety profile, our siRNA-LNP–anti-ROR1 therapeutic may combine the hematologic, infectious, and TLS-related risks of Venetoclax, the cardiac, bleeding, and malignancy risks of BTK inhibitors, the infusion, hepatic, renal, thrombotic, and injection-site toxicities of siRNA drugs, and the neuropathy, diarrhea, and dermatologic effects of anti-ROR1 antibodies.

This overlapping toxicity landscape suggests a high potential burden of life-threatening cytopenias, arrhythmias, thromboembolic and hemorrhagic events, hepatic/renal injury, and severe infections, requiring vigilant monitoring and risk mitigation strategies in early trials.

However, with the addition of selective spleen and lymph node localization and ROR1-directed antibody targeting, the safety profile of this siRNA therapy is expected to diverge meaningfully from that of classical BTK and BCL2 inhibitors. By reducing non-specific uptake in liver and kidney, the drug should limit the class-typical hepatotoxicity (transaminase elevations, vitamin A depletion) and renal impairment observed with existing siRNA/LNP products. Instead, pharmacologic activity will be concentrated in lymphoid tissues, enhancing the likelihood of on-target effects such as tumor lysis syndrome (TLS), cytopenias, and immunosuppression-driven infections. However, unlike the continuous target suppression seen with small-molecule BTK and BCL2 inhibitors, siRNA-mediated knockdown is transient and reversible, generally lasting weeks rather than months, which may mitigate the duration and cumulative burden of toxicities such as prolonged cytopenias, chronic cardiac arrhythmias, or persistent secondary malignancy risk. At the same time, the more focused targeting of ROR1-positive CLL cells should reduce systemic off-target exposure, but may increase the local intensity of ROR1-associated adverse events (neuropathy, diarrhea, neutropenia). In sum, while this approach shifts risk away from liver and kidney injury toward hematologic and immune-mediated complications, the transient nature of siRNA silencing offers a potential safety advantage, enabling dosing schedules and treatment interruptions to be used strategically to manage toxicity without permanently disrupting cellular signaling.

Finally, we ought to note that the aforementioned adverse reactions may be caused by components of the compared agents or other reasons that do not have relevance in the context of our own formulation. Conversely, clinical evaluation of our product may shine light on side effects that are currently not included in this list. Even so, this preliminary theoretical assessment may prove useful as a guideline for conducting preclinical and especially clinical research on this particular drug, potentially helping to explain experimental results, identify adverse reactions earlier, and protect the health of study participants ^{19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35} .

The ROR1 receptor is involved in nervous, cardio-respiratory, and musculoskeletal system development during embryonic life ^{36 37}. BTK expression is crucial for immune system maturation during late embryonic and postnatal development ³⁸. BCL2 expression is necessary for the development of the nervous system, cardiovascular system, and kidneys embryonically, and the immune system postnatally ^{39 40}. Thus, given the important role of the targets of this drug, it is not indicated for use during gestation, lactation, or early childhood, although these theoretical assumptions may need to be validated through experimental data. Additional use restrictions may arise after relevant preclinical and clinical studies.

Next experimental steps should follow a staged, data-driven progression from formulation to human testing. First, perform rigorous LNP validation: optimize composition, particle size (measured by dynamic light scattering), z potential, polydispersity, encapsulation efficiency, siRNA stability, serum stability, and manufacturability. Second, test in cell lines with the full nanoparticle (LNP + anti-ROR1 Fab’) using ROR1-positive CLL cell lines and relevant off-target cell lines: measure uptake, target mRNA knockdown (qPCR), protein suppression (Western/blot or flow cytometry), cell viability/apoptosis, and off-target effects. Third, advance to in vivo proof-of-concept in immunodeficient (SCID) mice grafted with human CLL/leukemic cells: perform biodistribution, pharmacokinetics and pharmacodynamics (siRNA persistence, target knockdown in spleen/lymph nodes vs liver/kidney), efficacy (tumor burden, survival, circulating malignant cells), and safety (CBCs, LFTs, renal panels, cytokines, histopathology of spleen/lymph nodes, liver, kidney, heart, and injection/infusion sites). Run dose-range finding and GLP toxicology studies, including evaluation of tumor lysis syndrome risk, thrombocytopenia, neutropenia, cardiac telemetry, and immunogenicity to the antibody and LNP. Finally, design first-in-human trials: a carefully staged clinical program beginning with a small, safety-focused, possibly open-label, dose-escalation phase (with TLS prophylaxis and step-up dosing), followed by randomized, double-blind efficacy trials in appropriately consented CLL patients once safety and dose are established; ensure rigorous monitoring (CBC, metabolic panel, ECG, infection surveillance), pre-specified stopping rules, and data safety monitoring board oversight ^{9 41 42 43}.