siRNA Therapeutics Research Articles

Biologics-based technologies for highly efficient and targeted RNA delivery

The demand for RNA-based therapeutics is increasing globally. However, their use is hampered by the lack of safe and effective delivery vehicles. Here, we developed technologies for highly efficient delivery of RNA cargo into programmable extracellular vesicle-mimetic nanovesicles (EMNV) by fabricating hybrid EMNVs-liposomes (Hyb). Tissue targeting is endowed by highly efficient genetic platforms based on truncated CD63 (ΔCD63) or PTGFRN proteins. For the first time we reveal their efficiency to functionalize EMNVs, resulting in >10-fold enhancement of NP internalization in vitro and >2-fold in vivo. RNA delivery using Hyb demonstrated efficiency of >85% in human and mouse cell lines. Comparative analysis of EMNVs and Hyb lysosome colocalization and stability suggested that Hyb enter the lysosomal compartment and escape over time, whereas EMNVs primarily avoid it. Finally, we used these technologies to generate liver-targeting Hyb loaded with therapeutic siRNA, and demonstrated the robust efficiency of this system in vitro and in vivo. These technologies can be adapted for manufacturing a wide range of next-generation vehicles for highly efficient, safe delivery of RNA into desired organs and tissues for therapeutic and prophylactic applications.

Design, synthesis, molecular dynamics and gene silencing studies of novel therapeutic HIF-1α siRNAs in hypoxic cancer cells

Hypoxia inducible factors (HIFs) are heterodimeric proteins that belong to a small group of transcription factors, which mainly regulates transcription of genes under hypoxic conditions. Particularly, oxygen sensing subunit of HIF-1α is a predominant subtype that heterodimerizes with oxygen-independent HIF-1β subunit, to trigger the transcription of hypoxia responsive genes. Due to poor supply of blood and rapid division of cancerous cells, tumor microenvironment exhibits low oxygen condition and therefore increased levels of HIF-1α. One of the promising therapeutic strategies to cancer is modulation of HIF-1α signaling pathway. Small interfering RNA (siRNA) mediated downregulation of HIF-1α has been reported to prevent growth and progression of various types of cancer and holds great promise in the cancer treatment. In this study, computational approaches were used to design potential siRNAs targeting HIF-1α and investigate their interaction with the human argonaute-2 (hAgo2). Molecular dynamic simulation of HIF-1α siRNAs-hAgo2 complexes revealed key interactions required for the efficient binding of guide strand to hAgo2 protein. Two siRNAs (S2 and S5) exhibiting strong binding with hAgo2 were further considered. Subsequently, we transfected the MCF-7 cell line with both standard HIF-1α and our designed siRNAs (S2 and S5). Following transfection, translation changes in the MCF-7 cells were assessed through western blotting. S2 and S5 efficiently reduced the expression of HIF-1α in hypoxic conditions. The aim of the present study is to understand the siRNA-hAgo2 interaction. It is also focused on the desiging of effective siRNA against HIF-1α.

Synthesis and characterization of chloroquine-modified albumin-binding siRNA-lipid conjugates for improved intracellular delivery and gene silencing in cancer cells.

siRNA therapeutics have considerable potential as molecularly-targeted therapeutics in malignant disease, but identification of effective delivery strategies that mediate rapid intracellular delivery while minimizing toxicity has been challenging. Our group recently developed and optimized an siRNA conjugate platform termed "siRNA-L 2 ," which harnesses non-covalent association with endogenous circulating albumin to extend circulation half-life and achieve tumor-selective delivery without the use of traditional cationic lipids or polymers for transfection. To improve intracellular delivery and particularly the endosomal escape properties of siRNA-L 2 towards more efficient gene silencing, we report synthesis of siRNA-CQ-L 2 conjugates, in which chloroquine (CQ), an endosomolytic quinoline alkaloid, is covalently incorporated into the branching lipid tail structure. We accomplished this via synthesis of a novel CQ phosphoramidite, which can be incorporated into a modular siRNA-L 2 backbone using on-column solid-phase synthesis through use of asymmetric branchers with levulinyl-protected hydroxide groups that allow covalent addition of pendant CQ repeats. We demonstrate that siRNA-CQ-L 2 maintains the ability to non-covalently bind albumin, and with multiple copies of CQ, siRNA-CQ-L 2 mediates higher endosomal disruption, cellular uptake/retention, and reporter gene knockdown in cancer cells. Further, in mice, the addition of CQ did not significantly affect circulation kinetics nor organ biodistribution and did not produce hematologic or organ-level toxicity. Thus, controlled, multivalent conjugation of albumin-binding siRNA-L 2 to endosomolytic small molecule compounds holds promise in improving siRNA-L 2 knockdown potency while maintaining albumin-binding properties and overall safety.

RNA Interference Unleashed: Current Perspective of Small Interfering RNA (siRNA) Therapeutics in the Treatment of Neuropathic Pain.

Neuropathic pain (NP) is one of the debilitating pain phenotypes that leads to the progressive degeneration of the central as well as peripheral nervous system. NP is often associated with hyperalgesia, allodynia, paresthesia, tingling, and burning sensations leading to disability, motor dysfunction, and compromised psychological state of the patients. Most of the conventional pharmacological agents are unable to improve the devastating conditions of pain because of their limited efficacy, undesirable side effects, and multifaceted pathophysiology of the diseased condition. A rapid rise in new cases of NP warrants further research for identifying the potential novel therapeutic modalities for treating NP. Recently, small interfering RNA (siRNA) approach has shown therapeutic potential in many disease conditions including NP. Delivery of siRNAs led to potential and selective downregulation of target mRNA and abolished the pain-related behaviors/pathophysiological pain response. The crucial role of siRNA in the treatment of NP by considering all of the pathways associated with NP that could be managed by siRNA therapeutics has been discussed. However, their therapeutic use is limited by several hurdles such as instability in systemic circulation due to their negative charge and membrane impermeability, off-target effects, immunogenicity, and inability to reach the intended site of action. This review also emphasizes several strategies and techniques to overcome these hurdles for translating these therapeutic siRNAs from bench to bedside by opening a new avenue for obtaining a potential therapeutic approach for treating NP.

Lipopolymer/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia.

The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro. Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.

Co-delivery of siRNA and cisplatin via electrospun Nanofibrous membranes for synergistic treatment of malignant melanoma

Tumor recurrence and metastasis remain formidable challenges in clinical oncology. Although surgery is an effective treatment for early-stage solid tumors, residual cancer cells can lead to subsequent recurrence or metastasis. Conventional treatments for melanoma, such as anti-tumor medications and gene therapy, have distinct limitations. The rapid systemic distribution of anti-tumor drugs poses a significant challenge, often resulting in notable side effects and inadequate drug concentrations at the tumor site. Melanoma (MM), a deadly form of skin cancer, is known for its high mortality rate. In this study, we propose a novel strategy for treating MM by combining the controlled release of chemotherapeutic drugs encapsulated within Metal-Organic Frameworks (MOFs) and liposomes with gene therapy targeting Minichromosome Maintenance Proteins 4 (MCM4) using electrospinning and surface modification techniques. In vitro and in vivo results confirmed that this hierarchical membrane system can effectively deliver therapeutic MCM4 siRNA and release cisplatin to inhibit tumor growth. Furthermore, we demonstrated that MCM4 silencing promoted the sensitivity of melanoma cells to ferroptosis both in vitro and in vivo. The proposed strategy, by allowing for a controlled and sustained release of medication, could alleviate the challenges in drug delivery and aid in prevent tumor recurrence.

Therapeutic siRNA targeting PLIN2 ameliorates steatosis, inflammation, and fibrosis in steatotic liver disease models

Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide. If left untreated, MASLD can progress from simple hepatic steatosis to metabolic dysfunction–associated steatohepatitis, which is characterized by inflammation and fibrosis. Current treatment options for MASLD remain limited, leaving substantial unmet medical needs for innovative therapeutic approaches. Here, we show that PLIN2, a lipid droplet protein inhibiting hepatic lipolysis, serves as a promising therapeutic target for MASLD. Hepatic PLIN2 levels were markedly elevated in multiple MASLD mouse models induced by diverse nutritional and genetic factors. The liver-specific deletion of Plin2 exhibited significant anti-MASLD effects in these models. To translate this discovery into a therapeutic application, we developed a GalNAc-siRNA conjugate with enhanced stabilization chemistry and validated its potent and sustained efficacy in suppressing Plin2 expression in mouse livers. This siRNA therapeutic, named GalNAc-siPlin2, was shown to be biosafe in mice. Treatment with GalNAc-siPlin2 for 6–8 weeks led to a decrease in hepatic triglyceride levels by approximately 60% in high-fat diet– and obesity-induced MASLD mouse models, accompanied with increased hepatic secretion of VLDL-triglyceride and enhanced thermogenesis in brown adipose tissues. Eight-week treatment with GalNAc-siPlin2 significantly improved hepatic steatosis, inflammation, and fibrosis in high-fat/high fructose–induced metabolic dysfunction–associated steatohepatitis models compared to control group. As a proof of concept, we developed a GalNAc-siRNA therapeutic targeting human PLIN2, which effectively suppressed hepatic PLIN2 expression and ameliorated MASLD in humanized PLIN2 knockin mice. Together, our results highlight the potential of GalNAc-siPLIN2 as a candidate MASLD therapeutic for clinical trials.

Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

Effects of Histidine Oligomers in Lipid Nanoparticles on siRNA Delivery.

In this study, histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) are developed as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) is greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of ≈65nm demonstrate a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibits a significant threefold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintain comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibit excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs.

ROS-Responsive Ferrocenyl Amphiphilic PAMAM Dendrimers for On-Demand Delivery of siRNA Therapeutics to Cancer Cells.

Small interfering RNA (siRNA) therapeutics, characterized by high specificity, potency, and durability, hold great promise in the treatment of cancer and other diseases. However, the clinic implementation of siRNA therapeutics critically depends on the safe and on-demand delivery of siRNA to the target cells. Here, we reported a family of ferrocenyl amphiphilic dendrimers (Fc-AmDs) for on-demand delivery of siRNA in response to the high ROS content in cancer cells. These dendrimers bear ROS-sensitive ferrocene moieties in the hydrophobic components and positively chargeable poly(amidoamine) dendrons as the hydrophilic entities, possessing favorable safety profiles and ROS responsive properties. One of these ferrocenyl amphiphilic dendrimers, Fc-C8-AmD 8A, outperforms in siRNA delivery, benefiting from its optimal balance of hydrophobicity and hydrophilicity. Its ROS feature facilitates specific and efficient disassembly of its complex with siRNA in ROS-rich cancer cells for effective siRNA delivery and gene silencing. Moreover, Fc-C8-AmD 8A also integrates the features and beneficial properties of both lipid and dendrimer vectors. Therefore, it represents a novel on-demand delivery system for cancer cell-specific siRNA delivery. This work opens new perspectives for designing self-assembly nanosystems for on-demand drug delivery.

Delivery Strategies of siRNA Therapeutics for Hair Loss Therapy.

Therapeutic needs for hair loss are intended to find small interfering ribonucleic acid (siRNA) therapeutics for breakthrough. Since naked siRNA is restricted to meet a druggable target in clinic,, delivery systems are indispensable to overcome intrinsic and pathophysiological barriers, enhancing targetability and persistency to ensure safety, efficacy, and effectiveness. Diverse carriers repurposed from small molecules to siRNA can be systematically or locally employed in hair loss therapy, followed by the adoption of new compositions associated with structural and environmental modification. The siRNA delivery systems have been extensively studied via conjugation or nanoparticle formulation to improve their fate in vitro and in vivo. In this review, we introduce clinically tunable siRNA delivery systems for hair loss based on design principles, after analyzing clinical trials in hair loss and currently approved siRNA therapeutics. We further discuss a strategic research framework for optimized siRNA delivery in hair loss from the scientific perspective of clinical translation.

Advances in nucleic acid-targeted therapies for cardiovascular disease prevention.

Nucleic acid-based therapies are being rapidly developed for prevention and management of cardiovascular diseases (CVD). Remarkable advancements have been achieved in the delivery, safety, and effectiveness of these therapeutics in the past decade. These therapies can also modulate therapeutic targets that cannot be sufficiently addressed using traditional drugs or antibodies. Among the nucleic acid-targeted therapeutics under development for CVD prevention are RNA-targeted approaches, including antisense oligonucleotides (ASO), small interfering RNAs (siRNA), and novel genome editing techniques. Genetic studies have identified potential therapeutic targets that are suggested to play a causative role in development and progression of CVD. RNA- and DNA-targeted therapeutics can be particularly well delivered to the liver, where atherogenic lipoproteins and angiotensinogen (AGT) are produced. Current targets in lipid metabolism include proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein A (ApoA), apolipoprotein C3 (ApoC3), angiopoietin-like 3 (ANGPTL3). Several large-scale clinical development programs for nucleic acid-targeted therapies in cardiovascular prevention are under way, which may also be attractive from a therapy adherence point of view, given the long action of these therapeutics. In addition to genome editing, the concept of gene transfer is presently under assessment in preclinical and clinical investigations as a potential approach for addressing low-density lipoprotein receptor deficiency. Furthermore, ongoing research is exploring the use of RNA-targeted therapies to treat arterial hypertension by reducing hepatic angiotensinogen (AGT) production. This review summarizes the rapid translation of siRNA and ASO therapeutics as well as gene editing into clinical studies to treat dyslipidemia and arterial hypertension for CVD prevention. It also outlines potential innovative therapeutic options that are likely relevant to the future of cardiovascular medicine.

Non-Coding RNA in Tumor Cells and Tumor-Associated Myeloid Cells-Function and Therapeutic Potential.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Flexible nano-liposomes-encapsulated recombinant UL8-siRNA (r/si-UL8) based on bioengineering strategy inhibits herpes simplex virus-1 infection

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection.

MDB-50. CAR-T CELL-DERIVED EXOSOMES LOADED WITH THERAPEUTIC SIRNA IS A PROMISING TECHNIQUE IN THE TREATMENT OF SUBGROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a childhood life-threatening tumor that occurs in the cerebellum. This tumor showed resistance against several types of immunotherapies like anti-PD1, cancer vaccines, monoclonal antibodies (mAbs), and even CAR-T cells. Subgroup 3 MB is the worst threatening subtype that poorly responds to treatment. Interestingly, our data showed that the HLX gene a transcription factor that regulates cell growth is highly expressed in G3-MB and associated with poor prognosis. The siRNA-HLX inhibited tumor growth in vitro and in vivo and promisingly showed long survival in the immunocompetent mouse model. We developed CAR-T-Exosomes that can pass brain-blood barriers (BBB) and deliver therapeutic siRNA to the brain tumor. Further analysis revealed that CAR-T-Exosomes contain T cell cytotoxicity materials that can destroy tumor cells and induce anti-tumor immune response. Promisingly, the expression of HLX was significantly inhibited in the tumor cells and the anti-tumor immune response was enhanced. CAR-T-Exosomes have high safety and can induce anti-tumor response without stimulation of immunotoxicity side effects like cytokine release syndrome (CRS). In this study, we are highlighting anti-EPHA2-CAR-Exosomes as a highly specific type of tumor CAR-T-Exos immunotherapy due to a very low expression of EPHA2 in normal cells and overexpression in medulloblastoma, particularly G3-MB. Therefore, we speculate significant inhibition of tumor growth and activation of CTL effector cells in the tumor microenvironment. This study provides a new biological technique for delivering tumor suppressor RNAs and developing specific anti-tumor immune responses. Indeed, the promising safety of anti-EPHA2-CAR-T cell exosomes compared to cell-based immunotherapy.

Comparison of multiple bioanalytical assay platforms for the quantitation of siRNA therapeutics.

Aim: Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and these methods have been compared extensively. However, few comparisons exist where the same analyte is evaluated by multiple assay platforms.Materials & methods: Hybrid LC-MS, SPE-LC-MS, HELISA and SL-RT-qPCR methods were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods.Results: All assay platforms provided comparable data, though higher concentrations were observed using the non-LC-MS assays. Hybrid LC-MS and SL-RT-qPCR were the most sensitive methodologies, and SL-RT-qPCR and HELISA demonstrated the highest throughput.Conclusion: Each assay platform is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on the prioritization of needs such as sensitivity, specificity and throughput.

Novel Lipid Nanocomplex Co-Carrying Bcl2 siRNA and Quantum Dots for EGF Receptor-Targeted Anti-Cancer Theranosis.

Many different types of nanoparticles have been suggested for tumor-targeted theranosis. However, most systems were prepared through a series of complicated processes and could not even overcome the blood-immune barriers. For the accurate diagnosis and effective treatment of cancers, herein we suggested the lipid micellar structure capturing quantum dot (QD) for cancer theranosis. The QD/lipid micelles (QDMs) were prepared using a simple self-assembly procedure and then conjugated with anti-epidermal growth factor receptor (EGFR) antibodies for tumor targeting. As a therapeutic agent, Bcl2 siRNA-cholesterol conjugates were loaded on the surface of QDMs. The EGFR-directed QDMs containing Bcl2 siRNA, so-called immuno-QDM/siBcl2 (iQDM/siBcl2), exhibited the more effective delivery of QDs and siBcl2 to target human colorectal cancer cells in cultures as well as in mouse xenografts. The effective in vivo targeting of iQDM/siBcl2 resulted in a more enhanced therapeutic efficacy of siBcl2 to the target cancer in mice. Based on the results, anti-EGFR QDM capturing therapeutic siRNA could be suggested as an alternative modality for tumor-targeted theranosis.

Evaluation of lipopolymer as therapeutic siRNA delivery agents in non-malignant and malignant primary cells.

e15189 Background: Targeting leukemia-driving oncogenes via short interfering RNA (siRNA) offers an alternative approach to leukemia treatment that can overcome the limitations of current therapies. Low molecular weight polyethyleneimine grafted with lipids (lipopolymers) have emerged as promising carriers for siRNA delivery to leukemia cells. Lipopolymers harboring select lipids were recently shown to downregulate target oncogene leading to significant anti-leukemic activity in both in vitro and in vivo cell line-based leukemia models. The goal of this study was to evaluate these lipopolymers in (i) PBMCs obtained from healthy donors to determine the potential impact on non-malignant cells, and (ii) primary leukemia patient cells to assess therapeutic efficacy. Methods: Target gene levels were determined by RT-qPCR and the effect of treatment on progenitor cells was assessed by measuring changes in colony forming ability. Results: At day 1 post-treatment, no changes in FLT3 mRNA levels were observed in all 6 PBMC samples with the 2 candidate lipopolymers (LeuFectB and LeuFectC) tested. Among 11 PBMC samples evaluated at day 2, only one sample showed a significant reduction in oncogene transcript levels on treatment with LeuFectB. The impact of lipopolymer/siRNA complex treatment on progenitor cells in the PBMC population was then assessed. Of the 7 PBMC samples tested, significant reduction in colony numbers was observed in one sample with LeuFectB and two samples with LeuFectC. On an average, the lipopolymer/siRNA complexes were well tolerated by normal healthy cells as changes observed in oncogene expression and colony forming ability were not consistent across the whole pool. Primary acute lymphoblastic leukemia (ALL) patient cells treated with complexes targeting STAT5A showed marked reduction in STAT5A transcript levels in 4 out of 7 samples with both the lipopolymers tested. The subsequent impact of STAT5A downregulation on the clonogenicity of leukemia stem cells was observed in the significant decrease in colony numbers seen in 3 samples (out of 8) with LeuFects. Additionally, combined downregulation of STAT5A and BCR-ABL genes in BCR-ABL+ ALL patient samples led to a notable decrease in cell proliferation and viability. Moreover,2 out of 3 acute myeloid leukemia (AML) patient samples harboring the FLT3-ITD mutation showed significant retardation in their colony forming ability on treatment with FLT3-targeting siRNA complexes. Collectively, the ALL and AML primary samples demonstrated promising preliminary results, suggesting a therapeutic effect on the stem cell population which is critical for mitigating relapse or prolonging remission. Conclusions: These findings provide further proof-of-concept for clinical translation of lipopolymer mediated siRNA therapy of leukemia, showing relative safety and selectivity of these lipopolymer/siRNA complexes.

The ADME characteristics of siRNA therapeutics and the opportunity to predict disposition in pregnant women.

Small interfering RNA (siRNA) therapeutics represent an emerging class of pharmacotherapy with the potential to address previously hard-to-treat diseases. Currently approved siRNA therapeutics include LNP-encapsulated siRNA and triGalNAc-conjugated siRNA. These siRNA therapeutics exhibit distinct pharmacokinetic characteristics and unique absorption, distribution, metabolism, and elimination (ADME) properties. As a new drug modality, limited clinical data are available for siRNA therapeutics in specific populations, including pediatrics, geriatrics, individuals with renal or hepatic impairment, and pregnant women, making dosing challenging. In this review, a mechanistic overview of the ADME properties of the five currently approved siRNA therapeutics is presented. A concise overview of the clinical data available for therapeutic siRNAs in special populations, focusing on the potential impact of physiological changes during pregnancy on siRNA disposition is provided. The utility of physiologically based pharmacokinetic (PBPK) modeling as a tool to elucidate the characteristics and disposition of siRNA therapeutics in pregnant women is explored. Additionally, opportunities to integrate known physiological alterations induced by pregnancy into PBPK models that incorporate siRNA ADME mechanisms to predict the effects of pregnancy on siRNA disposition are discussed. Clinical data regarding the use of therapeutic siRNA in special populations remains limited. Data for precise parameterization of maternal-fetal siRNA PBPK models is lacking presently and underscores the need for further research in this area. Addressing this gap in knowledge will not only enhance our understanding of siRNA pharmacokinetics during pregnancy but also advance possible development of siRNA therapeutics to treat pregnancy related conditions. Significance Statement This review proposes a framework on how siRNA disposition can be predicted in pregnancy based on mechanistic ADME information using physiologically based pharmacokinetic (PBPK) modeling. The mechanistic ADME information and available clinical data in special populations of currently FDA approved siRNA therapeutics are summarized. A detailed discussion on how physiological changes during pregnancy may affect siRNA disposition in pregnant women and on the opportunities to project siRNA disposition in pregnant women using PBPK modeling is provided.