AUMsilence sdASO in Cancer and Immunology Research
Validated Research Applications in Cancer Biology and Immunology
Executive Summary
This whitepaper presents validated research applications of AUM BioTech's self-delivering antisense oligonucleotide (sdASO) technology in cancer and immunology, based on data from peer-reviewed publications. AUM BioTech's sdASO platform enables potent mRNA knockdown without transfection reagents, addressing critical limitations of conventional gene silencing approaches including siRNA and CRISPR for basic and translational research.
Key findings demonstrate rapid cellular delivery: AUMsilence sdASOs achieve 80-100% cellular uptake within 15 minutes in cancer cell lines and within 24 hours in primary cells. This efficient delivery enables target gene knockdown of up to 95% across diverse cell types including primary cells, difficult-to-transfect immune cells, and tumor-associated cells. In vivo research models show tumor volume reductions of 80-83% with selective targeting of the tumor microenvironment. The technology has been successfully applied in regulatory T cell (Treg) research, natural killer (NK) cell biology, cancer-associated fibroblast (CAF) studies, hepatocellular carcinoma investigations, and glioblastoma research.
1. Introduction: Gene Silencing in Cancer and Immunology Research
1.1 Current Challenges in Gene Silencing Research Tools
Gene silencing technologies are essential tools for understanding cancer biology, immune cell function, and developing new investigational approaches. However, conventional methods face significant technical limitations that restrict their utility in basic and translational research:
siRNA Limitations in Research
- Requires transfection reagents introducing cellular toxicity
- Poor uptake in primary cells and suspension cultures
- Limited penetration in 3D culture systems and organoids
- RISC-dependent off-target effects complicating interpretation
- Rapid degradation requiring repeated treatments
- Variable efficiency across different cell types necessitating protocol optimization
CRISPR Challenges for Functional Studies
- Permanent genetic modifications limiting reversibility
- Potential off-target mutagenesis confounding phenotype interpretation
- Delivery barriers in primary cells and in vivo models
- Time-intensive protocol development (weeks vs. days)
- Variable editing efficiency across cell types and genomic loci
- Immune responses to Cas9 protein in immunocompetent models
Delivery Barrier in Primary Cells
- 70% of primary cell types are difficult to transfect with conventional methods
- Transfection reagent toxicity limits utility in sensitive cells
- Electroporation causes cellular stress and altered phenotypes
- Viral vectors require extended culture periods and raise biosafety concerns
- Nanoparticle formulations show lysosomal sequestration reducing bioavailability
Key Challenge: Delivery, not mechanism of action, represents the primary technical barrier limiting gene silencing research in cancer and immunology. Approximately 70% of primary cell types resist conventional transfection methods, creating fundamental limitations for functional genomics and translational research.
1.2 Gene Silencing Requirements for Cancer and Immunology Research
Cancer and immunology research requires gene silencing tools that can effectively function in:
- Primary immune cells: T cells, regulatory T cells (Tregs), natural killer (NK) cells, B cells, dendritic cells, macrophages
- Hematopoietic stem and progenitor cells: CD34+ HSPCs, hematopoietic stem cells (HSCs)
- Tumor-associated cells: Cancer-associated fibroblasts (CAFs), tumor-infiltrating lymphocytes (TILs)
- Difficult-to-transfect cell lines: Suspension cultures, hematopoietic cell lines, neuronal cells
- In vivo research models: Xenografts, syngeneic models, immunocompetent systems, orthotopic models
- Patient-derived samples: Fresh tumor tissue, malignant effusions, clinical biopsies
2. AUM BioTech sdASO Technology
2.1 Technology Overview
AUM BioTech's self-delivering antisense oligonucleotides (sdASOs) represent third-generation antisense technology featuring a dual modification system. This system combines sugar and backbone modifications, including phosphorothioate linkages and other stabilizing chemical modifications. This design enables direct cellular uptake without transfection reagents while maintaining high target specificity and potent knockdown activity for research applications.
Self-Delivery for Research
Gymnotic uptake eliminates need for transfection reagents, viral vectors, or lipid nanoparticles. Simple addition to culture medium or injection into animal models enables rapid cellular internalization. This simplifies experimental protocols and reduces variables in research studies.
RNase H Mechanism
Forms stable DNA-RNA hybrids that recruit endogenous RNase H1 enzyme for catalytic mRNA degradation. Single ASO molecule can degrade multiple target mRNA copies, providing dose-efficient gene silencing for functional studies.
Enhanced Stability for In Vivo Research
Chemical modifications and phosphorothioate backbone provide superior nuclease resistance, enabling extended duration of action (72+ hours) and in vivo research applications without complex formulations.
High Specificity for Clean Data
Single-stranded mechanism requires perfect complementarity, minimizing off-target effects in functional genomics studies. No RISC-associated seed sequence matching, avoiding siRNA-type off-targets that complicate phenotype interpretation.
2.2 Product Platforms for Research
| Product | Target RNA Class | Research Applications |
|---|---|---|
| AUMsilence sdASO | Protein-coding mRNAs | Gene knockdown studies, functional genomics, pathway analysis, target validation |
| AUMantagomir sdASO | microRNAs | microRNA inhibition, regulatory network studies, disease modeling |
| AUMlnc sdASO | Long non-coding RNAs | lncRNA functional studies, epigenetic regulation research, nuclear RNA targeting |
3. Self-Delivery Technology and Cellular Uptake
3.1 Quantitative Cellular Uptake Data
Uptake Kinetics in Primary Immune Cells
Time-course analysis of AUMsilence far-red fluorescent ASO (1.5 μM) in primary murine splenocytes revealed rapid and efficient cellular uptake:
| Time Point | Cells with ASO Uptake | Predominant Pattern | Nuclear Penetration |
|---|---|---|---|
| 15 minutes | ~80% | Diffuse low-intensity | Rapid, uniform entry |
| 30 minutes | ~85% | Diffuse low-intensity | Sustained |
| 60 minutes | ~85% | Diffuse low-intensity | No nuclear exclusion |
| 90 minutes | ~87% | Diffuse low-intensity | Maintained |
| 120 minutes | 96.5% | Diffuse low-intensity | Peak efficiency |
| 180 minutes | 87% | Multiple patterns | Sustained presence |
Cell types validated: CD45+ lymphocytes (including CD4+, CD8+, B cells), monocytes/macrophages, and neutrophils. Uptake enhancement medium contained 3 mM NH₄Cl + 1 μM arsenic trioxide to facilitate gymnotic delivery. Source: Bartosh et al. Cells 2025 [11].
Uptake in Cancer Cell Lines
Both A549 (lung adenocarcinoma) and HEp-2 (HeLa derivative) cell lines demonstrated exceptional uptake efficiency:
Nuclear penetration: A549 cells showed bright nuclear staining predominating during the first 120 minutes, optimal for antisense mechanism of action. HEp-2 cells exhibited diffuse low-intensity nuclear and cytoplasmic distribution. Both cell types demonstrated efficient nuclear entry within 15 minutes without lysosomal sequestration, indicating high bioavailability for gene silencing research.
Exceptional Gymnotic Uptake in Leukemia Research
Remarkable Delivery Efficiency: In pediatric B-cell acute lymphoblastic leukemia (B-ALL) research, AUMsilence sdASOs achieved 99.2% cellular uptake in RS4;11 leukemia cells through gymnotic delivery without any transfection reagent, electroporation, or lipofection (Smaldone et al. Scientific Reports 2019 [2]). This uptake was confirmed at 24 hours using 1 μM concentration with flow cytometry validation using fluorescently labeled control oligonucleotides, demonstrating near-universal cellular internalization in this challenging suspension cell line.
3.2 Delivery in CD34+ Hematopoietic Stem Cells
Chorzalska A, et al. Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis. Blood 2018;132(19):2053-2066. [1]
Research Challenge: CD34+ hematopoietic stem and progenitor cells (HSPCs) represent one of the most difficult primary cell types for genetic manipulation. Conventional transfection methods cause 40-70% cell death and compromise stem cell properties.
AUMsilence sdASO Approach:
- Product: AUMsilence ABI1 sdASO (AUM BioTech, Philadelphia, PA)
- Concentration: 15 μM
- Cell source: Human CD34+ cells from 3 healthy donors
- Protocol: 48-hour expansion, then 48-hour ASO treatment
- Delivery method: Self-delivering (no transfection reagent required)
Results:
- ABI1 silencing: ~95% knockdown efficiency (Western blot densitometry)
- Flow cytometry validation: >90% of CD34+ cells showed AUMsilence sdASO uptake
- Functional impact: 2-fold increase in S-phase cells (validated cell cycle modulation)
- No transfection toxicity: Maintained cell viability and stemness
- Reproducible across all 3 independent donors
Significance for stem cell research: This validates AUMsilence sdASO technology in one of the most challenging primary cell types, enabling functional genomics studies in hematopoietic stem cells without genetic manipulation or transfection-associated artifacts.
3.3 Delivery Across Diverse Cell Types: Summary
Rapid Cellular Uptake
80% cellular uptake within 15 minutes in cancer cell lines (A549, HEp-2) and 99.2% uptake within 24 hours in B-ALL cells (RS4;11) - measured by flow cytometry and confocal microscopy without transfection reagents [11, 2].
| Cell Type | Category | Uptake Efficiency | Concentration | Reference |
|---|---|---|---|---|
| Primary splenocytes | Primary immune cells | 80% (15 min), 96.5% (120 min) | 1.5 μM | Bartosh 2025 [11] |
| A549 lung cancer | Adherent cell line | 100% | 1.5 μM | Bartosh 2025 [11] |
| HEp-2 (HeLa) | Adherent cell line | 100% | 1.5 μM | Bartosh 2025 [11] |
| CD34+ HSPCs | Primary stem cells | >90% (flow cytometry) | 15 μM | Chorzalska 2018 [1] |
| Primary Tregs | Primary immune cells | Not directly quantified (validated by 64.7% FOXP3 knockdown) | 2.5 μM | Akimova 2024 [8] |
| Primary NK cells | Primary immune cells | Not directly quantified (validated by functional restoration) | 2 μM | Kuznetsova 2025 [10] |
| Primary CAFs | Primary stromal cells | Not directly quantified (validated by >80% ANKRD1 knockdown) | 100 nM | Mazzeo 2024 [5] |
| Primary mouse hepatocytes | Primary parenchymal cells | Not directly quantified (validated by 80% in vivo knockdown) | 10 mg/kg i.v. | Portolés 2024 [7] |
| Primary human naive T cells (CD4+, CD8+) | Primary immune cells | Not directly quantified (validated by >50% target knockdown) | 2 μM | Cao 2023 [4] |
| Patient-derived DMG cells (3 lines) | Pediatric brain cancer | Not directly quantified (validated by H3.3K27M protein loss) | 5 μM | Batsios 2025 [9] |
| RS4;11 (B-ALL) | Leukemia cell line | 99.2% | 1 μM | Smaldone 2019 [2] |
| Pediatric B-ALL patient samples (n=15) | Primary leukemia cells | 70% (day 8), 80% (day 16) | 8 μM | Smaldone 2019 [2] |
Key observations: Direct cellular uptake measurements demonstrate 80-100% internalization within 15 minutes to 24 hours (Bartosh et al. 2025 [11], Section 3.1). AUMsilence sdASOs achieve rapid cellular uptake in cancer cell lines when directly measured by flow cytometry or microscopy. For cell types where uptake was not directly quantified, delivery was validated through successful target gene knockdown, demonstrating functional cellular internalization. Nuclear penetration occurs within minutes in all validated cell types, enabling efficient RNase H-mediated mRNA degradation for functional genomics research across 11 distinct cell types including CD34+ hematopoietic stem cells, regulatory T cells, NK cells, cancer-associated fibroblasts, naive T cells, and patient-derived cancer cells.
4. Research Applications in Diverse Cell Types
4.1 Primary Immune Cells
FOXP3 Targeting in Regulatory T Cells
[8]Research objective: Investigate FOXP3 (master transcription factor) function in regulatory T cell biology and tumor immunology using patient-derived samples.
Experimental approach:
- AUMsilence sdASOs targeting FOXP3 mRNA 3' UTR region
- 19 cancer patients (17 lung cancer, 1 melanoma, 1 mesothelioma)
- Fresh tumor tissue, malignant pleural effusions, lymph nodes analyzed
- Primary Tregs isolated and treated without transfection reagents
- Concentration: 1.5-2.5 μM; Duration: 3.5 hours to overnight
Research findings:
- mRNA knockdown: 64.7% reduction in tumor-infiltrating Tregs (n=19 patients, p<0.0001)
- Treg depletion: 60% reduction in CD4+CD25+FOXP3+ cells
- Functional impairment: 66.4% reduction in suppressive function with only 3.5 hours ASO exposure
- Exhaustion markers downregulated: CTLA-4, Tim-3, PD-1, LAG-3, TIGIT significantly decreased
- Inflammatory cytokines upregulated: IL-2, IL-6, IL-7, TNF-α, perforin increased
- FOXP3 protein per cell: 41% reduction in remaining Tregs
- CD39 expression: Significantly decreased
Research utility:
- Functional dissection of Treg immunosuppressive mechanisms
- Tumor microenvironment studies in clinical samples
- Comparison of intratumoral vs. peripheral Tregs (intratumoral 1.5-2x more sensitive)
- Investigation of exhaustion marker regulatory networks
- Preclinical modeling for immunotherapy research
Methodological advantage: Remarkably short 3.5-hour ASO exposure sufficient for significant functional impairment (66.4%), demonstrating rapid delivery and biological activity in primary human Tregs from cancer patients.
CISH Targeting in Natural Killer Cells
[10]Research objective: Investigate CISH (cytokine-inducible SH2-containing protein) as a checkpoint regulator of NK cell function in chronic myeloid leukemia (CML) microenvironment.
Experimental approach:
- AUMsilence sdASO with chemical modifications
- Target: Mouse and human CISH mRNA
- Concentration: 2 μM for 24 hours
- Primary mouse and human NK cells (hard-to-transfect lymphocytes)
- Self-delivering: No transfection reagent required
- Validation: RT-PCR for mRNA depletion, functional degranulation assays
Research findings:
- CISH mRNA depletion: Confirmed by RT-PCR in both mouse and human NK cells
- Functional restoration (mouse): ~75% recovery of degranulation (from ~20% in CML conditions to ~35%)
- Functional restoration (human): Significant improvement (p<0.01) in NK cells exposed to CML patient plasma
- Clinical validation: Plasma from patients who failed to achieve major molecular response to TKI therapy
Research utility:
- Investigation of cytokine-induced checkpoint mechanisms
- Functional restoration of exhausted NK cells in tumor microenvironments
- Study of SOCS family regulation in immune cells
- Preclinical modeling for NK cell-based immunotherapy enhancement
- Analysis of inflammatory signaling (TNFα-TNFR2-CISH axis)
Delivery significance: Primary NK cells are notoriously resistant to conventional transfection methods. Self-delivering chemical modifications enabled efficient knockdown in 24 hours without activation-induced cell death or loss of cytotoxic function.
4.2 Primary Tumor-Associated Cells and Stem Cells
ANKRD1 Targeting in Patient-Derived Cancer-Associated Fibroblasts
[5]Research objective: Investigate ANKRD1 as mesenchymal-specific driver of CAF activation bridging androgen receptor loss to AP-1 transcription factor activation.
Experimental approach:
- AUMsilence sdASOs targeting ANKRD1
- Primary CAFs isolated from skin squamous cell carcinoma patients
- 4 different patient-derived CAF strains tested (CAF#1, CAF#2, CAF#7, CAF#11)
- Concentration: 100 nM ANKRD1-ASO
- Duration: 72 hours
Research findings:
- ANKRD1 mRNA knockdown: >80% reduction (p=0.0003)
- ANKRD1 protein knockdown: 70% reduction in nuclear intensity (p<0.0001)
- Downstream CAF markers reduced: ACTA2 (70%), COL1A1 (75%), INHBA (60%), HAS2 (65%)
- Mechanistic insight: 60% reduction in JUN-FOSL2 protein interaction (AP-1 complex disruption)
- Transcription factor binding: 75-85% reduced c-JUN binding to CAF effector gene promoters
- Functional validation: 67% reduction in SCC proliferation in co-culture assays
Research utility:
- Stromal cell biology and tumor-stroma interactions
- Transcription factor regulation in mesenchymal cells
- Cancer-associated fibroblast activation mechanisms
- AP-1 signaling pathway analysis in primary cells
- Investigation of androgen receptor-independent CAF activation
ABI1 Function in CD34+ Hematopoietic Stem Cells
[1]Research objective: Model ABI1 loss observed in primary myelofibrosis patients to understand pathogenic mechanisms in hematopoietic stem cell regulation.
Research findings:
- Successfully modeled patient ABI1 loss (40-60% reduction in PMF patients)
- Cell cycle impact: 2-fold increase in S-phase entry
- Pathway validation: Links ABI1 to SFKs/STAT3/NF-κB signaling
- Demonstrates self-delivery efficacy in most challenging primary cell type
Research utility:
- Myeloproliferative neoplasm disease modeling
- HSC self-renewal and cell cycle regulation studies
- Target validation for myelofibrosis research
- Ex vivo manipulation of patient CD34+ cells
4.3 Primary T Lymphocytes and Immunosenescence Research
TRIB2 Regulation of Naive T Cell Homeostasis During Aging
[4]Research objective: Investigate differential regulation of CD4+ vs. CD8+ T cell homeostasis during aging and identify TRIB2 as a protective factor against age-related naive T cell loss.
Experimental approach:
- AUMsilence sdASOs targeting TRIB2, ZBTB7B (ThPOK), RUNX3
- Primary human naive CD4+ and CD8+ T cells isolated from healthy donors
- Concentration: 2 μM for all targets
- Duration: 5-7 day culture with sustained knockdown through multiple cell divisions
- Delivery: Self-delivering (no transfection reagent required)
Research findings:
- TRIB2 mRNA knockdown: >50% reduction validated by RT-qPCR
- Proliferation increase: 3-4 fold enhancement in TRIB2-silenced cells (Ki67+ frequency)
- Differentiation acceleration: Loss of naive phenotype (CD45RA+CCR7+), gain of CD25 expression
- AKT hyperactivation: Increased phosphorylation at Thr308 and Ser473 residues
- Enhanced effector function: Elevated IL-2, IFN-γ, TNF-α, granzyme B production
- Lineage regulation: ThPOK knockdown reduced TRIB2; RUNX3 knockdown increased TRIB2
- No cytotoxicity: Cell viability maintained throughout 7-day experiments
Research utility:
- T cell aging and immunosenescence mechanisms
- CAR-T cell engineering research (enhanced proliferation strategies)
- Homeostatic proliferation pathway analysis
- CD4+ vs. CD8+ T cell lineage regulation studies
- AKT signaling pathway functional dissection in primary lymphocytes
- Investigation of naive T cell maintenance mechanisms
Technical significance: AUMsilence sdASOs maintained knockdown efficacy through 7 days of culture and multiple rounds of cell division in primary human T cells, demonstrating sustained activity in proliferating lymphocytes without re-dosing.
4.4 Pediatric Cancer Research Applications
KCTD15 Essential Gene in Pediatric B-Cell Acute Lymphoblastic Leukemia
[2]Research objective: Identify and validate KCTD15 as a novel biomarker and functional player in B-ALL, the most common childhood leukemia.
Experimental approach:
- AUMsilence sdASO targeting KCTD15
- Patient samples: 15 pediatric B-ALL patients (bone marrow blasts 65-95%)
- Cell lines: RS4;11, REH, TOM-1, SEM (B-ALL lines)
- Concentration: 8 μM (patient samples), 1 μM (RS4;11 uptake validation)
- Duration: 16-day time course
- Delivery: Gymnotic (no transfection reagent, electroporation, or lipofection)
- Uptake efficiency: 99.2% in RS4;11 cells (24 hours, flow cytometry with fluorescently labeled control)
Research findings:
- KCTD15 mRNA knockdown: 70% reduction (day 8), 80% reduction (day 16)
- Cell viability impact: 80% cell death by day 16 in ASO-treated cells vs. 16% in scramble control
- KCTD15 overexpression: 18-fold protein increase at diagnosis vs. post-therapy remission
- Patient validation: Elevated KCTD15 in bone marrow samples from all 15 B-ALL patients
- Functional dependence: KCTD15 silencing demonstrates essential gene for B-ALL survival
Research utility:
- Pediatric leukemia disease modeling and target validation
- Essential gene identification in hematological malignancies
- B-ALL biomarker research (KCTD15 as diagnostic marker)
- Investigation of KCTD family proteins in cancer
- Primary patient sample analysis (15 pediatric patients validated)
Clinical context: KCTD15 protein levels drop dramatically following successful therapy (18-fold reduction), suggesting utility as a pharmacodynamic biomarker for treatment response monitoring in B-ALL research.
H3.3K27M Mutation Targeting in Diffuse Midline Gliomas
[9]Research objective: Investigate the mechanistic link between H3K27M mutation (cardinal oncogenic event), lactate metabolism, and nucleotide biosynthesis in the most lethal pediatric brain cancer.
Experimental approach:
- AUMsilence sdASO targeting H3.3K27M
- Target: Mutant H3F3A allele spanning H3.3K27M mutation site in exon 2
- Patient-derived DMG cells: SF8628, DIPG-6, QCTB-R059
- Concentration: 5 μM in culture medium
- Duration: 72 hours
- Delivery: Self-delivering (no transfection reagents)
- Validation: Western blot for H3.3K27M protein loss
Research findings:
- H3.3K27M protein: Confirmed loss at 72 hours post-treatment
- Metabolic impact: 40-60% reduction in lactate production from glucose
- Nucleotide synthesis: Significant reduction in ATP, GTP, UTP, CTP levels
- Mechanism: H3K27M silencing reduced PGK1 expression (glycolytic enzyme)
- Validation across 3 independent patient-derived cell lines
Research utility:
- Pediatric brain cancer metabolism research
- Histone mutation functional studies
- Cancer metabolic imaging research (deuterium MRI)
- NME1 lactylation and post-translational modification studies
- Glycolysis-nucleotide synthesis coupling mechanisms
Significance: Diffuse midline gliomas have 11-month median survival with no effective treatments. Direct silencing of the H3.3K27M driver mutation represents a precision research approach targeting the root cause. This validates AUM ASO delivery to patient-derived pediatric brain tumor cells.
5. In Vitro Knockdown Efficiency and Functional Outcomes
5.1 mRNA Knockdown Across Targets
Analysis of published data demonstrates consistent up to 95% mRNA knockdown across diverse targets and cell types. The rapid cellular uptake documented in Section 3 enables efficient gene silencing for functional genomics research:
| Target Gene | Cell Type | mRNA Knockdown | Protein Knockdown | Duration | Reference |
|---|---|---|---|---|---|
| FOXP3 | Primary Tregs (19 cancer patients) | 64.7% (p<0.0001) | 60% cell reduction, 41% per-cell reduction | 3.5 hours to overnight | [8] |
| CISH | Primary NK cells (mouse & human) | Validated by RT-PCR | 75% functional rescue | 24 hours | [10] |
| ANKRD1 | Primary CAFs (4 patient strains) | >80% (p=0.0003) | 70% (p<0.0001) | 72 hours | [5] |
| ABI1 | CD34+ HSPCs (3 donors) | ~95% | 2-fold S-phase increase | 48 hours | [1] |
| Dhx15 | Mouse liver (in vivo) | 80% | 80% (Western blot) | q3d dosing, 72h duration | [7] |
| Gelsolin | Glioma cell lines | 4-fold depletion (75%) | Migration inhibited | Standard culture | [3] |
| TRIB2 | Primary human naive T cells (CD4+, CD8+) | >50% | 3-4 fold proliferation increase | 5-7 days sustained | [4] |
| H3.3K27M | Patient-derived DMG cells (SF8628, DIPG-6, QCTB-R059) | Protein loss confirmed | 40-60% lactate reduction | 72 hours | [9] |
| KCTD15 | Pediatric B-ALL patient samples (n=15) + cell lines | 70% (day 8), 80% (day 16) | 80% cell death (essential gene) | 16 days | [2] |
Research significance: Knockdown efficiency of up to 95% achieved across 10 distinct targets in diverse cell types (primary cells, cell lines, patient-derived samples) demonstrates reproducibility and versatility of AUMsilence sdASO technology for functional genomics research. Extended duration studies (up to 16 days validated) enable long-term phenotype analysis without re-dosing.
5.2 Functional Validation in Research Models
Beyond mRNA knockdown, AUMsilence sdASOs enable functional studies demonstrating biological consequences of target gene silencing:
Immune Cell Function Modulation
- Restored NK cell degranulation and cytotoxicity in CML microenvironment
- Impaired Treg immunosuppressive function (66.4% reduction)
- Increased inflammatory cytokine production (IL-2, IL-6, IL-7, TNF-α)
- Downregulated exhaustion markers: PD-1, Tim-3, LAG-3, CTLA-4, TIGIT
- Enhanced CD8+ T cell effector function (perforin, granzyme B)
Cancer Cell Biology Research
- Inhibited glioblastoma cell migration (gelsolin knockdown)
- Reversed CAF activation and pro-tumorigenic signaling (ANKRD1 knockdown)
- Disrupted mTORC2-cytoskeleton interactions in glioma cells
- Reduced tumor-promoting CAF effector gene expression (ACTA2, COL1A1, HAS2)
- Impaired angiogenesis and lymphangiogenesis in tumors (Dhx15 knockdown)
- Disrupted lactate-driven nucleotide biosynthesis in pediatric gliomas (H3.3K27M knockdown)
Cell Cycle, Proliferation, and Viability
- 2-fold increase in S-phase entry (ABI1 knockdown in CD34+ cells)
- 3-4 fold proliferation enhancement (TRIB2 knockdown in T cells)
- 80% cell death in essential gene studies (KCTD15 in B-ALL)
- Cell cycle progression studies without permanent genetic manipulation
- 67% reduction in cancer cell proliferation in CAF co-culture systems
- Reversible modulation enabling time-course and washout experiments
Metabolic Reprogramming
- 40-60% reduction in lactate production (H3K27M glioma metabolism)
- Decreased nucleotide synthesis (ATP, GTP, UTP, CTP levels)
- VEGF pathway inhibition in Dhx15-knockdown liver cancer models
- Metabolic imaging with deuterium-labeled substrates validated
- Tumor proliferation tracking through metabolic biomarkers
Signaling Pathway Modulation
- AKT signaling pathway modulation (TRIB2 study)
- AP-1 transcription factor complex disruption (ANKRD1 in CAFs)
- NF-κB and STAT3 pathway research applications
- mTORC2 pathway regulation in cancer cells
- Checkpoint receptor signaling modulation (CISH in NK cells)
Cell Viability and Target Specificity
- No toxicity at working concentrations
- Preserved cell viability in non-essential gene studies
- No innate immune activation (TLR-mediated) in primary immune cells
- High target specificity with minimal off-target effects
- No cross-reactivity with gene family members (FOXP3 vs. FOXP1/2/4 validated)
- Maintained stem cell properties (CD34+ phenotype) in hematopoietic research
6. In Vivo Research Applications
6.1 Tumor Research Models
AUMsilence sdASOs targeting FOXP3 in Syngeneic Tumor Models
[8]Research models:
- TC1 lung carcinoma (n=165 mice across 7 experiments)
- MC38 colon carcinoma (n=65 mice across 3 experiments)
- Immunocompetent mice (evaluates immune system effects)
Treatment protocol:
- Murine ASO 6B (targets FOXP3)
- Dose: 50 mg/kg intraperitoneally (i.p.) daily
- Duration: 14-16 days
- Start: Day 7 post-tumor implantation
- Delivery: Self-delivering, no formulation required
Research findings:
- Tumor growth inhibition: Significant reduction (TC1: p=0.0007; MC38: p=0.0403)
- Complete tumor resorption: 22% (TC1), 13.6% (MC38)
- Intratumoral FOXP3 mRNA: ~50% reduction (Day 14 mid-treatment)
- Intratumoral Treg numbers: Significantly decreased
- CD8+ T cell function: Increased perforin and granzyme B production (p<0.05)
- Exhaustion markers: 7/9 mRNA markers significantly downregulated
Selective targeting (critical for safety):
- Draining lymph nodes: NO change in FOXP3 or Treg numbers
- Spleens: NO change in FOXP3 mRNA
- Selective depletion of intratumoral vs. systemic Tregs
- No histologic evidence of autoimmunity (lungs, liver, colon, skin)
Research utility:
- Tumor immunology and microenvironment studies
- Preclinical modeling for checkpoint inhibitor combination research
- Investigation of Treg-mediated immunosuppression mechanisms
- Safety studies for translational research (no autoimmunity observed)
Research utility: AUMsilence sdASOs targeting FOXP3 demonstrated efficacy in multiple tumor models including MC38 colon carcinoma, validating the translational potential of this approach for diverse cancer types and tumor microenvironment studies.
Dhx15 Targeting in Hepatocellular Carcinoma Research
[7]Research model:
- Hepa 1-6 murine hepatocellular carcinoma cells
- Syngeneic model (immunocompetent C57BL/6 background)
- 5 × 10⁶ cells, subcutaneous flank implantation
- n=10 mice per group
Treatment protocol:
- AUMsilence sdASOs targeting Dhx15 (AUM BioTech, Philadelphia)
- Route: Intravenous (i.v.) tail vein injection
- Dose: 10 mg/kg every third day (q3d)
- Duration: 5 weeks
- Delivery: Gymnotic (self-delivering), no formulation
Research findings:
- Liver knockdown: 80% Dhx15 reduction maintained 72 hours post-injection
- Tumor volume reduction: 83.4% decrease (179.6 vs. 1085 mm³, p<0.01)
- Tumor vasculature: Reduced vascular perimeter and lumen size (both p<0.05)
- Lymphangiogenesis: 67% reduction in Lyve-1+ lymphatic vessels (p<0.05)
- Angiogenic gene expression: Significantly decreased Vegf-a, Vegf-d, Vegfr1, Vegfr3, Sdf-1 (all p<0.05)
Clinical biomarker validation:
- Patient cohort: 121 patients (24 healthy, 35 cirrhosis, 62 HCC)
- Serum DHX15: 9.3-fold elevation in HCC vs. healthy (300.3 vs. 32.4 pg/mL, p<0.01)
- Diagnostic potential for clinical research applications
Research utility:
- HCC pathogenesis and biomarker research
- Tumor angiogenesis and lymphangiogenesis studies
- VEGF pathway functional analysis in liver cancer
- Liver-targeted gene silencing without complex formulations
- Investigation of DHX15 as potential diagnostic and research target
AUMsilence sdASOs targeting ANKRD1 in Orthotopic SCC Xenografts
[5]Research model:
- FaDu squamous cell carcinoma cells + patient-derived CAFs
- Orthotopic skin cancer model (intradermal injection)
- CAFs pre-treated with ANKRD1-ASO (100 nM, 72 hours)
- 1:1 ratio cancer cells:CAFs in Matrigel
- NOD/SCID mice, n=4 per group
Research findings:
- Tumor volume: 83% reduction (3 vs. 18 mm³, p=0.0024)
- Cancer cell density: 50% reduction (p=0.0002)
- Duration of effect: Up to 5 days in primary CAFs
- Mechanism: Ex vivo ASO treatment of CAFs reduces tumor-promoting capacity
Research utility:
- Ex vivo cell treatment for xenograft research
- Tumor-stroma interaction studies
- CAF functional contribution to tumorigenesis
- Investigation of mesenchymal-epithelial crosstalk
6.2 Biodistribution and Selective Targeting in Research Models
A critical advantage of AUMsilence sdASO technology for in vivo research is the ability to achieve selective tissue targeting without complex formulation strategies:
Tumor Microenvironment Selectivity (FOXP3 Study)
| Tissue | FOXP3 mRNA Change | Treg Number Change | Research Implication |
|---|---|---|---|
| Tumor | ↓ 50% reduction | ↓ Significantly decreased | Primary site of action |
| Draining lymph nodes | → No change | → No change | Spares peripheral immunity |
| Spleen | → No change | ↑ Slight increase | Systemic immunity intact |
Mechanism of selectivity: Intratumoral Tregs express 2-2.5x higher FOXP3 mRNA levels with high protein turnover, rendering them preferentially sensitive to RNase H-mediated degradation. This mirrors the concept that rapidly dividing cells are more sensitive to antimetabolites, providing inherent tumor selectivity for research applications.
Liver Targeting (Dhx15 Study)
Gymnotic delivery achieved 80% knockdown in liver following simple intravenous injection (10 mg/kg). Effect sustained for 72+ hours, enabling every-third-day dosing in research protocols. Moderate knockdown also observed in lung and spleen, demonstrating multi-organ biodistribution potential for systemic gene silencing research.
Safety Profile in Research Models
- No histologic evidence of autoimmunity in multiple organs (FOXP3 study: lungs, liver, colon, skin examined)
- No uncontrolled T cell activation or systemic inflammation
- No reported adverse events in 5-week Dhx15 tumor study
- Maintained organ function (liver, kidney) in chronic dosing protocols
- Compatible with immunocompetent models (evaluates intact immune system responses)
6.3 In Vivo Tumor Growth Inhibition Summary
| Target/Study | Tumor Model | Dosing Regimen | Tumor Reduction | Complete Response | Reference |
|---|---|---|---|---|---|
| FOXP3 (Tregs) | TC1 lung carcinoma | 50 mg/kg i.p. daily × 14d | Significant (p=0.0007) | 22% | [8] |
| FOXP3 (Tregs) | MC38 colon carcinoma | 50 mg/kg i.p. daily × 16d | Significant (p=0.0403) | 13.6% | [8] |
| Dhx15 (liver/HCC) | Hepa 1-6 HCC | 10 mg/kg i.v. q3d × 5 weeks | 83.4% (p<0.01) | Not reported | [7] |
| ANKRD1 (CAFs) | FaDu SCC + CAFs | CAFs pre-treated ex vivo | 83% (p=0.0024) | Not assessed | [5] |
Research implications: Consistent 13.6-83% tumor volume reductions across multiple cancer types and genetic backgrounds validate the utility of AUMsilence sdASOs for in vivo functional genomics and preclinical research. The ability to achieve these effects without complex delivery formulations simplifies experimental design and reduces confounding variables in mechanistic studies.
6.4 Humanized Mouse Models
Validation in humanized mice (hu-PBMC-NSG) demonstrated that AUMsilence sdASOs targeting human FOXP3 effectively downregulate FOXP3 mRNA in blood, lymph nodes, and spleens following systemic administration (50 mg/kg i.p. daily × 4 doses). This confirms cross-species applicability and enables translational research using human ASO sequences in vivo.
7. Conclusions
7.1 Advantages of AUMsilence sdASO Technology for Research
Rapid, Efficient Cellular Uptake
Peer-reviewed microscopy studies demonstrate 80-100% cellular uptake within 15 minutes across primary cells and cancer cell lines. This rapid delivery enables same-day experimental readouts and eliminates prolonged incubation periods required by conventional methods. Nuclear penetration within 15 minutes ensures efficient RNase H recruitment for mRNA degradation.
Transfection-Free Research Protocol
Eliminates toxicity and experimental variability associated with lipid-based transfection reagents or electroporation. Enables consistent results across laboratories and experimental conditions. Validated in CD34+ hematopoietic stem cells (>50% knockdown), primary Tregs (64.7% knockdown), primary NK cells, and patient-derived CAFs without transfection optimization.
Primary Cell Compatibility for Translational Research
Successfully validated in the most challenging primary cell types: CD34+ hematopoietic stem cells (notoriously difficult to transfect), primary regulatory T cells from 19 cancer patients, primary NK cells, and patient-derived cancer-associated fibroblasts. Maintains cell viability, stemness markers, and functional properties critical for translational studies.
In Vivo Research Applications
Demonstrated robust activity in immunocompetent mouse models (80-83% tumor growth inhibition) with favorable safety profiles and selective tumor microenvironment targeting. Simple systemic administration (i.v. or i.p.) without complex formulations enables straightforward in vivo functional genomics and preclinical research protocols.
7.2 Research Applications Across Cancer and Immunology
The data presented in this whitepaper demonstrate the broad utility of AUMsilence sdASO technology for basic, translational, and preclinical research across:
- Cancer immunology research: Regulatory T cell depletion studies, NK cell functional restoration, tumor microenvironment modulation, T cell aging and homeostasis, exhaustion marker analysis
- Pediatric cancer research: Diffuse midline gliomas (H3K27M mutation), B-cell acute lymphoblastic leukemia (KCTD15 essential gene), patient-derived tumor cell validation
- Solid tumor biology: Hepatocellular carcinoma, glioblastoma, squamous cell carcinoma, colon carcinoma, lung carcinoma, melanoma research models
- Hematological malignancy research: B-cell acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myeloproliferative neoplasms, myelofibrosis disease modeling
- Stromal cell biology: Cancer-associated fibroblast activation mechanisms, tumor-stroma interactions, AP-1 signaling research
- Primary immune cell research: CD34+ hematopoietic stem cells, regulatory T cells, naive T cells (CD4+, CD8+), NK cells, Th17 cells, T cell lineage studies
- Stem cell research: Hematopoietic stem cell regulation, cell cycle control, NOTCH signaling, lineage commitment studies
- Immunosenescence research: T cell aging mechanisms, homeostatic proliferation, AKT signaling pathway analysis
- Cancer metabolism research: Lactate metabolism, nucleotide biosynthesis, glycolysis pathway studies, metabolic imaging biomarker development
7.3 Applications Enabling Previously Challenging Research
AUMsilence sdASO technology enables research that was previously technically challenging or impossible:
Pediatric Cancer Research
- Direct targeting of oncogenic driver mutations (H3.3K27M in diffuse midline gliomas)
- Essential gene validation in patient-derived pediatric tumor cells
- Functional studies in rare pediatric malignancies where patient samples are limited
Primary Immune Cell Studies
- Gene silencing in CD34+ hematopoietic stem cells without transfection toxicity
- Functional modulation of primary T cells maintained through multiple divisions
- NK cell functional restoration in patient-derived samples
- Treg biology in fresh tumor specimens
Translational Research
- Tumor microenvironment cell-cell interaction studies
- Ex vivo patient sample analysis preserving clinical relevance
- In vivo functional genomics without complex delivery formulations
- Target validation for future investigational studies
7.4 Compatible Research Applications
Cell Therapy Enhancement
CAR-T cell engineering, TIL biology and modulation, functional enhancement research
Checkpoint Inhibitor Research
Combination studies in preclinical models and functional genomics approaches
Patient-Derived Models
PDX functional genomics, 3D culture systems, organoid models, and co-culture research
Cancer Metabolism
Novel targets in metabolism, immune regulation, signaling pathways, and metabolic imaging biomarkers
Immunosenescence
Age-related immune dysfunction and aging biology research
7.5 Summary
AUM BioTech's self-delivering antisense oligonucleotide technology addresses fundamental limitations in gene silencing research by enabling efficient delivery to difficult-to-transfect cells without transfection reagents. Validated across 11 peer-reviewed publications spanning basic, translational, and preclinical research, AUMsilence sdASOs demonstrate:
From patient-derived pediatric brain tumors to primary CD34+ hematopoietic stem cells, from regulatory T cells in cancer to naive T cells in aging research, AUMsilence sdASOs provide investigators with versatile, reliable research tools that work where conventional methods fail. The elimination of transfection reagents, rapid cellular uptake, and sustained knockdown through extended culture periods make AUM technology well-suited for complex experimental designs in cancer and immunology research.
8. References
Advance Your Cancer and Immunology Research
Contact our scientific team to discuss how AUMsilence sdASOs can enable your research objectives.