AUMsilence™ sdASO™ 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 for a stable knockout
• Variable editing efficiency across cell types and genomic loci
• Immune responses to Cas9 protein in immunocompetent models

Delivery barrier in primary cells

• Many primary cell types are difficult to transfect with conventional methods
• Transfection reagent toxicity limits utility in sensitive cells
• Electroporation can cause cellular stress and altered phenotypes
• Viral vectors require extended culture periods and raise biosafety concerns
• Nanoparticle formulations show lysosomal sequestration reducing bioavailability

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

2.1 Technology overview

AUM BioTech's self-delivering antisense oligonucleotides (sdASOs) carry 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 the single-stranded mechanism keeps silencing to sequences that match the target.

Self-delivery for research

Gymnotic uptake eliminates need for transfection reagents, viral vectors, or lipid nanoparticles. Addition to culture medium or injection into animal models enables rapid cellular internalization. No transfection step is run, so fewer variables enter a research study.

RNase H1 mechanism

Forms stable DNA-RNA hybrids that recruit endogenous RNase H1 for catalytic mRNA degradation. A single ASO can degrade multiple target mRNA copies, providing dose-efficient gene silencing for functional studies.

Enhanced stability for in vivo research

Chemical modifications and a phosphorothioate backbone give greater nuclease resistance than an unmodified oligonucleotide, which is the comparison the papers make. In the one study that measured how long the effect lasts, a single intravenous dose held 80% liver silencing up to 72 hours.

Specificity

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

3.1 Quantitative cellular uptake data

Uptake kinetics in primary immune cells

Uptake in cancer cell lines

Both A549 (lung adenocarcinoma) and HEp-2 (HeLa derivative) cell lines took up the oligonucleotide in every cell scored, at the earliest time point measured:

Gymnotic uptake in leukemia research

3.2 Delivery in CD34+ hematopoietic stem cells

• 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)

• ABI1 silencing: more than 50% efficiency after 48 hours incubation, by immunoblotting for Abi-1 protein
• Cellular penetrance confirmed by confocal microscopy and fluorescence-activated cell sorting; the paper gives no uptake percentage
• Functional impact: a nearly 2-fold increase in CD34+ cells in S-phase, by EdU incorporation
• No transfection reagent was required, so no reagent toxicity; the paper reports no viability or stemness measurement for this arm

3.3 Delivery across diverse cell types: Summary

4.1 Primary immune cells

FOXP3 targeting in regulatory T cells [8]

• AUMsilence sdASO 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 μM for the 5-day gene expression and flow cytometry work, 2.5 μM for the 3.5-hour suppression assay; duration 3.5 hours, overnight, or 5 days depending on the readout

• mRNA knockdown: 64.7% reduction across cancer samples, tumor, pleural effusion, distant tumor-free lung and lymph node, from 11 patients in 7 experiments (p < 0.0001)
• Treg depletion: 60.0% decrease of Treg numbers in the same cancer samples
• Functional impairment: after only 3.5 hours of exposure, suppressive function fell to 66.4% of the Scramble control, a loss of about a third. The 66.4% is the residual, which Figure 2C's axis and its one-sample test against a mean of one both settle
• Exhaustion markers downregulated: mRNA expression of five of the nine markers tested fell significantly, CTLA-4 (p = 0.0034), Tim-3 (p = 0.0001), PD-1 (p < 0.0001), LAG-3 (p = 0.0048) and TIGIT (p = 0.0284), all printed on Figure 3E
• Inflammatory cytokines upregulated: 7 of 11 rose significantly, IL-2 (p < 0.0096), IL-6 (p < 0.0312), IL-7 (p < 0.0187), IL-10 (p < 0.0421), IL-18 (p < 0.0040), TNF-α (p < 0.0332) and Perforin (p < 0.0375), all printed on Figure 4C
• FOXP3 protein per cell: the leftover Tregs that were still FOXP3+ after 5 days carried less FOXP3 protein per cell, and the decrease was greater in cancer samples than in PBMC samples. The paper states no percentage
• CD39 expression: Significantly decreased

• Functional dissection of Treg immunosuppressive mechanisms
• Tumor microenvironment studies in clinical samples
• Comparison of intratumoral vs. peripheral Tregs; the paper concludes the intratumoral cells were more sensitive, and states no ratio
• Investigation of exhaustion marker regulatory networks
• Preclinical modeling for immunotherapy research

CISH targeting in natural killer cells [10]

• 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

• CISH mRNA depletion: Confirmed by RT-PCR in both mouse and human NK cells
• Functional restoration (mouse): the paper reports "a partial restoration of NK degranulation capacity in CML serum-treated CishKD NK cells". Read it as partial: Figure 4C annotates that comparison 0.089, which is above the paper's own significance threshold
• Functional restoration (human): "CISH knockdown potentiated the degranulation capacity of NK cells exposed to CML plasma". Figure 5I marks that comparison with a single asterisk, and its key is P ≤ .05
• Clinical validation: Plasma from patients who failed to achieve major molecular response to TKI therapy

• 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)

4.2 Primary tumor-associated cells and stem cells

ANKRD1 targeting in patient-derived cancer-associated fibroblasts [5]

• AUMsilence sdASO targeting ANKRD1
• Primary CAFs isolated from discarded skin samples of squamous cell carcinoma, in parallel with normal fibroblasts from flanking skin
• Four patient-derived CAF strains in the RT-qPCR, keyed on Figure 8a as CAF1, CAF2, CAF7 and CAF11; the imaging panels use CAF#7 and the mouse experiment CAF#2
• Concentration: 100 nM of the ANKRD1-targeting oligonucleotide, against a scrambled control at the same concentration
• Duration: experiments performed after 72 hours of incubation
• Delivery: transfected. This paper is the one exception on this page. Its Methods state that the cells were transfected with the antisense oligonucleotides purchased from AUM BioTech, using 10 μL of HiPerFect transfection reagent mixed with 100 nM of oligonucleotide in DMEM only, incubated at room temperature for 15 minutes, with the medium changed after 6 hours. Nothing in this paper is evidence about delivery without a reagent.

• ANKRD1 and the CAF effector genes were downmodulated together, by RT-qPCR across four patient-derived CAF strains over 6 biological replicates, with the p values printed on Figure 8a: ANKRD1 p = 0.0003, ACTA2 p = 0.0006, COL1A1 p = 0.0002, INHBA p = 0.0013 and HAS2 p = 0.0028
• ANKRD1 protein and αSMA both fell in CAF#7 by immunofluorescence, each at p < 0.0001 over 3 independent experiments with more than 10 fields per condition and more than 100 cells per field (Figure 8b), with no effect on CAF proliferation (Supplementary Figure 7)
• The association of c-JUN with FOSL2 in these cells was significantly reduced, p = 0.0037, counted as proximity-ligation puncta per cell in CAF#7 over 2 independent experiments and more than 100 cells per condition (Figure 8c)
• Binding of the c-JUN protein was strongly suppressed at three sites in ACTA2 (p < 0.0001, p < 0.0001, p = 0.000232) and two in HAS2 (p = 0.000168, p = 0.013407), by chromatin immunoprecipitation with Tn5 tagging across three CAF strains over 4 biological replicates, expressed as enrichment folds over non-immune IgG (Figure 8d)
• Squamous cell carcinoma cell expansion was significantly reduced in co-culture with CAFs pre-treated with ANKRD1-ASO, p = 0.0039, measured as Pan-keratin positive area over 4 biological replicates (Figure 8e)
• The paper states no knockdown percentage for any of these readouts, so this page carries none; the p values above are the ones printed on its panels

• 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]

• Successfully modeled patient ABI1 loss: granulocytes from 36 patients with primary myelofibrosis, and 9 with secondary myelofibrosis post-polycythaemia vera, showed a 40% to 60% decrease in ABI1 mRNA against 16 controls, and CD34+ cells from bone marrow were down by approximately 40%
• Cell cycle impact: 2-fold increase in S-phase entry
• Pathway validation: Links ABI1 to SFKs/STAT3/NF-κB signaling
• Silencing in CD34+ hematopoietic stem cells with no transfection reagent

• 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]

• AUMsilence sdASO targeting TRIB2, ZBTB7B (ThPOK), RUNX3
• Primary human naive CD4+ and CD8+ T cells isolated from healthy donors
• Concentration: 2 μM for all targets
• Duration: 7 days with IL-7 and plate-bound anti-CD28, or 5 days with plate-bound anti-CD3/anti-CD28
• Delivery: Self-delivering (no transfection reagent required)

• TRIB2 silencing confirmed in Figure S1B; the paper states no knockdown percentage
• Proliferation increase: TRIB2 deficiency increased IL-7-induced proliferation to a level closer to that of naive CD8+ T cells, measured by CellTrace Violet dilution over 7 days, and promoted TCR-induced proliferation, measured as Ki67+ frequency over 5 days. The paper states no fold change
• Differentiation acceleration: around 50% of TRIB2-silenced CD4+ cells had lost CD45RA expression after 7 days, and the cells started to gain CD25 expression
• AKT activation: TRIB2 silencing increased IL-7-induced AKT phosphorylation, measured at Thr308 and Ser473, and the paper attributes the downstream effects to that sustained activation
• 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

• T cell aging and immunosenescence mechanisms
• CAR-T cell engineering research
• 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

4.4 Pediatric cancer research applications

KCTD15 essential gene in pediatric B-cell acute lymphoblastic leukemia [2]

• 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: 4 and 8 μM tested in RS4;11 cells and 8 μM chosen; 1 μM of the labeled control for the uptake check. No patient sample in this paper received an oligonucleotide
• Duration: 16-day time course
• Delivery: no transfection reagent appears anywhere in this paper's methods, and the Supplementary Figure 2 legend records internalization “without the use of electroporation or liposome mixture”. Gymnotic is AUM's word for that, not the authors'
• Uptake, and whose: 99.2% of RS4;11 cells at 24 hours by flow cytometry, measured on the fluorescently labeled control oligonucleotide at 1 μM (Supplementary Figure 2). The KCTD15 oligonucleotide's own uptake was not measured

• KCTD15 silencing in RS4;11: a progressive decrease of both KCTD15 mRNA and protein from day 8 to day 16, by real-time PCR, western blot and flow cytometry. The paper states no knockdown percentage
• Cell viability impact: dead RS4;11 cells rose from 26.3% at day 8 to about 80% at day 16; cells treated with the scrambled variant did not show appreciable increases in mortality
• KCTD15 at diagnosis against day 33 after therapy: transcript levels 3.6 times higher at diagnosis (p = 0.002), and protein falling from a median 5.66 × 10⁴ to 3.1 × 10³ MFI (p = 0.0005), both by Wilcoxon matched-pairs test
• Patient material was used for expression profiling only: elevated KCTD15 in bone marrow samples across the 15-patient cohort of Table 1, with the diagnosis against day 33 comparison made in 12 of them and the transcript analysis in 10
• Functional dependence: KCTD15 silencing demonstrates essential gene for B-ALL survival

• Pediatric leukemia disease modeling and target validation
• Essential gene identification in hematological malignancies
• B-ALL biomarker research (KCTD15 as a candidate marker)
• Investigation of KCTD family proteins in cancer
• Primary patient sample analysis (15 pediatric patients validated)

H3.3K27M mutation targeting in diffuse midline gliomas [9]

• 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

• H3.3K27M protein: Confirmed loss at 72 hours post-treatment by western blotting
• Metabolic impact: 13C labeling of the glycolysis metabolites, including lactate, was significantly reduced in silenced cells relative to mutant cells in all models. The paper states no percentage
• Nucleotide synthesis: Significant reduction in IMP, AMP, ATP, GMP, GTP, UMP, UTP and CTP labeling
• Mechanism: H3K27M silencing reduced PGK1 expression (glycolytic enzyme)
• Validation across 3 independent patient-derived cell lines

• 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

5.1 mRNA knockdown across targets

Knockdown across these targets is reported in whatever terms each paper used. Several state a percentage, several state a direction and a significance, and several state only that the target went down. This table gives each one as its paper gives it, and says so where no magnitude exists:

5.2 Functional validation in research models

Immune cell function modulation

• Partially restored NK cell degranulation in the CML microenvironment (CISH)
• Impaired Treg immunosuppressive function, falling to 66.4% of the Scramble control
• Increased inflammatory cytokine mRNA: IL-2, IL-6, IL-7, IL-10, IL-18, TNF-α and Perforin-1
• Downregulated exhaustion marker mRNA: CTLA-4, Tim-3, PD-1, LAG-3, TIGIT
• More perforin and granzyme B produced by intratumoral T cells in treated mice

Cancer cell biology research

• Reversed CAF activation and pro-tumorigenic signaling (ANKRD1 knockdown)
• Disrupted mTORC2-cytoskeleton interactions in glioma cells (gelsolin knockdown): without gelsolin, RICTOR was no longer associated with the plasma membrane and was present only in cytoplasm and nuclei, and the F-actin network was maximally disrupted at 10.0 μM
• 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

• A nearly 2-fold increase in S-phase entry (ABI1 knockdown in CD34+ cells)
• Increased proliferation with no fold change stated (TRIB2 knockdown in T cells)
• 80% cell death in essential gene studies (KCTD15 in B-ALL)
• Cell cycle progression studies without permanent genetic manipulation
• Significantly reduced cancer cell expansion in CAF co-culture systems, with no percentage stated (ANKRD1)

Metabolic reprogramming

• Significantly reduced glycolytic labeling, lactate included, with no percentage stated (H3K27M glioma metabolism)
• Decreased nucleotide biosynthesis labeling (IMP, AMP, ATP, GMP, GTP, UMP, UTP, CTP)
• 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

• Where viability was measured it was preserved: ASO FOXP3 had no effects on cell viability or cell division, TRIB2 knockdown did not affect naive T cell survival, and ANKRD1 knockdown had no effect on CAF proliferation
• The FOXP3 screen set explicit cut-offs rather than assuming none: candidates had to decrease FOXP3+ cell numbers by at least 15%, cause no more than a 10% decrease in cell viability, and inhibit CD4+ and CD8+ T cell division by no more than 20%. Eleven of nineteen candidates passed all three
• No cross-reactivity with gene family members: ASO FOXP3 did not significantly downregulate any other member of the FOXP family
• Reported not to activate Toll-like receptor signaling, which Akimova et al. state in their discussion citing earlier work rather than measuring it here; none of the studies on this page measured it

6.1 Tumor research models

AUMsilence™ sdASO™ targeting FOXP3 in syngeneic tumor models [8]

• TC1 adenocarcinoma (n=165 mice across 7 experiments)
• MC38 colon carcinoma (n=65 mice across 3 experiments)
• Immunocompetent mice (evaluates immune system effects)

• 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

• Tumor growth inhibition: significant in both models, against Scramble at p = 0.0007 for TC1 and p = 0.0040 for MC38, and against PBS at p < 0.0001 in both
• Complete tumor resorption: 22% (TC1), 13.6% (MC38)
• Intratumoral FOXP3 mRNA: ~50% reduction (Day 14 mid-treatment)
• Intratumoral Treg numbers: Significantly decreased
• T cell function: intratumoral T cells in treated mice produced more perforin and granzyme B, and CD4-CD8- non-T cells had increased IL-2 and IFN-γ
• Exhaustion markers: 7 of 9 significantly downregulated at mRNA level, 4 of 6 at protein level

• Draining lymph nodes: NO change in FOXP3 or Treg numbers
• Spleens: no change in FOXP3 mRNA, and Treg numbers significantly increased (Figure 8C)
• Selective depletion of intratumoral vs. systemic Tregs
• No histologic evidence of autoimmunity (lungs, liver, colon, skin)

• 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)

Dhx15 targeting in hepatocellular carcinoma research [7]

• Hepa 1-6 murine hepatocellular carcinoma cells
• Syngeneic model in wild-type mice
• 5 × 10⁶ cells, subcutaneous flank implantation
• n=10 mice per group

• AUMsilence sdASO 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

• Liver knockdown: 80% Dhx15 silencing after a single intravenous dose, maintained up to 72 hours post-injection
• Primary tumor volume at five weeks: 179.6 ± 80.06 mm³ in the Dhx15 group against 1085 ± 277.1 mm³ in the scramble group (p < 0.01)
• Tumor vasculature: significantly reduced vascular perimeter and lumen of blood vessels, by endomucin immunostaining
• Lymphangiogenesis: significant reduction in lymphatic vessels, by Lyve-1 immunostaining. The paper states no percentage for the treated group
• Angiogenic gene expression in the livers of treated mice: decreased Vegf-a, Vegf-d, Vegfr1 and Vegfr3

• Cohort, from the Figure 6C legend: 62 patients with hepatocellular carcinoma, 35 with cirrhosis, and 24 healthy controls
• Serum DHX15 in hepatocellular carcinoma against healthy controls: 300.3 ± 88.2 vs. 32.4 ± 27.7 pg/mL (p < 0.01)
• Against cirrhosis without tumor the difference was a trend only: 300.3 ± 88.2 vs. 132.0 ± 46.2 pg/mL (p = 0.095)
• Potential as a marker in research

• 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 a candidate marker and research target

AUMsilence™ sdASO™ targeting ANKRD1 in orthotopic SCC xenografts [5]

• FaDu squamous cell carcinoma cells + patient-derived CAFs (CAF#2)
• Orthotopic skin cancer model (intradermal injection into contralateral mouse back skin)
• CAFs pre-treated with the ANKRD1-targeting oligonucleotide or a scrambled control (100 nM, 72 hours), transfected with HiPerFect
• 2.5 × 10⁵ cancer cells admixed with an equal number of CAFs, resuspended in 70 μL of Matrigel
• NOD/SCID mice, 6-10 weeks old, n=4
• Mice sacrificed 10 days after injection for this experiment

• Tumor size: cancer cells admixed with CAFs pre-treated with ANKRD1-ASO produced smaller lesions than those admixed with control CAFs, p = 0.0024 (Figure 8f). The paper states no volume and no percentage
• Cancer cell density: lesser, p = 0.0002, quantified as Pan-keratin positive cells per field over 25 fields for Scrambled and 27 for ANKRD1-ASO (Figure 8g). The paper states no percentage
• Duration of effect: the paper disagrees with itself. Its Figure 8e legend says CAFs were transfected for 72 hours and then co-cultured for 5 days; its body text says pre-treated for 48 hours and co-cultured for seven days
• Mechanism: ex vivo ASO treatment of CAFs reduces tumor-promoting capacity

• 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

Tumor microenvironment selectivity (FOXP3 study)

Liver targeting (Dhx15 study)

Gymnotic delivery achieved 80% Dhx15 silencing in the liver after a single intravenous injection (10 mg/kg), maintained up to 72 hours post-injection, which is what the every-third-day dosing in the tumor study rests on. A milder deletion was also seen in the lungs and spleen, so the biodistribution is not liver-only.

Safety profile in research models

• No histologic evidence of autoimmunity in multiple organs (FOXP3 study: lungs, liver, colon, skin examined by a pathologist)
• No signs of apparent autoimmunity or inflammation, either from ex-Treg subsets or from non-Treg cells, in tumor bearing mice or in RAG1-/- mice (FOXP3 study)
• The Dhx15 study makes no safety claim: its own words are that "[e]valuation of chronic toxicity, routes of administration, and dosage are still pending to establish robust conclusions about the biosafety" of that treatment, and its liver function panel was run in heterozygous mice rather than treated ones
• Compatible with immunocompetent models (evaluates intact immune system responses)

6.3 In vivo tumor growth inhibition summary

6.4 Humanized mouse models

7.1 Advantages of AUMsilence™ sdASO™ technology for research

1. 01

   Step 1: Rapid cellular uptake

   Microscopy reported 80-100% cellular uptake within 15 minutes in the three cell types where uptake was imaged: primary murine splenocytes at approximately 80%, and A549 and HEp-2 at 100%. ASO fluorescence was detectable in the nuclei of both cell lines at the same 15 minutes, and diffuse through primary cells with no nuclear exclusion at early time points. The papers that imaged it did so in medium supplemented with ammonium chloride and arsenic to enhance gymnosis, and none of them measured a gene knockdown on the day of dosing, so the speed reported here is the speed of uptake rather than of a readout.
2. 02

   Step 2: Transfection-free research protocol

   Where no transfection reagent is used, its toxicity and its batch-to-batch variability are not in the experiment. Applied without a transfection reagent in CD34+ hematopoietic stem cells (more than 50% ABI1 silencing at 48 hours), primary Tregs (64.7% FOXP3 mRNA reduction in cancer samples) and primary NK cells. The cancer-associated fibroblast paper on this page is not evidence for this claim: its Methods name a commercial transfection reagent.
3. 03

   Step 3: Primary cell compatibility for translational research

   Applied in some of the most challenging primary cell types: CD34+ hematopoietic stem cells (difficult to transfect), primary regulatory T cells from cancer patients, primary NK cells, and patient-derived cancer-associated fibroblasts, the last of these with a transfection reagent. Where the papers measured viability and function they found them preserved: ASO FOXP3 had no effects on cell viability or cell division, TRIB2 knockdown did not affect naive T cell survival, and ANKRD1 knockdown had no effect on CAF proliferation.
4. 04

   Step 4: In vivo research applications

   Activity in immunocompetent mouse models: significant inhibition of tumor growth in both the TC1 and MC38 FOXP3 models, and in the Dhx15 hepatocellular carcinoma model a primary tumor volume of 179.6 ± 80.06 mm³ against 1085 ± 277.1 mm³ in the scramble group at five weeks. Systemic administration (i.v. or i.p.) without complex formulations enables in vivo functional genomics and preclinical research protocols.

7.2 Research applications across cancer and immunology

• 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, 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, T cell lineage studies
• Stem cell research: Hematopoietic stem cell regulation, cell cycle control
• 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

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 sustained to the end of a 7-day culture
• NK cell function studied under patient-derived plasma
• 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 engineering research. 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

1. 01Chorzalska et al. Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis. Blood 2018;132(19):2053-2066.
2. 02Smaldone et al. KCTD15 is overexpressed in human childhood B-cell acute lymphoid leukemia. Scientific Reports 2019;9:20108.
3. 03Chantaravisoot et al. mTORC2 interactome and localization determine aggressiveness of high-grade glioma cells through association with gelsolin. Scientific Reports 2023;13:7037.
4. 04Cao et al. TRIB2 safeguards naive T cell homeostasis during aging. Cell Reports 2023;42(3):112195.
5. 05Mazzeo et al. ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation. Nature Communications 2024;15:1038.
6. 06Thambyrajah et al. Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate. Nature Communications 2024;15:1604.
7. 07Portolés et al. Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice. International Journal of Molecular Sciences 2024;25(7):3716.
8. 08Akimova et al. Antisense targeting of FOXP3+ Tregs to boost anti-tumor immunity. Frontiers in Immunology 2024;15:1426657.
9. 09Batsios et al. Lactylation fuels nucleotide biosynthesis and facilitates deuterium metabolic imaging of tumor proliferation in preclinical models of H3K27M-mutant gliomas. Science Translational Medicine 2026;18(836):eadw0834. DOI: 10.1126/scitranslmed.adw0834
10. 10Kuznetsova et al. Chronic inflammation deters natural killer cell fitness and cytotoxicity in myeloid leukemia. Blood Advances 2025;9(4):759-773.
11. 11Bartosh et al. New Approaches to Old Techniques in Cell Handling for Microscopy. Cells 2025;14:1271.