What siRNA & RNA Interference Knockdown got wrong, from 34 theses

Lost to a baseline

Transfection of 1 nM siRNA with RNAiMAX achieved ~50% luciferase knockdown in Neuro2a cells, outperforming NHS 10,000x siRNA-barcoded EVs which achieved ~25% knockdown.

Understanding extracellular vesicle biology for the development of bioinspired nanotechnology platforms · Oxford · PhD

Lost to a baseline

In siRNA nanoparticle uptake assays in P815 cells, KITag14 functionalization (2.38-fold uptake) performed below mouse SCF-functionalized nanoparticles.

Expanding the Designable Landscape of Protein Interfaces through Computational Binder Design · University of Washington · Doctorate

Lost to a baseline

Lipofectamine RNAiMAX achieved 68% GFP knockdown at 100 nM siRNA, whereas siRNA-loaded protein vesicles showed 0% gene silencing at 50-100 nM

Engineering Recombinant Protein Vesicles for Delivery Applications · Georgia Tech · PhD

Lost to a baseline

Lipofectamine RNAiMAX positive control achieved ~82% IL-8 gene suppression compared to ~75% by UCNP-siRNA nanoplexes.

Nanochemistry for Nanomedicine: Design, Synthesis, and Applications · University at Buffalo · PhD

Tried and failed

intermediate charge ratio complexation in lipid nanoparticles applied to siRNA delivery and gene silencing. Outcome: worse than baseline. Reason: intermediate charge ratios exhibited slowest release kinetics, reducing downregulation efficacy

Lipid-based nanoparticles for drug delivery · UT Austin · Doctorate

Tried and failed

mTOR inhibitor pretreatment to enhance delivery applied to lipid nanoparticle siRNA delivery in vitro. Outcome: worse than baseline. Reason: mTOR inhibition failed to enhance silencing and impaired functional delivery at higher doses

The impact of the metabolic state of a cell on nucleic acid therapeutics · Georgia Tech · PhD

Tried and failed

engineered protein vesicle delivery vehicle applied to intracellular siRNA delivery for gene silencing. Outcome: no signal. Reason: endosomal entrapment and lack of intracellular cargo release even with pH-sensitive peptides

Engineering Recombinant Protein Vesicles for Delivery Applications · Georgia Tech · PhD

Tried and failed

increasing hydrophobicity in ph-responsive cationic polymer carriers applied to sirna delivery and gene knockdown. Outcome: no signal. Reason: excessive hydrophobicity from diisopropylamino substitution hindered efficient intracellular sirna release or endosomal escape

A biodegradable pH-responsive carrier system for targeted siRNA delivery in inflammatory bowel diseases and cancer · UT Austin · PhD

Tried and failed

non-liposomal chemical transfection of siRNA applied to primary and immortalised myometrial cells. Outcome: no signal. Reason: failed to achieve reliable, effective target gene knockdown compared to electroporation

PKA and EPAC: cyclic AMP effectors in the regulation of myometrial inflammatory responses in parturition · Imperial · PhD

Lost to a baseline

Polymer substrate laser-based transfection achieved only up to 20% efficiency for 150 kDa dextrans and 9% for siRNA with ~60% viability, performing worse than gold nanoparticles or plasmonic pyramids.

Visualization and manipulation of repair and regeneration in biological systems using light · Leibniz Universität Hannover · PhD

Considered and rejected

Considered and rejected: Rejected direct 'naked' siRNA injections due to blood-brain barrier restrictions and poor neuronal uptake in vivo.

Role of Electrical Synapses in the Rat Inferior Olive: Assessment During Postnatal Development and After Knockdown of Connexin 36 · Tübingen · PhD

Considered and rejected

Considered and rejected: Covalent conjugation of endosomal escape domains (EED, GFWFG) directly to siRNA was rejected due to inferior knockdown efficiency compared to transfection reagents and risks of hydrophobic collapse and immune activation.

Multiple strategies to enhance efficacy of oligonucleotide therapeutics · Oxford · PhD

Considered and rejected

Considered and rejected: Rejected chemical/lipofectamine siRNA transfection due to poor PKAc knockdown efficiency compared to nucleofection electroporation.

PKA and EPAC: cyclic AMP effectors in the regulation of myometrial inflammatory responses in parturition · Imperial · PhD

Lost to a baseline

Without CaCl2, W1R9 + siRNA (25-50 nM) caused 0% GRP78 knockdown, losing to Lipofectamine RNAiMAX which achieved 25-35% protein knockdown.

Novel Peptide Nanoparticles Designed for Tumour Cell Entry Via Glucose Regulated Protein 78 Dependent Targeting · Carleton University · Master's

Tried and failed

shRNA-mediated gene knockdown applied to modeling complete knockout phenotypes. Reason: partial depletion failed to trigger lineage plasticity or phenotypic switching caused by complete knockout

Integrative multi-omics dissection of cancer cell states and susceptibility · MIT · PhD

Considered and rejected

Considered and rejected: Rejected siRNA knockdowns for CRISPR screen candidate validation due to incomplete target knockdown and failure of siCDK2 to outgrow siCTRL after HU treatment.

Targeting the Cell Cycle in Oral, Head, and Neck Squamous Cell Carcinoma · University of Washington · PhD

Considered and rejected

Considered and rejected: Rejected using siRNA knock-downs alone due to incomplete knockdown efficiency and cell-to-cell variability, transitioning to CRISPR/Cas9 knock-out cell lines.

Organisation and dynamics of mitochondria and their RNA. · EPFL · PhD

Considered and rejected

Considered and rejected: Rejected shRNA and siRNA knockdown systems in favor of constitutive pLentiCRISPRv2 knockout because incomplete depletion of ALG3 masked the growth and ER stress phenotypes.

The Glycosyltransferase alpha-1,3-mannosyltransferase (ALG3) is an AKT Substrate that Regulates Protein N-glycosylation in Breast Cancer Cells · Harvard · PhD

Considered and rejected

Considered and rejected: Transfection time of 4 hours for siRNA in WJ-MSCs was rejected due to insufficient knockdown (87.79%), requiring prolongation to 24 hours (achieving 95.6%).

The effect of gestational diabetes on human umbilical mesenchymal stem cells and their ability to influence endothelial permeability · Nottingham · PhD

Tried and failed

dsRNA-mediated RNA interference gene knockdown applied to specific serine protease immune genes. Reason: poor gene silencing efficiency and failure to successfully generate targeted dsRNA constructs

Dissecting The Melanization Immune Response In The Malaria Vector Anopheles Gambiae · Penn · PhD

Considered and rejected

Considered and rejected: Rejected using siRNA for nuclear retrocopy silencing in favor of antisense LNA gapmeRs due to siRNA inefficiency on nuclear transcripts.

Powiązania pomiędzy retrogenami i nowotworami – kontekst ewolucyjny Relationships between retrogenes and cancer – evolutionary context · Adam Mickiewicz University · PhD

Tried and failed

shRNA-mediated gene knockdown applied to cultured immortalised neuroblastoma cell line. Outcome: no signal. Reason: knockdown failed to replicate the Golgi morphology phenotype observed in knockout primary neurons

Disease Mechanisms and Therapeutic Strategies in Munc18-1 Encephalopathies · Cornell · PhD

Tried and failed

siRNA knockdown of target genes applied to mammary epithelial cell lines. Outcome: no signal. Reason: failed to achieve statistically significant mRNA reduction with high replicate variability

Distribution and role of TET-mediated DNA and RNA hydroxymethylation in breast cancer and normal breast epithelium · Imperial · PhD

Tried and failed

siRNA knockdown confirmed by mRNA quantification applied to target protein depletion in endothelial cells. Outcome: no signal. Reason: mRNA depletion did not translate to significant protein reduction on Western blot

Disrupted iron homeostasis in the vascular endothelium: implications for health and disease · Imperial · PhD

Tried and failed

siRNA-mediated gene knockdown applied to target gene mRNA depletion in cancer cells. Outcome: no signal. Reason: Designed siRNAs failed to produce measurable target mRNA knockdown despite downstream assay signal

Identifying Non-canonical, Keap1-independent Regulators of Nrf2 Degradation in Non-small Cell Lung Cancer · Harvard · Master's

Tried and failed

siRNA knockdown of upstream regulatory pathway targets applied to cancer cell redox metabolism pathways. Reason: Knockdown caused unexpected compensatory off-target increases in downstream protein expression

Multiscale Computational Modeling of ROS-Generating Chemotherapies in Head and Neck Squamous Cell Carcinoma · Georgia Tech · PhD

Lost to a baseline

In JL-1 cells, transfection of scrambled (SCR) siRNA caused a 32% reduction in cell count, outperforming several specific siRNAs

Potential der siRNA-Transfektion auf chemotherapieresistente NSCLC- und Pleuramesotheliomzellen - Auswirkungen auf Viabilität und Wachstumsverhalten · Tübingen · PhD

Considered and rejected

Considered and rejected: Rejected using siRNA and shRNA systems in favor of CRISPR/Cas9 due to higher off-target effects in RNAi

Histone Lysine Demethylase KDM4A as a Therapeutic Target for Small Cell Lung Cancer · UT Southwestern · PhD

Considered and rejected

Considered and rejected: Rejected using 2.1 pmols siRNA despite higher transfection efficiency due to materials released into media and potential cytotoxicity at 2.4 pmols

Unravelling Molecular Pathways of Neurodegeneration in Hereditary Spastic Paraplegias Associated with ER-Shaping Defects · University College Dublin · PhD

Tried and failed

pooled siRNA gene knockdown validation applied to host innate immune response phenotyping. Outcome: did not generalise. Reason: Observed phenotype was caused by off-target silencing from an individual siRNA within the pool.

The Relationship Between Herpes Simplex Virus 1 and the Cellular DNA Damage Response · Harvard · PhD

Tried and failed

constitutive gene knockdown via RNA interference applied to stage-specific embryonic morphogenesis. Outcome: unstable. Reason: strong knockdown caused lethal defects in earlier developmental stages before the target process occurred

Organizing morphogenesis: Mechanisms of actomyosin patterning by RhoGTPase signaling · MIT · PhD

Considered and rejected

Considered and rejected: Rejected siRNA knockdown for generating protein-depleted CDMs because siRNA produces only transient knockdowns (2-7 days) insufficient for the 10-14 day matrix deposition period and achieves incomplete knockout.

Engineering ‘extracellular matrix factories’ to study how the extracellular microenvironment regulates gene expression · Nottingham · PhD

Tried and failed

simultaneous siRNA co-transfection without pre-depletion applied to prime editing efficiency enhancement. Outcome: no signal. Reason: siRNA knockdown kinetics were too slow to deplete target protein before editing occurred

Advancing precision genome and transcriptome engineering · Harvard · PhD

Considered and rejected

Considered and rejected: Rejected stable shRNA knockdown of PCAF in HEK293T cells due to cell death/lack of proliferation, opting to switch to transient siRNA knockdown.

NUTRIENT STRESS INDUCED LIPID METABOLISM: EXPLORING THE SIRT2-ACSS2 INTERSECTION · Cornell · PhD

Considered and rejected

Considered and rejected: Stable transfection with shRNA hairpins for FOXC1 knockdown was rejected in favor of transient siRNA transfection due to lack of stable, maintained silencing.

Identification of chemotherapy resistance drivers in breast cancer cells · Imperial · PhD

Tried and failed

layer-by-layer polymer coating for sustained oligonucleotide delivery applied to in vivo target gene knockdown in wounds. Outcome: unstable. Reason: Transient initial knockdown was lost over extended timepoints due to inadequate sustained release or rapid target recovery

Systematic engineering of controlled, localized oligonucleotide delivery systems for wound angiogenesis · MIT · PhD

Left open

Test whether E-Ras knockdown via siRNAs in ESCs or ectopic expression in MEFs regulates EV-mediated reprogramming. Blocker: Requires a wet lab with mammalian cell culture, siRNA transfection, and EV isolation capabilities

EXTRACELLULAR VESICLES IN STEM CELL BIOLOGY · Cornell · PhD

Left open

Improve MAP4 knockdown efficiency in mature mammalian cardiomyocytes using multiple targeted siRNA or CRISPR interference. Blocker: Requires a wet biology lab, cardiomyocyte cell cultures, and gene editing reagents (siRNA/CRISPRi).

Studying the role of microtubule network in beta-adrenergic receptors mRNA localisation in cardiomyocytes using single molecule techniques · Imperial · PhD

Left open

Evaluate connectosomes for intracellular delivery of small interfering RNAs (siRNAs) and single-stranded nucleic acids. Blocker: Requires wet-lab experimental facilities to culture cells, generate connectosomes, and test intracellular delivery of nucleic acids.

Engineering liposomal particles for direct, intracellular delivery of therapeutic molecules · UT Austin · PhD

Left open

Synthesize siRNA-SLNs using DLin-MC3-DMA instead of DOTAP and evaluate their cytotoxicity and proinflammatory properties. Blocker: Requires wet lab chemical synthesis and biological testing assays

Lipid-based nanoparticles for drug delivery · UT Austin · Doctorate

Left open

Investigate how nanogel crosslinking density affects siRNA core penetration using synthesis and characterization experiments. Blocker: Requires wet lab synthesis of nanogels with varied crosslinking density and experimental assays to measure siRNA penetration

A biodegradable pH-responsive carrier system for targeted siRNA delivery in inflammatory bowel diseases and cancer · UT Austin · PhD

Left open

Optimize siRNA loading into ClyA-ZZ bacterial outer membrane vesicles to achieve luminal internalization rather than surface aggregation. Blocker: Requires wet-lab experimental facilities, biological materials, and vesicle formulation equipment

Multimodal Engineering Approaches to Circumvent P-glycoprotein-Mediated Multidrug Resistance in Cancer · Cornell · PhD

Left open

Evaluate an in vivo liver-targeting lipid nanoparticle delivery system containing siRNAs against SDS. Blocker: Requires wet lab facilities, animal models (in vivo), and nanoparticle formulation capabilities

DEFINING THE METABOLIC LANDSCAPE OF FIBROLAMELLAR CARCINOMA · Cornell · PhD

Left open

Perform in vivo delivery experiments of siRNA, ASO, mRNA, or CRISPR editors using functionalized wireframe DNA nanoparticles in non-cancer disease models. Blocker: Requires wet lab facilities, synthesized DNA origami nanoparticles, and animal disease models for in vivo testing

Development of a Structural Nucleic Acid Delivery Vector Technology · MIT · PhD

Left open

Perform siRNA knockdown of RNU4-1, RNU4-2, and VTRNA1-3 to assess their role in viral replication or host antiviral defense. Blocker: Requires wet lab facilities, cell lines, and siRNA reagents to conduct physical knockdown assays

Oncolytic viruses in malignant pleural mesothelioma · Imperial · PhD

Left open

Evaluate the effect of LINC01013 knockdown on SNAI1 and WNT/beta-catenin signalling pathways in cardiac fibroblasts. Blocker: Requires wet lab experiments (siRNA knockdown, cell culture, molecular assays)

The role of small open reading frames in myocardial fibrosis and genetic disease · Imperial · PhD