dna-barcode-based-multiplex-immunofluorescence-imaging-to-analyze

DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma

Presented here is an emerging DNA-barcode-based multiplex imaging technique based on Co-Detection-by-indEXing that analyzes the spatial proteomics of ...

Johns Hopkins University

Current standard of care for many CNS tumors involves surgical resection followed by chemotherapy and/or radiation. Some pediatric brain tumor types are infiltrative and diffuse in nature, which reduces the role for surgery. Furthermore, children are extremely vulnerable to neurological sequelae from surgery and radiation therapy, thus alternative approaches are in critical need. As molecular targets underlying various cancers become more clearly defined, there is an increasing push for targeted gene therapies. Viral vectors and nonviral nanoparticles have been thoroughly investigated for gene delivery and show promise as vectors for gene therapy for pediatric brain cancer. Here, we review inorganic and organic materials in development for nanoparticle gene delivery to the brain with a particular focus on safety.

Safety considerations for nanoparticle gene delivery in pediatric brain tumors.

Clinical translation of polymer-based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA-mediated knockdown in both glioma and melanoma cells in high-serum conditions compared to non-crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue-mediated siRNA delivery beyond the liver, unlike conventional nanoparticle-based delivery. These attributes of XbNPs facilitate robust siRNA-mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid-based therapeutics.

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy.

Glioblastoma (GBM) is an aggressive central nervous system cancer with a dismal prognosis. The standard of care involves surgical resection followed by radiotherapy and chemotherapy, but five-year survival is only 5.6% despite these measures. Novel therapeutic approaches, such as immunotherapies, targeted therapies, and gene therapies, have been explored to attempt to extend survival for patients. Nanoparticles have been receiving increasing attention as promising vehicles for non-viral nucleic acid delivery in the context of GBM, though delivery is often limited by low blood-brain barrier permeability, particle instability, and low trafficking to target brain structures and cells. In this review, nanoparticle design considerations and new advances to overcome nucleic acid delivery challenges to treat brain cancer are summarized and discussed.

Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies.

Transient transfection is an essential tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and the resulting protein yield are limited by the polymer's toxicity. This study investigated the use of a class of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection in comparison to linear 25 kDa PEI, a commonly used transfection reagent.

Poly(Beta-Amino Ester)s as High-Yield Transfection Reagents for Recombinant Protein Production.

Nonviral nanoparticles have emerged as an attractive alternative to viral vectors for gene therapy applications, utilizing a range of lipid-based, polymeric, and inorganic materials. These materials can either encapsulate or be functionalized to bind nucleic acids and protect them from degradation. To effectively elicit changes to gene expression, the nanoparticle carrier needs to undergo a series of steps intracellularly, from interacting with the cellular membrane to facilitate cellular uptake to endosomal escape and nucleic acid release. Adjusting physiochemical properties of the nanoparticles, such as size, charge, and targeting ligands, can improve cellular uptake and ultimately gene delivery. Applications in the central nervous system (CNS; i.e., neurological diseases, brain cancers) face further extracellular barriers for a gene-carrying nanoparticle to surpass, with the most significant being the blood-brain barrier (BBB). Approaches to overcome these extracellular challenges to deliver nanoparticles into the CNS include systemic, intracerebroventricular, intrathecal, and intranasal administration. This review describes and compares different biomaterials for nonviral nanoparticle-mediated gene therapy to the CNS and explores challenges and recent preclinical and clinical developments in overcoming barriers to nanoparticle-mediated delivery to the brain. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Nonviral nanoparticle gene delivery into the CNS for neurological disorders and brain cancer applications.

Hepatocellular carcinoma (HCC) has limited treatment options, and modest survival after systemic chemotherapy or procedures such as transarterial chemoembolization (TACE). There is therefore a need to develop targeted therapies to address HCC. Gene therapies hold immense promise in treating a variety of diseases, including HCC, though delivery remains a critical hurdle. This study investigated a new approach of local delivery of polymeric nanoparticles (NPs) via intra-arterial injection for targeted local gene delivery to HCC tumors in an orthotopic rat liver tumor model.

Non-Viral Gene Delivery to Hepatocellular Carcinoma via Intra-Arterial Injection.

Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due its self-adjuvating and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles have been successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window would benefit from further improvement. To investigate alternatives to lipid nanoparticles, we developed a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM  and  as assessed by measuring expression of SAM encoding reporter proteins. We evaluated the ability of these polymers to deliver SAM intramuscularly in mice, and identified a polymer-based formulation that yielded up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicited superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.

Biodegradable Polyester Nanoparticle Vaccines Deliver Self-Amplifying mRNA in Mice at Low Doses.

Tumor immunotherapy is a promising anticancer strategy; however, tumor cells may employ resistance mechanisms, including downregulation of major histocompatibility complex (MHC) molecules to avoid immune recognition. Here, we investigate reprogramming nanoparticles (NPs) that deliver immunostimulatory genes to enhance immunotherapy and address defective antigen presentation in skin cancer  and . We use a modular poly(beta-amino ester) (PBAE)-based NP to deliver DNA encoding 4-1BBL, IL-12, and IFNγ to reprogram human Merkel cell carcinoma (MCC) cells  and mouse melanoma tumors  to drive adaptive antitumor immune responses. Optimized NP formulations delivering 4-1BBL/IL-12 or 4-1BBL/IL-12/IFNγ DNA successfully transfect MCC and melanoma cells  and , respectively, resulting in IFNγ-driven upregulation of MHC class I and II molecules on cancer cells. These NPs reprogram the tumor immune microenvironment (TIME) and elicit strong T-cell-driven immune responses, leading to cancer cell killing and T-cell proliferation  and slowing tumor growth and improving survival rates . Based on expected changes to the tumor immune microenvironment, particularly the importance of IFNγ to the immune response and driving both T-cell function and exhaustion, next-generation NPs codelivering IFNγ were designed. These offered mixed benefits, exchanging improved polyfunctionality for increased T-cell exhaustion and demonstrating higher systemic toxicity . Further profiling of the immune response with these NPs provides insight into T-cell exhaustion and polyfunctionality induced by different formulations, providing a greater understanding of this immunotherapeutic strategy.

Biomaterial-Mediated Genetic Reprogramming of Merkel Cell Carcinoma and Melanoma Leads to Targeted Cancer Cell Killing  and .

Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 μl of NPs containing 19.2 μg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 μl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 μl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial  sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.

Suprachoroidal gene transfer with nonviral nanoparticles in large animal eyes.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms  monocyte programming.

Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming.

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is CoDetection by indEXing, recently renamed as the PhenoCycler system. This is a highly multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh frozen tissues. Formalin-fixed paraffin-embedded blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine formalin-fixed paraffin-embedded tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of 3 gene delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift toward intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of interferon gamma, in addition to 4-1BBL and IL-12, not only further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of interferon gamma therapeutic efficacy.

Highly Multiplexed Immunofluorescence PhenoCycler Panel for Murine Formalin-Fixed Paraffin-Embedded Tissues Yields Insight Into Tumor Microenvironment Immunoengineering.

Despite aggressive therapy, glioblastoma (GBM) recurs in almost all patients and treatment options are very limited. Despite our growing understanding of how cellular transitions associate with relapse in GBM, critical gaps remain in our ability to block these molecular changes and treat recurrent disease. In this study we combine computational biology, forward-thinking understanding of miRNA biology and cutting-edge nucleic acid delivery vehicles to advance targeted therapeutics for GBM. Computational analysis of RNA sequencing from clinical GBM specimens identified TGFβ type II receptor (TGFBR2) as a key player in the mesenchymal transition associated with worse outcome in GBM. Mechanistically, we show that elevated levels of TGFBR2 is conducive to reduced temozolomide (TMZ) sensitivity. This effect is, at least partially, induced by stem-cell driving events coordinated by the reprogramming transcription factors Oct4 and Sox2 that lead to open chromatin states. We show that blocking TGFBR2 via molecular and pharmacological approaches decreases stem cell capacity and sensitivity of clinical recurrent GBM (rGBM) isolates to TMZ in vitro. Network analysis uncovered miR-590-3p as a tumor suppressor that simultaneously inhibits multiple oncogenic nodes downstream of TGFBR2. We also developed novel biodegradable lipophilic poly(β-amino ester) nanoparticles (LiPBAEs) for in vivo microRNA (miRNAs) delivery. Following direct intra-tumoral infusion, these nanomiRs efficiently distribute through the tumors. Importantly, miR-590-3p nanomiRs inhibited the growth and extended survival of mice bearing orthotopic human rGBM xenografts, with an apparent 30% cure rate. These results show that miRNA-based targeted therapeutics provide new opportunities to treat rGBM and bypass the resistance to standard of care therapy.

Multipronged SMAD pathway targeting by lipophilic poly(β-amino ester) miR-590-3p nanomiRs inhibits mesenchymal glioblastoma growth and prolongs survival.

Article	Year
Safety considerations for nanoparticle gene delivery in pediatric brain tumors. Nanomedicine (London, England)\| PubMed ID: 32698671	2020
Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy. Advanced functional materials\| PubMed ID: 34650390	2021
Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies. Advanced drug delivery reviews\| PubMed ID: 34715258	2021
Poly(Beta-Amino Ester)s as High-Yield Transfection Reagents for Recombinant Protein Production. International journal of nanomedicine\| PubMed ID: 36176585	2022
Nonviral nanoparticle gene delivery into the CNS for neurological disorders and brain cancer applications. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology\| PubMed ID: 36193561	2023
Non-Viral Gene Delivery to Hepatocellular Carcinoma via Intra-Arterial Injection. International journal of nanomedicine\| PubMed ID: 37197026	2023
Biodegradable Polyester Nanoparticle Vaccines Deliver Self-Amplifying mRNA in Mice at Low Doses. Advanced therapeutics\| PubMed ID: 37743930	2023
Biomaterial-Mediated Genetic Reprogramming of Merkel Cell Carcinoma and Melanoma Leads to Targeted Cancer Cell Killing and . ACS biomaterials science & engineering\| PubMed ID: 37797944	2023
Suprachoroidal gene transfer with nonviral nanoparticles in large animal eyes. Science advances\| PubMed ID: 38457512	2024
Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 39172792	2024
Highly Multiplexed Immunofluorescence PhenoCycler Panel for Murine Formalin-Fixed Paraffin-Embedded Tissues Yields Insight Into Tumor Microenvironment Immunoengineering. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 39490742	2025
Multipronged SMAD pathway targeting by lipophilic poly(β-amino ester) miR-590-3p nanomiRs inhibits mesenchymal glioblastoma growth and prolongs survival. Signal transduction and targeted therapy\| PubMed ID: 40301302	2025

Kathryn M. Luly

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