Inflammatory recruitment of healthy hematopoietic stem and progenitor cells in the acute myeloid leukemia niche.

Inflammation in the bone marrow (BM) microenvironment is a constitutive component of leukemogenesis in acute myeloid leukemia (AML). Current evidence suggests that both leukemic blasts and stroma secrete proinflammatory factors that actively suppress the function of healthy hematopoietic stem and progenitor cells (HSPCs). HSPCs are also cellular components of the innate immune system, and we reasoned that they may actively propagate the inflammation in the leukemic niche. In two separate congenic models of AML we confirm by evaluation of the BM plasma secretome and HSPC-selective single-cell RNA sequencing (scRNA-Seq) that multipotent progenitors and long-lived stem cells adopt inflammatory gene expression programs, even at low leukemic infiltration of the BM. In particular, we observe interferon gamma (IFN-γ) pathway activation, along with secretion of its chemokine target, CXCL10. We show that AML-derived nanometer-sized extracellular vesicles (EVAML) are sufficient to trigger this inflammatory HSPC response, both in vitro and in vivo. Altogether, our studies indicate that HSPCs are an unrecognized component of the inflammatory adaptation of the BM by leukemic cells. The pro-inflammatory conversion and long-lived presence of HSPCs in the BM along with their regenerative re-expansion during remission may impact clonal selection and disease evolution.

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation.

Introduction: Colorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations. Methods: We generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells. Results: Using flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture. Discussion: We conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

Rational biomarker development for the early and minimally invasive monitoring of AML.

Recurrent disease remains the principal cause for treatment failure in acute myeloid leukemia (AML) across age groups. Reliable biomarkers of AML relapse risk and disease burden have been problematic, as symptoms appear late and current monitoring relies on invasive and cost-ineffective serial bone marrow (BM) surveillance. In this report, we discover a set of unique microRNA (miRNA) that circulates in AML-derived vesicles in the peripheral blood ahead of the general dissemination of leukemic blasts and symptomatic BM failure. Next-generation sequencing of extracellular vesicle-contained small RNA in 12 AML patients and 12 controls allowed us to identify a panel of differentially incorporated miRNA. Proof-of-concept studies using a murine model and patient-derived xenografts demonstrate the feasibility of developing miR-1246, as a potential minimally invasive AML biomarker.

Extracellular Vesicles and Chemotherapy Resistance in the AML Microenvironment.

Extracellular vesicle (EV) trafficking provides for a constitutive mode of cell-cell communication within tissues and between organ systems. Different EV subtypes have been identified that transfer regulatory molecules between cells, influencing gene expression, and altering cellular phenotypes. Evidence from a range of studies suggests that EV trafficking enhances cell survival and resistance to chemotherapy in solid tumors. In acute myeloid leukemia (AML), EVs contribute to the dynamic crosstalk between AML cells, hematopoietic elements and stromal cells and promote adaptation of compartmental bone marrow (BM) function through transport of protein, RNA, and DNA. Careful analysis of leukemia cell EV content and phenotypic outcomes provide evidence that vesicles are implicated in transferring several known key mediators of chemoresistance, including miR-155, IL-8, and BMP-2. Here, we review the current understanding of how EVs exert their influence in the AML niche, and identify research opportunities to improve outcomes for relapsed or refractory AML patients.

In vitro bioassay model for screening non-viral neurotrophic factor gene delivery systems for glaucoma treatment.

The feasibility of a two-layer contact-independent 3D neuronal co-culture model to test the bioactivity of brain-derived neurotrophic factor (BDNF), produced by non-virally transfected A7 astrocytes (trA7), on neurite growth in a second cell population of SH-SY5Y (CRL-2266) neuroblastoma cells with (oxSH-SY5Y) or without oxidative damage (SH-SY5Y) was evaluated. Transfection of A7 astrocytes was carried out with BDNF-encoding plasmid using K2® nanoparticle gene delivery system (K2-NPs). The physicochemical characteristics of K2-NPs, transfection efficiency, and BDNF production were evaluated using dynamic light scattering, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), respectively. Neurite counts and length measurements were performed after anti-neuron-specific ß-III tubulin antibody immunostaining using confocal laser scanning microscopy. Transfection efficiency of A7 astrocytes by K2-NPs (diameter 83.9 ± 0.4 nm, zeta potential +57.3 ± 2.8 mV) was 39.5 ± 4.6 % with cell viability of 73 ± 2 %. BDNF levels produced were 3750.8 ± 251.1, 9052.6 ± 1391.2, and 10,367.1 ± 390.8 pg/mL at 24, 48, and 72 h, respectively. The increased number of neurites with higher neurite lengths confirmed the bioactivity of BDNF secreted from the transfected A7 astrocytes over 72 h. Neurite count comparisons showed that both trA7/oxSH-SY5Y and trA7/SH-SY5Y consistently produced higher neurite counts compared to A7/oxSH-SY5Y and oxSH-SY5Y only experimental conditions. The results of this study demonstrate that neurite outgrowth quantitation in astrocyte-SH-SY5Y cell co-culture is a suitable bioassay model for evaluating non-viral gene delivery systems. Furthermore, it also demonstrates a proof-of-concept for nanoparticle-based neurotrophic factor gene delivery to astrocytes and stimulation of neurite outgrowth.

Non-viral gene therapy: Gains and challenges of non-invasive administration methods.

Gene therapy is becoming an influential part of the rapidly increasing armamentarium of biopharmaceuticals for improving health and combating diseases. Currently, three gene therapy treatments are approved by regulatory agencies. While these treatments utilize viral vectors, non-viral alternative technologies are also being developed to improve the safety profile and manufacturability of gene carrier formulations. We present an overview of gene-based therapies focusing on non-viral gene delivery systems and the genetic therapeutic tools that will further revolutionize medical treatment with primary focus on the range and development of non-invasive delivery systems for dermal, transdermal, ocular and pulmonary administrations and perspectives on other administration methods such as intranasal, oral, buccal, vaginal, rectal and otic delivery.

A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation.

BACKGROUND: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin. The aim of this study was to evaluate non-viral gene delivery by a series of dicationic gemini surfactant-phospholipid nanoparticles (GL-NPs) and to explore their mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes. METHODS: NPs containing pCMV-tdTomato plasmid encoding red fluorescent protein (RFP) were prepared using 12 different gemini surfactants (m-s-m, with m = 12, 16 and 18C alkyl tail and s = 3 and 7C polymethylene spacer group and 7C substituted spacers with 7NH and 7NCH3) and dioleoylphosphatidylethanolamine helper lipid. RFP gene expression and cell viability status were evaluated using flow cytometry. MitoTracker Deep Red mitochondrial stain and the cell impermeable Sytox red nuclear stain were used as indicators of cell viability and cell membrane integrity, respectively. RESULTS: No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs. Compared to Lipofectamine Plus, 18-3-18 GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Flow cytometric analysis of PAM212 cells stained with Sytox red revealed two cell populations with low and high fluorescent intensity, representing cells with partially-porated and highly-porated membranes, respectively. Additional combined staining with MitoTracker and ethidium homodimer showed that that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and had mitochondrial activity. CONCLUSION: Taken together, this study demonstrated that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the interaction of 18-3-18 GL-NPs with PAM212 cell membranes involves a permeability increase, possibly through the formation of nanoscale pores, which could explain efficient gene delivery. This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.