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.

BDNF gene delivery to the retina by cell adhesion peptide-conjugated gemini nanoplexes in vivo.

Retinal ganglion cell (RGC) neurodegeneration in glaucoma is not prevented by controlling the elevated intraocular pressure alone. Neuroprotective gene therapy approaches could be an essential part of a combination treatment. Five cell adhesion peptide (CAP)-gemini surfactants (18-7N(p1-5)-18) were synthesized as building blocks for brain-derived neurotrophic factor (BDNF) gene carrier nanoparticles (CAP-NPXs). The composition of CAP-NPXs was optimized, physicochemically characterized and evaluated for in vitro transfection efficiency (TE) in A7 astrocytes, 3D retinal neurospheres and for gene expression in vivo in CD1 mice using RFP reporter gene and BDNF levels after intravitreal (IVT) injection. The IgSF-binding 18-7N(pFASNKL)-18 pNPXs treated cells demonstrated 1.4-fold higher TE compared to integrin-binding 18-7N(pRGD)-18 pNPXs and parent 18-7NH-18 NPXs with overall viability between 86 and 95%. The 18-7N(pFASNKL)-18 pNPXs selectively transfected RGCs in 3D MiEye8 neurospheres. In the in vivo CD1 mouse model 18-7N(pFASNKL)-18 pNPXs administered by IVT injection delivered tdTomato/BDNF plasmid to retinal cells and produced higher gene expression than the 18-7N(pRGD)-18 pNPXs, the parent 18-7NH-18 NPXs and Lipofectamine® 3000 as demonstrated by confocal microscopy of whole mount retinas. The BDNF gene expression, assessed by ELISA, showed significantly high levels of BDNF with 18-7N(pFASNKL)-18 (422.60 ± 42.60 pg/eye), followed by 18-7N(pRGD)-18 pNPXs (230.62 ± 24.47 pg/eye), 18-7NH-18 NPXs (245.90 ± 39.72 pg/eye), Lipofectamine® 3000 (199.99 ± 29.90 pg/eye) and untreated controls (131.33 ± 20.30 pg/eye). In summary, the 18-7N(pFASNKL)-18 pNPXs induced 3.4-fold higher BDNF level compared to controls and 2-fold higher than 18-7N(pRGD)-18 pNPXs. The in vivo efficacy of 18-7N(pFASNKL)-18 NPXs to produce BDNF at pharmacologically relevant levels supports further studies.

Gene therapy strategies for glaucoma from IOP reduction to retinal neuroprotection: Progress towards non-viral systems.

Glaucoma is the result of the gradual death of retinal ganglion cells (RGCs) whose axons form the optic nerve. Elevated intraocular pressure (IOP) is a major risk factor that contributes to RGC apoptosis and axonal loss at the lamina cribrosa, resulting in progressive reduction and eventual anterograde-retrograde transport blockade of neurotrophic factors. Current glaucoma management mainly focuses on pharmacological or surgical lowering of IOP, to manage the only modifiable risk factor. Although IOP reduction delays disease progression, it does not address previous and ongoing optic nerve degeneration. Gene therapy is a promising direction to control or modify genes involved in the pathophysiology of glaucoma. Both viral and non-viral gene therapy delivery systems are emerging as promising alternatives or add-on therapies to traditional treatments for improving IOP control and providing neuroprotection. The specific spotlight on non-viral gene delivery systems shows further progress toward improving the safety of gene therapy and implementing neuroprotection by targeting specific tissues and cells in the eye and specifically in the retina.

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.

Multipotent stem cell-derived retinal ganglion cells in 3D culture as tools for neurotrophic factor gene delivery system development.

Non-viral neurotrophic factor (NF) gene therapy is a new paradigm in glaucoma treatment with the potential for neuroprotection and regeneration of damaged retinal ganglion cells (RGCs). To improve nanoparticle gene delivery systems and generate a suitable RGC cell model to facilitate in vitro investigations, we have developed mouse multipotent retinal stem cell (MRSC)-derived RGCs (XFC-3 cells) that express key RGC characteristics as demonstrated through biomarker expression profiling and stimuli-inducible neurite extension evaluation. Dicationic gemini surfactant-, single-walled carbon nanotube-, and K2-lipopolyamine polymer-based gene delivery systems were formulated and evaluated in three-dimensional (3D) A7/XFC-3 and XFC-3/XFC-3 co-cultures to validate the model for transfection efficiency (TE) and brain-derived neurotrophic factor (BDNF) bioactivity measurements, which helped identify the K2-NPs as having high TE (63.1% ±â€¯1.4%) and high cell viability (94.4% ±â€¯0.4%). Overall, XFC-3 cells are suitable for the construction of 3D in vivo-like tissue models and enable the screening of RGC-aimed gene delivery systems for neuroprotective treatment of glaucoma.

Tuning optimum transfection of gemini surfactant-phospholipid-DNA nanoparticles by validated theoretical modeling.

Gemini nanoparticles (NPs) are a family of non-viral gene delivery systems with potential for applications in non-invasive gene therapy. Translation of these non-viral gene delivery systems requires improvement of transfection efficiency (TE) through fine-tuning of their physicochemical properties such as electric charge and exact ratios of their components. Since high-throughput experimental screening of minute differences in NP compositions is not routinely feasible, we have developed a coarse-grained model to quantitatively study the energetics of the formation of gene delivery complexes with cationic gemini surfactants (G) (m-s-m type) and helper lipids (H) (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DOPE/1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)), in order to use it as a tool to predict effective compositions. The model is based on the polymorphic structural conformational flip of NPs and incorporates the electrostatic, entropic and elastic energies, to predict the formation energy and stability of different polymorphic structures as a function of the electric charge of cationic surfactants and concentration of constituent helper lipids. Our results show that these two factors are intertwined in determining the behavior of gene delivery vectors. Specifically, we show that increasing H/G lowers free energy per DNA base pair and increases the stability of the complex. At pH 7, low H/G and charge ratio (ρ±), where the lamellar structure is favored, the formation free energy per DNA base pair is between 0 and -14kBT. At higher values of H/G (2-3) and ρ±, where HII and cubic structures are formed, the formation free energy drops down to values ≈-50kBT, indicating the stable existence of these polymorphic structures in the NPs. At pH 5, the structural transformation of NPs in the endosomes to the lamellar/HII structure with free energy values of about -40kBT is beneficial for endosomal escape, and correlates with increased transfection efficiency. The theoretical model is supported by transfection data in A7 astrocytes with a panel of 16-3-16 gemini NPs, which validates the mathematical model and supports the hypothesis that the NP polymorphic phase transition increases transfection efficiency.

Three-Dimensional Co-Culture Bioassay for Screening of Retinal Gene Delivery Systems.

Herein we describe a three-dimensional co-culture bioassay protocol designed to assess the therapeutic potential of the proteins expressed from gene delivery transfected cells through the evaluation of expressed protein bioavailability and bioactivity. Using a combination of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent-based neurite length profiling methodologies, the bioavailability of the secreted therapeutic protein in the medium can be quantitated, and the bioactivity of the secreted therapeutic protein can also be evaluated through neurite length profiling, respectively. The versatility and rationale of this bioassay could serve as a useful screening tool in the development of retinal gene delivery systems.

The intricacies of neurotrophic factor therapy for retinal ganglion cell rescue in glaucoma: a case for gene therapy.

Regeneration of damaged retinal ganglion cells (RGC) and their axons is an important aspect of reversing vision loss in glaucoma patients. While current therapies can effectively lower intraocular pressure, they do not provide extrinsic support to RGCs to actively aid in their protection and regeneration. The unmet need could be addressed by neurotrophic factor gene therapy, where plasmid DNA, encoding neurotrophic factors, is delivered to retinal cells to maintain sufficient levels of neurotrophins in the retina. In this review, we aim to describe the intricacies in the design of the therapy including: the choice of neurotrophic factor, the site and route of administration and target cell populations for gene delivery. Furthermore, we also discuss the challenges currently being faced in RGC-related therapy development with special considerations to the existence of multiple RGC subtypes and the lack of efficient and representative in vitro models for rapid and reliable screening in the drug development process.

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.

Selective nuclear localization of siRNA by metallic versus semiconducting single wall carbon nanotubes in keratinocytes.

BACKGROUND: The potential use of carbon nanotubes (CNTs) in gene therapy as delivery systems for nucleic acids has been recently recognized. Here, we describe that metallic versus semiconducting single-wall CNTs can produce significant differences in transfection rate and cellular distribution of siRNA in murine PAM212 keratinocytes. RESULTS/METHODOLOGY: The results of cell interaction studies, coupled with supportive computational simulations and ultrastructural studies revealed that the use of metallic single wall CNTs resulted in siRNA delivery into both the cytoplasm and nucleus of keratinocytes, whereas semiconducting CNTs resulted in delivery only to the cytoplasm. CONCLUSION: Using enriched fractions of metallic or semiconducting CNTs for siRNA complex preparation may provide specific subcellular targeting advantages.