Delivery of transgenically modified adult bone marrow cells to the rodent central nervous system

A major challenge in the treatment of neurological disorders is the effective delivery of molecules into the CNS and, more so, to the lesion site. The blood–brain barrier restricts the delivery of therapeutic molecules into the CNS when injected intravenously. This difficulty is further compounded by the short half-life of certain therapeutic agents. Organ-targeted protein delivery could circumvent these difficulties, provided that the corresponding cDNA can be delivered and expressed in the target tissue effectively and safely. Recent studies from a number of laboratories indicate that a subset of adult bone marrow cells home to the CNS among other organs and can be engineered to deliver and express therapeutic proteins into the CNS. This article, which is focused on work from the authors’ research, reviews the opportunities and difficulties presented by this approach.     

Delivery of transgenically modified adult bone marrow cells to the rodent central nervous system

A major challenge in the treatment of neurological disorders is the effective delivery of molecules into the CNS and, more so, to the lesion site. The blood-brain barrier restricts the delivery of therapeutic molecules into the CNS when injected intravenously. This difficulty is further compounded by the short half-life of certain therapeutic agents. Organ-targeted protein delivery could circumvent these difficulties, provided that the corresponding cDNA can be delivered and expressed in the target tissue effectively and safely. Recent studies from a number of laboratories indicate that a subset of adult bone marrow cells home to the CNS among other organs and can be engineered to deliver and express therapeutic proteins into the CNS. This article, which is focused on work from the authors' research, reviews the opportunities and difficulties presented by this approach.     

Coronary thrombosis and cell adhesion molecules]

A fundamental role of cell adhesion molecules are implicated for the disease process of acute coronary syndromes. Among adhesion molecules, platelet membrane glycoprotein Ib binds to the von Willebrand factor and thereby activates glycoprotein IIb/IIIa, resulting in platelet aggregation. P-selection is rapidly translocated onto the cell surface within minutes and adheres to a sialylated fucosylated carbohydrate structure, sialyl Lewis(x), on leukocytes. P-selectin mediates adhesion of leukocytes to the activated platelets. Thus, these platelet glycoproteins play a active role in cell-extracellular matrix interaction and cell-cell interaction at the first step of the thrombus formation at the culprit lesion of the coronary artery. The more comprehensive understandings of adhesion molecules and their functions may promote the more effective therapeutic strategies for the acute coronary syndromes.     

Human leukocyte differentiation antigens as therapeutic targets: the CD molecules and CD antibodies

The cell membrane presents an attractive target in a number of different disease situations. Most obviously, malignant cells may be killed by damaging their cell membranes. There are also more subtle, though effective, ways of rendering cells harmless by engaging proteins at the cell surface. The cells of the immune system may be targeted, for example to stop a damaging immune reaction, such as acute inflammation or rejection of a transplanted organ. If we are to make the best use of the opportunities to modulate disease by targeting the cell membrane, we need a detailed understanding of the many proteins, glycoproteins and glycolipids that are attached to or inserted in the cell membrane. The CD (cluster of differentiation) Workshops, more properly known as the HLDA (Human Leukocyte Differentiation Antigens) Workshops have, since 1982, focussed on the study of the membrane molecules of leukocytes, including the major cells of the immune system and malignant cells derived from them. The scope has extended to molecules on endothelium which are important in interaction with leukocytes. Many of the molecules characterised as leukocyte antigens are also expressed on other tissue. The approaches developed by the HLDA Workshops are useful in the study of the molecular composition and function of cells of other organ systems. Some of the antibodies produced in order to study the CD molecules have proved useful as therapeutic agents. This review describes the CD system, how it has developed and what it means and introduces the field of therapy based on antibodies against CD or similar molecules. The author is responsible for organising the next (8th) HLDA Workshop and invites readers to suggest ways in which the therapeutic relevance of the Workshop may be enhanced.     

The influence of the foreign body response evoked by fibroblast transplantation on soluble factor diffusion in surrounding brain tissue

The transplantation of genetically engineered fibroblasts has been shown to be an effective approach for achieving continuous and site-specific delivery of therapeutic molecules to various regions of the central nervous system. However, to our knowledge no one has asked whether soluble factors released from the transplanted fibroblasts influence the delivery of therapeutic molecules from the engrafted cells. To address this issue, we used a newly developed cell encapsulation device to study the functional consequence of the foreign body response on soluble factor delivery from fibroblasts transplanted into adult brain tissue. We found that transplanted fibroblasts increased the level of inflammation and glial cell encapsulation at the transplantation site, and reduced the diffusion of a 70 kDa dextran probe through the reactive tissue. The response, however, did not prevent the diffusion of the 70 kDa dextran test probe indicating that the approach appears suitable for the delivery of neurotrophins and other therapeutic molecules with a molecular weight less than 70 kDa. The results suggest that less reactive cell types may be better suited for sustained delivery of therapeutic molecules into brain tissue.     

Tissue engineering with the aid of inkjet printers

Tissue engineering holds the promise to create revolutionary new therapies for tissue and organ regeneration. This emerging field is extremely broad and eclectic in its various approaches. However, all strategies being developed are based on the therapeutic delivery of one or more of the following types of tissue building-blocks: cells; extracellular matrices or scaffolds; and hormones or other signaling molecules. So far, most work has used essentially homogenous combinations of these components, with subsequent self-organization to impart some level of tissue functionality occurring during in vitro culture or after transplantation. Emerging 'bioprinting' methodologies are being investigated to create tissue engineered constructs initially with more defined spatial organization, motivated by the hypothesis that biomimetic patterns can achieve improved therapeutic outcomes. Bioprinting based on inkjet and related printing technologies can be used to fabricate persistent biomimetic patterns that can be used both to study the underlying biology of tissue regeneration and potentially be translated into effective clinical therapies. However, recapitulating nature at even the most primitive levels such that printed cells, extracellular matrices and hormones become integrated into hierarchical, spatially organized three-dimensional tissue structures with appropriate functionality remains a significant challenge.     

Tissue engineering with the aid of inkjet printers

Tissue engineering holds the promise to create revolutionary new therapies for tissue and organ regeneration. This emerging field is extremely broad and eclectic in its various approaches. However, all strategies being developed are based on the therapeutic delivery of one or more of the following types of tissue building-blocks: cells; extracellular matrices or scaffolds; and hormones or other signaling molecules. So far, most work has used essentially homogenous combinations of these components, with subsequent self-organization to impart some level of tissue functionality occurring during in vitro culture or after transplantation. Emerging ‘bioprinting’ methodologies are being investigated to create tissue engineered constructs initially with more defined spatial organization, motivated by the hypothesis that biomimetic patterns can achieve improved therapeutic outcomes. Bioprinting based on inkjet and related printing technologies can be used to fabricate persistent biomimetic patterns that can be used both to study the underlying biology of tissue regeneration and potentially be translated into effective clinical therapies. However, recapitulating nature at even the most primitive levels such that printed cells, extracellular matrices and hormones become integrated into hierarchical, spatially organized three-dimensional tissue structures with appropriate functionality remains a significant challenge.     

Multiple drug delivery hydrogel system for spinal cord injury repair strategies

The multifactorial pathological progress of spinal cord injury (SCI) is probably the main reason behind the absence of efficient therapeutic approaches. Hence, very recent highlights suggest the use of new multidrug delivery systems capable of local controlled release of therapeutic agents. In this work, a biocompatible hydrogel-based system was developed as multiple drug delivery tool, specifically designed for SCI repair strategies. Multiple release profiles were achieved by loading gel with a combination of low and high steric hindrance molecules. In vitro, in vivo and ex vivo release studies showed an independent combination of fast diffusion-controlled kinetics for smaller molecules together with slow diffusion-controlled kinetics for bigger ones. A preserved functionality of loaded substances was always achieved, confirming the absence of any chemical stable interactions between gel matrix and loaded molecules. Moreover, the relevant effect of the cerebrospinal fluid flux dynamics on the drug diffusion in the spinal cord tissue was here revealed for the first time: an oriented delivery of the released molecules in the spinal cord tract caudally to the gel site is demonstrated, thus suggesting a more efficient gel positioning rostrally to the lesion.     

Targeting Axl With an High-affinity Inhibitory Aptamer

Axl is a tyrosine kinase receptor that was first identified as a transforming gene in human myeloid leukemia. Recent converging evidence suggests its implication in cancer progression and invasion for several solid tumors, including lung, breast, brain, thyroid, and pancreas. In the last decade, Axl has thus become an attractive target for therapeutic development of more aggressive cancers. An emerging class of therapeutic inhibitors is now represented by short nucleic acid aptamers. These molecules act as high affinity ligands with several advantages over conventional antibodies for their use in vivo, including their small size and negligible immunogenicity. Furthermore, these molecules can easily form conjugates able to drive the specific delivery of interfering RNAs, nanoparticles, or chemotherapeutics. We have thus generated and characterized a selective RNA-based aptamer, GL21.T that binds the extracellular domain of Axl at high affinity (12 nmol/l) and inhibits its catalytic activity. GL21.T blocked Axl-dependent transducing events in vitro, including Erk and Akt phosphorylation, cell migration and invasion, as well as in vivo lung tumor formation in mice xenografts. In this respect, the GL21.T aptamer represents a promising therapeutic molecule for Axl-dependent cancers whose importance is highlighted by the paucity of available Axl-specific inhibitory molecules.     

Chemical modification of therapeutic drugs or drug vector systems to achieve targeted therapy: looking for the grail

Most therapeutic drugs distribute to the whole body, which results in general toxicity and poor acceptance of the treatments by patients. The targeted delivery of chemotherapeutics to defined cells, either stromal or cancer cells in cancer lesions, or defined inflammatory cells in immunological disorders, is one of the main challenges and a very active field of research in the development of treatment strategies to minimize side-effects of drugs. Disease-associated cells express molecules, including proteases, receptors, or adhesion molecules, that are different or differently expressed than their normal counterparts. Therefore one goal in the field of targeted therapies is to develop chemically derivatized drugs or drug vectors able to target defined cells via specific recognition mechanisms and also able to overcome biological barriers. This article will review the approaches which have been explored to achieve these goals and will discuss in more detail three examples (i) the use of nanostructures to take advantage of increased vascular permeability in some human diseases, (ii) the targeting of therapeutic drugs to an organ, the brain, protected against foreign molecules by the blood-brain barrier, and (iii) the use of the folate receptor to target either tumor cells or activated macrophages.     

Ribozymes and siRNA for the treatment of diseases of the nervous system

Recent advances in our understanding of RNA biology have focused attention on the potential of developing RNA-based strategies to treat human disease. Naturally occurring catalytic RNA molecules (ribozymes), their synthetic DNA counterparts (deoxyribozymes or DNAzymes), as well as the exciting, emerging technology of small interfering RNA which utilizes the highly conserved cellular RNA interference pathway, are being developed for therapeutic gene silencing purposes. The challenges for the application of this technology to neurological disease will be to identify appropriate disease targets, and to optimize the function, and particularly delivery of these RNA-based therapeutic molecules within the complex environment of the nervous system. This review will assess the potential of these RNA-based therapeutic strategies and the challenges ahead in their application to the treatment of neurological disease.     

Present and future of cell-penetrating peptide mediated delivery systems: "is the Trojan horse too wild to go only to Troy?".

During the last decade, small peptides (10 to 15 amino acids) derived from the HIV-1 Tat protein and from the drosophila Antennapedia homeodomain have been used to internalize various types of molecules into the cells. The way these peptides enter cells is still under investigation and the object of strong controversy. The main discussions rely on whether these peptides are internalized or not in an energy-independent fashion, and, depending on the situation, whether they follow one pathway instead of another. At present, we find in the literature a very large number of data with, at times, some contradictory results. Indeed the diversity of employed peptide sequences, the cell type used, the attachment or not of a cargo molecule, the chemical nature of this cargo itself, and the followed protocol during the experimental process do not simplify the comparison and hence final conclusions about the mechanism of cell entry. However, one common feature emerges with these cell-penetrating peptides: most of them do not show any cell specificity. Despite their demonstrated efficiency in delivering biologically active molecules in in vitro experiments, their use for a therapeutic application in vivo has been the object of a relatively little number of studies, probably because of the quite important amounts of CPP-cargo that needs to be prepared for an accurate and complete in vivo study, but more likely, because of the massive spreading of the cargo all around the body. However, it appears from recent studies that an increased targeting ability of these CPPs is possible, making the use of CPP mediated delivery compatible with an in vivo therapeutic approach.     

IRES-based Vector Coexpressing FGF2 and Cyr61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE^−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia.     

Adipokines and melanocortins in the hepatic manifestation of metabolic syndrome: nonalcoholic fatty liver disease

Metabolic syndrome is associated with nonalcoholic fatty liver disease and its more aggressive form, nonalcoholic steatohepatitis. Adipokines produced by white adipose tissue possess broad physiological activity and play an important autocrine role in obesity-associated complications, including metabolic syndrome, nonalcoholic fatty liver disease and cardiovascular disease. Various adipokines may have beneficial or harmful effects. Other tissues, particularly stomach and intestine, produce active molecules that can influence the function of adipocytes and, possibly, the levels of adipokine secretion. In some cases, the production sites of these molecules remain unknown. The review focuses on our current understanding of the disease-related effects of the adipokines and the melanocortins on various peripheral tissues, and discusses some of their potential interactions with each other. Potential therapeutic applications are also considered.     

Engineering of monoclonal antibodies and antibody-based fusion proteins: successes and challenges

Monoclonal antibodies (mAbs) and antibody-based fusion molecules have now come of age as therapeutics. Eighteen mAbs and two fusion molecules are on the market. mAbs directed against new targets are progressing at a rapid rate with the help of proteomics and genomics approaches. Many technical efforts have been made to generate a second-generation mAb with decreased immunogenicity and with optimised effector functions. The development of molecular engineering techniques applied to antibody molecules has also made it possible to design fusion molecules exhibiting different modules with bifunctional activities. Different approaches developed over the last two decades to generate and optimise therapeutic antibodies and antibody-based fusion molecules are described, with a particular focus on antibodies and fusion proteins used in oncology and inflammatory diseases. Some current technical challenges and trends are also discussed.     

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy

RNA interference (RNAi) entails the potential for novel therapeutic strategies through the silencing of disease-causing genes in vivo. However, recent studies have raised an issue regarding applicable routes of administration for small interfering RNA (siRNA) molecules as therapeutics. In this study, we demonstrate that liposomally formulated siRNA molecules, the so-called siRNA-lipoplexes, but not naked siRNAs, are delivered to the tumor endothelial cells in vivo by microscopy. In addition, functional intracellular delivery of formulated siRNA targeting the tumor suppressor PTEN is shown in endothelial cells of the liver and tumor. Finally, the therapeutic potential of systemically administered siRNA(CD31)-lipoplexes is established by inhibition of tumor growth in two different xenograft mouse models. Our findings corroborate the applicability of this liposomal siRNA delivery technology for inducing RNAi to modulate gene expression levels in angiogenesis-dependent processes. In addition, our results advocate CD31 as a promising therapeutic target for antiangiogenic intervention. Therefore, our study provides a basis for the development of antiangiogenic cancer therapies based on RNAi.     

Epigenetic polypharmacology: A new frontier for epi-drug discovery

Recently, despite the great success achieved by the so-called “magic bullets” in the treatment of different diseases through a marked and specific interaction with the target of interest, the pharmacological research is moving toward the development of “molecular network active compounds,” embracing the related polypharmacology approach. This strategy was born to overcome the main limitations of the single target therapy leading to a superior therapeutic effect, a decrease of adverse reactions, and a reduction of potential mechanism(s) of drug resistance caused by robustness and redundancy of biological pathways. It has become clear that multifactorial diseases such as cancer, neurological, and inflammatory disorders, may require more complex therapeutic approaches hitting a certain biological system as a whole. Concerning epigenetics, the goal of the multi-epi-target approach consists in the development of small molecules able to simultaneously and (often) reversibly bind different specific epi-targets. To date, two dual histone deacetylase/kinase inhibitors (CUDC-101 and CUDC-907) are in an advanced stage of clinical trials. In the last years, the growing interest in polypharmacology encouraged the publication of high-quality reviews on combination therapy and hybrid molecules. Hence, to update the state-of-the-art of these therapeutic approaches avoiding redundancy, herein we focused only on multiple medication therapies and multitargeting compounds exploiting epigenetic plus nonepigenetic drugs reported in the literature in 2018. In addition, all the multi-epi-target inhibitors known in literature so far, hitting two or more epigenetic targets, have been included.     

Recent developments in RNA-based strategies for cancer gene therapy

The introduction of RNA into mammalian cells is a relatively straightforward procedure with many therapeutic applications. An advantage of using mRNA is that protein expression can be achieved in post-mitotic or quiescent cells where there is usually little or no gene expression with non-viral DNA delivery systems. Furthermore, the cleavage of mRNA by catalytic RNA molecules, or ribozymes, is a useful strategy to downregulate aberrant gene expression. The purpose of this review is to provide an update of current applications that use RNA molecules such as mRNA and ribozymes as a basis for gene therapy strategies targeting the initiation and progression of cancer. In particular, we focus on recent developments that improve the delivery and stability of RNA molecules to achieve therapeutic efficacy.     

Current advances in ultrasound-combined nanobubbles for cancer-targeted therapy: a review of the current status and future perspectives

The non-specific distribution, non-selectivity towards cancerous cells, and adverse off-target side effects of anticancer drugs and other therapeutic molecules lead to their inferior clinical efficacy. Accordingly, ultrasound-based targeted delivery of therapeutic molecules loaded in smart nanocarriers is currently gaining wider acceptance for the treatment and management of cancer. Nanobubbles (NBs) are nanosize carriers, which are currently used as effective drug/gene delivery systems because they can deliver drugs/genes selectively to target sites. Thus, combining the applications of ultrasound with NBs has recently demonstrated increased localization of anticancer molecules in tumor tissues with triggered release behavior. Consequently, an effective therapeutic concentration of drugs/genes is achieved in target tumor tissues with ultimately increased therapeutic efficacy and minimal side-effects on other non-cancerous tissues. This review illustrates present developments in the field of ultrasound-nanobubble combined strategies for targeted cancer treatment. The first part of this review discusses the composition and the formulation parameters of NBs. Next, we illustrate the interactions and biological effects of combining NBs and ultrasound. Subsequently, we explain the potential of NBs combined with US for targeted cancer therapeutics. Finally, the present and future directions for the improvement of current methods are proposed.

NBs combined with ultrasound demonstrated the ability to enhance the targeting of anticancer agents and improve the efficacy.