Juan M. Benito

Preorganized, Macromolecular, Gene‐Delivery Systems

Viruses represent a paradigmatic example of multicomponent, self-organized supramolecular systems specialized in the delivery and replication of their genetic material. Mimicking their functioning by artificial synthetic molecules represents a fantastic challenge that will lead to the future development of gene therapy. This is only possible if general approaches towards the construction of nanoscale vehicles for DNA are developed and the key rules governing their capacity to compact genetic material and its active transport/delivery through cell membranes are understood. In this area of research, synthetic organic chemistry plays an important role by providing tools to create tailor-made molecules of increasing complexity. Preorganization of functional elements onto macromolecular platforms has the potential to allow control of the self-assembling behavior of discrete architectures to produce nanometric objects that can be programmed to complex, compact, deliver, and release plasmid DNA in a target cell.

Pharmacological chaperone therapy for Gaucher disease: a patent review

Introduction: Mutations in the gene encoding for acid β-glucosidase (β-glucocerebrosidase, GlcCerase) are seen in Gaucher disease (GD), which give rise to significant protein misfolding effects and result in progressive accumulation of glucosyl ceramide. The main treatment for GD is enzyme replacement therapy (ERT). The iminosugar glycosidase inhibitor N-(n-butyl)-1-deoxynojirimycin (miglustat, Zavesca™) is used in a second treatment modality known as substrate reduction therapy. At the beginning of the 21st century, a third therapeutic paradigm was launched, namely, pharmacological chaperone therapy (PCT). This therapeutic strategy relies on the capability of such inhibitors to promote the correct folding and stabilize mutant forms of lysosomal enzymes, such as GlcCerase, as they pass through the secretory pathway. Areas covered: This review summarizes the different approaches used to implement the concept of PCT for GD. It discusses the relevant research, patents and patent applications filed in the last decade. Expert opinion: While the significance of PCT remains a matter of debate, the great interest gathered regarding it in a relatively few years reflects its broad potential scope, well beyond GD. The fact that pharmacological chaperones can be designed to cross the blood brain barrier (BBB) make them candidates for the treatment of neuronopathic forms of GD that are not responsive to ERT. Combined therapies offer even broader possibilities that deserve to be fully explored.

Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression.

A molecular-diversity-oriented approach for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene-delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic-hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure-activity relationship studies. Gel electrophoresis revealed that paCDs self-assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70-150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL-CL2 and COS-7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen-bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space-oriented dendritic polycationic constructs.

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.

Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin A.

The affinities of the mannose‐specific lectin concanavalin A (Con A) towards D‐glucose‐centred mannosyl clusters differing in the anomeric configuration of the monosaccharide core, nature of the bridging functional groups and valency, have been measured by a competitive enzyme‐linked lectin assay. Pentavalent thioether‐linked ligands (5 and 7) were prepared by radical addition of 2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐α‐D‐mannopyranose to the corresponding penta‐O‐allyl‐α‐ or ‐β‐D‐glucopyranose, followed by deacetylation. The distinct reactivity of the anomeric position in the D‐glucose scaffold was exploited in the preparation of a tetravalent cluster (10) that keeps a reactive aglyconic group for further manipulation, including incorporation of a reporter group or attachment to a solid support. Hydroboration of the double bonds in the penta‐O‐allyl‐α‐D‐glucopyranose derivative and replacement of the hydroxy groups with amine moieties gave a suitable precursor for the preparation of pentavalent and 15‐valent mannosides through the thiourea‐bridging reaction (17 and 20, respectively). The diastereomeric 1‐thiomannose‐coated clusters 5 and 7 were demonstrated to be potent ligands for Con A, with IC50 values for the inhibition of the Con A–yeast mannan association indicative of 6.4‐ and 5.5‐fold increases in binding affinity (valency‐corrected values), respectively, relative to the value for methyl α‐D‐mannopyranoside. The tetravalent cluster 10 exhibited a valency‐corrected relative lectin‐binding potency virtually identical to that of the homologous pentavalent mannoside 7. In sharp contrast, replacement of the 1‐thiomannose wedges of 5 with α‐D‐mannopyranosylthioureido units (17) virtually abolished any multivalent or statistic effects, with a dramatic decrease of binding affinity. The 15‐valent ligand 20, possessing classical O‐glycosidic linkages, exhibited a twofold increase in lectin affinity relative to the penta‐O‐(thioglycoside) 5; it is less efficient based on the number of mannose units. The results illustrate the potential of carbohydrates as polyfunctional platforms for glycocluster construction and underline the importance of careful design of the overall architecture in optimising glycocluster recognition by specific lectins.

Insights in cellular uptake mechanisms of pDNA–polycationic amphiphilic cyclodextrin nanoparticles (CDplexes)

It is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70nm). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell internalization and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated beta-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol- and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection.

Cyclodextrin‐Scaffolded Glycoclusters

Fully carbohydrate glycoclusters featuring a hydrophobic cavity and an outer seven-fold symmetric saccharide patch (see picture) have been efficiently prepared in two steps from readily available sugar isothiocyanates and per-(6-amino-6-deoxy)-β-cyclodextrin. The flexiblility of this strategy, compatible with the use of alkyl or aryl spacers, and its potential for the design of specific-site-delivery drug carriers are outlined. R = H, β-D-Glcp, β-D-Galp

Dependence of Concanavalin A Binding on Anomeric Configuration, Linkage Type, and Ligand Multiplicity for Thiourea-Bridged Mannopyranosyl–β-Cyclodextrin Conjugates

Concanavalin A (Con A), the mannose-specific lectin from Concanavalia ensiformis, has long been used as a model for carbohydrate ± protein interactions. Its commercial availability and the rather extensive structural knowledge currently available make it attractive for assessing and optimizing the functional parameters that affect its affinity for mannose neoglycoconjugates. Understanding these key elements may facilitate the development of new therapeutic strategies based on specific recognition events such as targeting of drugs. Con A binds a-D-mannopyranosides preferentially over the corresponding b anomers. The affinity for monosaccharide ligands is low; however, this is a rather common feature when considering protein ± carbohydrate interactions. Carbohydrate ± protein binding events usually involve several simultaneous contacts between carbohydrates that are clustered on cell surfaces and protein receptors that contain multiple carbohydrate-binding sites. Based on this concept, one could anticipate that multiplication of the saccharide epitope on the surface of the carrier may lead to a greater affinity than predicted from the sum of the constitutive one-to-one interactions Ðthe soThe authors are grateful to Sylvia Löbermann for expert technical assistance. This work received financial support by the Max-PlanckGesellschaft, Evotec Biosystems AG, BMBF together with Roche Diagnostics (grant 0311003), and the DFG (grant Ei 411/1-1), which is gratefully acknowledged.

Symmetry Complementarity-Guided Design of Anthrax Toxin Inhibitors Based on β-Cyclodextrin: Synthesis and Relative Activities of Face-Selective Functionalized Polycationic Clusters

Three new series of potential anthrax toxin inhibitors based on the β‐cyclodextrin (βCD) scaffold were developed by exploiting face‐selective CuI‐catalyzed azide–alkyne 1,3‐cycloadditions, amine–isothiocyanate coupling, and allyl group hydroboration–oxidation/hydroxy → amine replacement reactions. The molecular design follows the “symmetry–complementarity” concept between homogeneously functionalized polycationic βCD derivatives and protective antigen (PA), a component of anthrax toxin known to form C7‐symmetric pores on the cell membrane used by lethal and edema factors to gain access to the cytosol. The synthesis and antitoxin activity of a collection of βCD derivatives differing in the number, arrangement, and face location of the cationic elements are reported herein. These results set the basis for a structure–activity relationship development program of new candidates to combat the anthrax threat.

Host-Guest-Mediated DNA Templation of Polycationic Supramolecules for Hierarchical Nanocondensation and the Delivery of Gene Material.

Only a few examples of monodisperse molecular entities that can compact exogenous nucleic acids into nanocomplexes, protect the cargo from the biological environment, facilitate cell internalization, and promote safe transfection have been reported up to date. Although these species open new venues for fundamental studies on the structural requirements that govern the intervening processes and their application in nonviral gene-vector design, the synthesis of these moieties generally requires a relatively sophisticated chemistry, which hampers further development in gene therapy. Herein, we report an original strategy for the reversible complexation and delivery of DNA based on the supramolecular preorganization of a β-cyclodextrin-scaffolded polycationic cluster facilitated by bisadamantane guests. The resulting gemini-type, dual-cluster supramolecules can then undergo DNA-templated self-assembly at neutral pH value by bridging parallel DNA oligonucleotide fragments. This hierarchical DNA condensation mechanism affords transfectious nanoparticles with buffering capabilities, thus facilitating endosomal escape following cell internalization. Protonation also destabilizes the supramolecular dimers and consequently the whole supramolecular edifice, thus assisting DNA release. Our advanced hypotheses are supported by isothermal titration calorimetry, NMR and circular dichroism spectroscopic analysis, gel electrophoresis, dynamic light scattering, TEM, molecular mechanics, molecular dynamics, and transfection studies conducted in vitro and in vivo.