Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces.

Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide.

Intravitreal air tamponade after AAV2 subretinal injection modifies retinal EGFP distribution.

The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.

Regio- and Stereoselectivity of the Norrish-Yang Photocyclization of Dialkyl 1,2-Diketones: Solution versus Solid State Photochemistry of Two Polymorphs.

As shown by X-ray crystallography, crystals of 3ß-acetoxy-16,17-seco-17,20-dioxopregn-5-ene-16-nitrile are dimorphic. The regioselectivity of the Norrish-Yang type II photocyclization under visible light of this steroidal 1,2-diketone, which bears primary, secondary, and tertiary nonequivalent abstractable γ-hydrogens, dramatically increases in the crystalline state of both polymorphs. X-ray crystallography and molecular mechanics calculations reveal crystal structure-solid state photochemistry relationships.

Electrochemistry-coupled to liquid chromatography-mass spectrometry-density functional theory as a new tool to mimic the environmental degradation of selected phenylurea herbicides.

In vitro and in vivo experimental models, mainly based on cell cultures, animals, healthy humans and clinical trials, are useful approaches for identifying the main metabolic pathways. However, time, cost, and matrix complexity often hinder the success of these methods. In this study, we propose an alternative non-enzymatic method, using electrochemistry (EC) coupled to liquid chromatography (LC) - high resolution mass spectrometry (HRMS) - DFT theoretical calculations (EC/LC-MS/DFT) for the mimicry/simulation of the environmental degradation of phenylurea herbicides, and for the mechanism elucidation of this class of herbicides. Fenuron, monuron, isoproturon, linuron, monolinuron, metoxuron and chlortoluron were selected as relevant model compounds. The intended compounds are oxidized by EC, separated by LC and detected using electrospray ionization HRMS. The main oxidation products were hydroxylated compounds obtained by substitution and addition reactions. Unstable quinone imines/methines, rarely observed by conventional methods, have been identified during the oxidative degradation of phenylurea herbicides for the first time in this study. Some were directly observed and the others were trapped by glutathione GSH. Reactions such as hydrolytic substitutions (-Cl/+OH and -C3H7/+OH and -CH3/+OH and -OCH3/+OH), aromatic hydroxylation, alkyl carbon hydroxylation, dehydrochlorination/dehydromethylation/dehydromethoxylation and conjugation have been successfully mimicked. The obtained results, supported by theoretical calculations, are useful for simulating/understanding and predicting the oxidative degradation pathways of pesticides in the environment.

Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins.

New methods for chemo-selective modifications of peptides and native proteins are important in chemical biology and for the development of therapeutic conjugates. Less abundant and uncharged amino-acid residues are interesting targets to form less heterogeneous conjugates and preserve biological functions. Phenylurazole (PhUr), N-methylphenylurazole (NMePhUr) and N-methylluminol (NMeLum) derivatives were described as tyrosine (Y) anchors after chemical or enzymatic oxidations. Recently, we developed the first electrochemical Y-bioconjugation method coined eY-click to activate PhUr in biocompatible media. In this work, we assessed the limitations, benefits and relative efficiencies of eY-click conjugations performed with a set of PhUr, NMePhUr and NMeLum derivatives. Results evidenced a high efficiency of NMeLum that showed a complete Y-chemoselectivity on polypeptides and biologically relevant proteins after soft electrochemical activation. Side reactions on nucleophilic or heteroaromatic amino-acids such as lysine or tryptophan were never observed during mass spectrometry analysis. Myoglobine, bovine serum albumin, a plant mannosidase, glucose oxidase and the therapeutically relevant antibody trastuzumab were efficiently labelled with a fluorescent probe in a two-step approach combining eY-click and strain-promoted azide-alkyne cyclization (SPAAC). The proteins conserved their structural integrity as observed by circular dichroism and the trastuzumab conjugate showed a similar binding affinity for the natural HER2 ligand as shown by bio-layer interferometry. Compared to our previously described protocol with PhUr, eY-click with NMeLum species showed faster reaction kinetics, higher (complete) Y-chemoselectivity and reactivity, and offers the interesting possibility of the double tagging of solvent-exposed Y.

Using electrochemistry coupled to high resolution mass spectrometry for the simulation of the environmental degradation of the recalcitrant fungicide carbendazim.

Currently, there is a growing interest in the study of environmental degradation pathways of organic contaminants such as pesticides, with the objective to better understand their potential risk for environmental systems and living organisms. In this context, DFT (conceptual density functional theory) and predictive methods may systematically be used to simplify and accelerate the elucidation of environmental degradation. We report herein the electrochemical behavior/degradation of the carbendazim (CBZ) fungicide widely used to treat cereal and fruit crops. Oxidative degradation of CBZ was studied using an electrochemical flow-through cell directly coupled to a mass spectrometer for rapid identification of CBZ degradation products. The structural elucidation of CBZ oxidation products was based on retention time, accurate mass, isotopic distribution and fragmentation pattern by using LC-HRMS an LC-HRMS2. The most important chemical reactions found to occur in the transformation of CBZ were hydrolysis and hydroxylation. EC-LC-MS and EC-MS analysis has made it possible to highlight the identification of degradation products of CBZ. In addition to previously known transformation products common to those observed during environmental degradation (monocarbomethoxyguanidine, benzimidazole-isocyanate, 2-aminobenzimidazole, hydroxy-2-aminobenzimidazole, hydroxycarbendazim, CBZ-CBZ dimer), two new degradation products were identified in this work: a quinone imine and a nitrenium ion. Electrochemistry mass spectrometry hyphenated techniques represent an accessible, rapid and reliable tool to elucidate the oxidative degradation of CBZ, including reactive degradation products and conjugates.

Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases*.

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.

Tyrosine Conjugation Methods for Protein Labelling.

Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalise the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody-drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates, and PEG conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for the specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues.

Chemical modification of the adeno-associated virus capsid to improve gene delivery.

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling.

Electrochemically Promoted Tyrosine-Click-Chemistry for Protein Labeling.

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

Physiochemical Tuning of Potent Escherichia coli Anti-Adhesives by Microencapsulation and Methylene Homologation.

Thiazolylaminomannosides (TazMan) are FimH antagonists with anti-adhesive potential against adherent-invasive Escherichia coli (AIEC) promoting gut inflammation in patients with Crohn's disease. The lead TazMan is highly potent in vitro, but shows limited in vivo efficiency, probably due to low pH stability and water solubility. We recently developed a second generation of stable TazMan, but the anti-adhesive effect was lower than the first. Herein we report a co-crystal structure of the lead TazMan in FimH, revealing that the anomeric NH group and the second thiazole moiety provide a positive hydrogen bonding interaction with a trapped water molecule, and π-stacking with Tyr48 of FimH, respectively. Consequently, we developed NeoTazMan homologated with a methylene group for low-pH and mannosidase stability with a conserved NH group and bearing various heterocyclic aglycones. Microencapsulation of the lead NeoTazMan in γ-cyclodextrin dramatically improved water solubility without disrupting the affinity for FimH or the anti-adhesive effect against AIEC isolated from patients with Crohn's disease.

Radical-Mediated C-H Functionalization: A Strategy for Access to Modified Cyclodextrins.

A simple and efficient radical C-H functionalization to access modified cyclodextrins (CDs) has been developed. The well-defined conformation of glycosidic and aglyconic bonds in α-, ß-, and γ-CDs favors the intramolecular 1,8-hydrogen atom transfer (HAT) promoted by the 6I-O-yl radical, which abstracts regioselectively the hydrogen at C5II of the contiguous pyranose. The C5II-radical evolves by a polar crossover mechanism to a stable 1,3,5-trioxocane ring between two adjacent glucoses or alternatively triggers the inversion of one α-d-glucose into a 5-C-acetoxy-ß-l-idose unit possessing a 1C4 conformation. The 6I,IV- and 6I,III-diols of α- and ß-CDs behave similarly to the monoalcohols, forming mostly compounds originating from two 1,8-HAT consecutive processes. In the case of 6I,II-diols the proximity of the two 6-O-yl radicals in adjacent sugar units allows the formation of unique lactone rings within the CD framework via a 1,8-HAT-ß-scission tandem mechanism. X-ray diffraction carried out on the crystalline 1,4-bis(trioxocane)-α-CD derivative shows a severe distortion toward a narrower elliptical shape for the primary face.

Differentiation of Crohn's Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force.

Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn's disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Development of Heptylmannoside-Based Glycoconjugate Antiadhesive Compounds against Adherent-Invasive Escherichia coli Bacteria Associated with Crohn's Disease.

UNLABELLED: The ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasive Escherichia coli (AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives of n-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were tested in vitro in IEC infected with the AIEC LF82 strain and in vivo by oral administration to CEACAM6-expressing mice infected with LF82 bacteria. In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacy in vivo in decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or ß-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effects in vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators. IMPORTANCE: Current treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cells in vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observed in vivo in AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.

Surface Plasmon Resonance (SPR) for the Evaluation of Shear-Force-Dependent Bacterial Adhesion.

The colonization of Escherichia coli (E. coli) to host cell surfaces is known to be a glycan-specific process that can be modulated by shear stress. In this work we investigate whether flow rate changes in microchannels integrated on surface plasmon resonance (SPR) surfaces would allow for investigating such processes in an easy and high-throughput manner. We demonstrate that adhesion of uropathogenic E. coli UTI89 on heptyl α-d-mannopyranoside-modified gold SPR substrates is minimal under almost static conditions (flow rates of 10 µL·min⁻¹), and reaches a maximum at flow rates of 30 µL·min⁻¹ (≈30 mPa). This concept is applicable to the investigation of any ligand-pathogen interactions, offering a robust, easy, and fast method for screening adhesion characteristics of pathogens to ligand-modified interfaces.

Easy access to modified cyclodextrins by an intramolecular radical approach.

A simple method to modify the primary face of cyclodextrins (CDs) is described. The 6(I)-O-yl radical of α-, ß-, and γ-CDs regioselectively abstracts the H5(II), located in the adjacent D-glucose unit, by an intramolecular 1,8-hydrogen-atom-transfer reaction through a geometrically restricted nine-membered transition state to give a stable 1,3,5-trioxocane ring. The reaction has been extended to the 1,4-diols of α- and ß-CD to give the corresponding bis(trioxocane)s. The C2-symmetric bis(trioxocane) corresponding to the α-CD is a stable crystalline solid whose structure was confirmed by X-ray diffraction analysis. The calculated geometric parameters confirm that the primary face is severely distorted toward a narrower elliptical shape for this rim.