In this study, we present an ultrafast, efficient, and broadly applicable strategy for cell membrane modification via tyrosine-selective bioconjugation using diazonium salt derivatives. This chemical approach enables both one-step and two-step functionalization of adherent, suspension, and primary cells with a wide range of ligands, including imaging probes, carbohydrates, biotin, and proteins, without inducing cytotoxicity or immune activation. Cell membrane engineering through bioconjugation has emerged as a powerful tool in biomedical research, given the central role of the membrane in signaling, transport, and cell-cell interactions. In contrast to conventional glyco-engineering methods, which typically require multiday incubations and can induce cellular stress, our approach achieves rapid, precise, and high-density surface functionalization in less than one hour, with improved reproducibility and biological compatibility. We further demonstrate the applicability of this strategy across diverse cell types, including immortalized cell lines and clinically relevant primary cells such as peripheral blood mononuclear cells (PBMCs) and human natural killer (NK) cells. Notably, surface grafting of an EGFR-targeting Nanofitin enhances the cytotoxic activity of NK cells against EGFR-positive cancer cells. In addition, we show that the bioconjugated signal progressively diminishes over successive cell divisions, providing a self-limiting and transient alternative to permanent genetic modifications such as CAR-based engineering, thereby potentially reducing the risk of prolonged immune activation. Finally, the ability to store pre-functionalized cells at -80 °C increases the practicality of this platform for future ready-to-use applications. Overall, this versatile and non-genetic bioconjugation strategy offers a compelling alternative to existing technologies for applications in targeted therapy, diagnostics, and cell-based immunotherapy.

Carbohydrates play essential roles in biology and medicine, yet their direct conjugation to proteins and cell surfaces remains technically challenging. Here we report a broadly applicable electrochemical strategy for the rapid and chemoselective grafting of native and synthetic complex carbohydrates onto proteins and living cells. A bifunctional N-methylluminol (NML) linker bearing a terminal oxyamine was designed to couple unprotected complex carbohydrates at their reducing end. The resulting glycan–NML conjugates undergo electrobioconjugation with solvent-exposed tyrosine residues of the model protein α-chymotrypsinogen A through selective electrooxidation of the NML anchor at ~+800 mV vs. Ag/AgCl. This operationally simple approach proceeds in aqueous buffer under mild conditions and preserves the structural integrity of both protein and glycan partners. Using a model protein, we demonstrate efficient installation of a chemically diverse panel of glycans, including biologically relevant sialylated and tumor-associated epitopes, with retention of lectin-binding specificity. Extending this chemistry to proteins in cell membranes, we achieve high-density, covalent display of millions of carbohydrate ligands per cell within minutes, without compromising viability. Notably, the method enables direct engineering of immune cell surfaces with sialylated ligands that selectively engage SIGLEC-2, a clinically relevant receptor on B lymphocytes and lymphomas. By providing the first electrochemical route to protein and cell-surface pseudo-glycosylation, this work establishes a versatile platform for probing glycan function and for the development of carbohydrate-based therapeutics in immunology, oncology, and vaccination.

We report the chemical conjugation of a recombinant Adeno Associated Virus (rAAV) capsid with various functionalities, including proteins, using a bioorthogonal strategy. rAAVs were azido-coated or dibenzylcyclooctyne (DBCO)-coated by chemically modifying lysine or tyrosine residues. Lysine residues were modified using a phenyl isothiocyanate anchor, and tyrosine residues using either an aryl diazonium salt or a N-methyl luminol derivative. We demonstrate anchor-dependent labeling levels, as observed with biochemical assays and mass spectrometry. Strain-promoted azide-alkyne cycloaddition (SPAAC) was then implemented and evaluated on the rAAV to append functionalities such as fluorescein, biotin, and carbohydrates to the azido-coated capsids. We confirmed the efficiency of the bioorthogonal reaction and observed a stronger reactivity with dibenzylcyclooctyne (DBCO) compared to bicyclononyne (BCN). The optimized SPAAC reaction was finally used to label the viral vectors with two relevant nanobodies targeting specific immune cell receptors (CD62L and CD45). In vitro transduction assays conducted with one rAAV-nanobody conjugate demonstrated the promising targeting properties of these chemically modified vectors. Thus, we anticipate that this strategy will positively impact the field of rAAV capsid engineering and contribute in tissue-specific targeting for the optimization of gene therapy treatments.

Nanoblades are viral particles loaded with the Cas9 protein complexed with gRNA, which allowed efficient gene editing in hematopoietic stem and progenitor cells (HSPCs). Combined with recombinant adeno-associated vector (rAAV) 6 containing two homologous arms to a gene locus resulted in 50% of expression cassette knockin into HSPCs. However, high effective doses of rAAV6 induced HSPC cell death. Here, we demonstrated that, at high doses, rAAV2 was much less toxic for template DNA delivery and allowed transduction levels in HSPCs equivalent to rAAV6. To improve donor template delivery, rAAV2 and rAAV6 were chemically bio-conjugated with a mannose ligand, via the lysine or tyrosine amino acid residues exposed at the adeno-associated vector (AAV) capsid surface. High-level transduction of HSPCs with mannose-coupled rAAV6 vectors accompanied by a remarkable lower toxicity was achieved as compared to control rAAV6 in correlation with highly reduced p53 pathway activation. Mannose-conjugated rAAV6 combined with nanoblades allowed efficient gene knockin and increased survival of HSPCs from 10% to 80% as compared to the unmodified rAAV6 even in the most immature CD34+CD38lowCD90+ hematopoietic stem cell (HSC) population. Summarizing, mannose-conjugated rAAV6 maintained high-level donor mediated gene knockin when combined with nanoblades without inducing significant toxicity for the HSPCs, an important feature for clinical translation of HSPC gene-editing strategies.

The remodeling of microorganism surfaces with biomolecules is a powerful tool to study the role of membrane receptors in chemical biology and to develop drug delivery systems in gene therapy using viral vectors and cell-based therapies. Methods for direct covalent ligation of these surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. In the latter case, a tagged precursor must first be enzymatically metabolized and delivered to the outer cell membrane to become available for chemo-selective labeling. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. This would also need to be compatible with organisms showing poor levels of precursor assimilation or lacking the metabolic function. 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 a 750 mV vs Ag/AgCl electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The electro-coupling was performed with NML anchors bearing a bioorthogonal azide, biotin, or carbohydrate (mannose and N-acetyl galactosamine) handles. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), E. coli (Gram-) and S. epidermis (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. As NML activation immediately stops if there is no current, the method offers reproducible temporal control on the degree of surface functionalization. Thus, click-electrochemistry should significantly expand the scope of bioconjugation methods.

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.

Osteoarthritis (OA), the most common form of joint disease, affects more than 500 million people worldwide. This painful, and debilitating disease imposes a huge socioeconomic cost worldwide. Despite years of promising research, no etiological drug has been successfully introduced into daily clinical practice. In this context, gene therapy (GT) is emerging as a tool capable of meeting an increasingly specialized medical need. Five GT drugs for OA are currently under clinical evaluation, demonstrating the relevance of this tool. However, the widespread use of GT is still limited by considerations of safety, long-term efficacy, controlled and specific targeting, and the presence of neutralizing immune responses. Cartilage, a tissue of interest to target in OA, is a complex tissue to penetrates with the various GT vectors. Thus, this manuscript reviews current clinical trials involving DNA-based GT for OA and suggests ways to improve recombinant adenoviral and adeno-associated viral vectors including capsid engineering and transgene sequence optimization to achieve long term long-term expression of a given transgen exclusively in the target joint tissue, including cartilage. This review then highlights that the use of hybrid serotypes and/or chemical modifications of capsids are promising for improved tissue targeting. In addition, the choice of promoter and type of vectorized nucleic acid (single- or double-stranded DNA) appears to be critical for efficient transgene expression. Finally, the combination of increasing knowledge about biocompatible materials and viral vectors should also be a way to improve transduction efficiency, increase the stability of transgene expression, and allow escape from neutralizing antibodies.

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.

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.

Parvoviridae are small viruses composed of a 4–6 kb linear single-stranded DNA protected by an icosahedral capsid. The viral genes coding non-structural (NS), capsid, and accessory proteins are flanked by intriguing sequences, namely the telomeres. Telomeres are essential for parvovirus genome replication, encapsidation, and integration. Similar (homotelomeric) or different (heterotelomeric) at the two ends, they all contain imperfect palindromes that fold into hairpin structures. Up to 550 nucleotides in length, they harbor a wide variety of motifs and structures known to be recognized by host cell factors. Our study aims to comprehensively analyze parvovirus ends to better understand the role of these particular sequences in the virus life cycle. Forty Parvoviridae terminal repeats (TR) were publicly available in databases. The folding and specific DNA secondary structures, such as G4 and triplex, were systematically analyzed. A principal component analysis was carried out from the prediction data to determine variables signing parvovirus groups. A special focus will be put on adeno-associated virus (AAV) inverted terminal repeats (ITR), a member of the genus Dependoparvovirus used as vectors for gene therapy. This chapter highlights the diversity of the Parvoviridae telomeres regarding shape and secondary structures, providing information that could be relevant for virus-host interactions studies.

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.

New methodologies for the chemo-selective modifications of peptides and native proteins are of great importance in chemical biology and for the developm ent 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 oxydations. 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 offer the interesting possibility for the double tagging of solvent-exposed Y.