Three recent approvals and over 100 ongoing clinical trials make adeno‐associated virus (AAV)‐based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic‐biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine‐tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic‐biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic‐biological approaches. The concept of upgrading adeno‐associated viral (AAV) vectors by means of synthetic‐biological engineering is initiating a new era in gene therapy. The development of strategies to control and fine‐tune transduction mechanisms or transgene expression gives rise to a new generation of viral vectors. This review discusses recent trends in the field and particularly focuses on synthetic‐biological AAV engineering. Depending on the target cells, two different approaches are mainly applied: stem cells or dividing precursor cells are treated ex vivo with viral vectors, such as retroviral or lentiviral vectors, capable of stably inserting the therapeutic gene into the target cell genome. As a consequence, the therapeutic gene is preserved in progenitor cells. One major concern associated with integrating vectors is the potential for insertional mutagenesis. Postmitotic, nondividing cells are therefore typically targeted in vivo by nonintegrating vectors derived from, e.g., adeno‐associated virus (AAV), adenovirus, or herpes simplex virus. The genetic information encoded in these vectors is maintained as episomal DNA for prolonged periods of time.Gene therapy aims at the transfer of therapeutic genes into a patient's cells to treat diseases on the genetic level.2 In m Although the drug was not commercially successful due to low demand and its extremely high cost and is therefore no longer on the market, it clearly demonstrated the safety and functionality of AAV‐based therapeutic approaches. Since then, Voretigene neparvovec (Luxturna) for the treatment of Leber's congenital amaurosis and Onasemnogene abeparvovec (Zolgensma) for the treatment of spinal muscular atrophy received U.S. Food and Drug Administration (FDA) approval in 2017 and 2019, respectively. A major factor in the success of AAV as a therapeutic gene delivery vehicle is its low immunogenicity and lack of pathogenicity. Current clinical research focuses on the treatment of monogenetic diseases by gene replacement, gene silencing, or gene editing, as well as on gene addition approaches using AAV‐based vectors . AAV also holds promise for cancer therapies, the development of novel types of vaccines, and vectored immunoprophylaxis.With three approvals and over 100 ongoing clinical trials, AAV vectors have become one of the leading gene delivery vehicles for gene therapy. In 2012, Alipogene tiparvovec (Glybera) was the first AAV therapy approved in Europe for the treatment of lipoprotein lipase deficiency.7 AlthIn recent years, increasing knowledge of the structure and biology of AAV has led to the targeted modification of its proteinaceous capsid, which has opened the door to viral vectors with enhanced tissue specificity, cellular uptake, transgene expression, and protection from neutralizing antibodies. The engineering of the capsid has been complemented by sophisticated design of the packaged vector genome to modulate the time, location, and strength of transgene expression. In addition, synthetic‐biological principles have extended the toolbox for upgrading AAV‐based vectors at various levels of the transduction cycle.In this review, we summarize recent advances in the engineering of recombinant AAVs (rAAVs) with an emphasis on synthetic‐biological approaches. We will first give an overview of the biology and infection cycle of AAV. Then, we will describe engineering strategies to control distinct stages of AAV transduction—at the capsid and at the vector genome level. Although the therapeutic potential of most of these designed vectors has yet to be evaluated in the clinical setting, they represent novel concepts that will likely contribute to both the development of advanced gene delivery vehicles and the uncovering of fundamental AAV biology‐related mechanisms.2Parvoviridae . As the genus name implies, AAV replication and production of progenitor viral particles depend on coinfection with a helper virus such as adenovirus, herpes virus, or vaccinia virus. In the absence of these helper viruses, latent infection with AAV occurs by integration into the host genome, preferentially at the AAVS1 locus on chromosome position 19q13.AAVs are small (≈25 nm in diameter), nonenveloped, single‐stranded DNA (ssDNA) viruses. They are classified as dependoviruses—a genus belonging to the family of r small).27 As 2.1rep, cap, aap, and the newly identified maap . They are produced from two alternatively spliced mRNAs whose expression is controlled by the two promoters p5 and p19 , VP2 (72 kDa), and VP3 (62 kDa)  in an alternative open reading frame with a nonconventional CUG start codon. AAP localizes in the nucleus, where it plays an essential role for most serotypes  in promoting the capsid assembly of newly synthesized viral proteins. Recently, an intriguing study employed a codon‐scanning approach to detect hidden genes in alternative reading frames. The authors identified an additional gene product in the VP1 region coding for a membrane‐associated accessory protein (MAAP) with an as‐yet undefined function. Interestingly, individual MAAP mutations had no negative impact on viral production, but production of MAAP mutants was markedly impaired in the presence of a competitive wild‐type AAV genome. In addition, the authors questioned the existence of the gene X putatively encoded on an alternative reading frame in the 3’ region of cap.The 4.7 kb ssDNA genome of AAV is encapsidated in both (+) and (−) single‐strand orientation. It is composed of inverted terminal repeats (ITRs), which are structural elements required for second strand synthesis, genome amplification, integration into the chromosome, and ssDNA packaging. The ITRs flank the four genes 2.2 AAV1 shows a skeletal and cardiac muscle tropism and was used for Glybera to deliver a gene encoding a gain‐of‐function variant of the lipoprotein lipase (LPLS447X) to muscle cells for the treatment of LPL deficiency. AAV9 preferentially binds to galactose as its primary receptor, which is thought to contribute to its ability to cross the blood–brain barrier. As a result, this serotype efficiently targets motor neurons upon intravenous administration, and it has been successfully used in clinical trials for gene delivery to the brain and spinal cord in the case of the recently approved Zolgensma. Moreover, AAV9 efficiently infects muscle tissues. Consequently, this serotype is a preferential choice for muscle‐directed gene therapies in preclinical and clinical trials. So far, most AAV serotypes have been discovered in contaminated adenovirus stocks, human clinical samples, or primates. However, the identification and investigation of AAV isolates from other animal species has also attracted interest with respect to their tropism, serological profile, and compatibility with human tissues. For example, a recent study reported a porcine AAV isolate, AAVpo1, that robustly transduces muscle tissues and, in comparison with AAV9, is remarkably detargeted from the liver.Up to date, more than ten AAV serotypes have been isolated from human and nonhuman primate tissues. In general, these serotypes show a broad tropism, though the different serotypes have preferences for distinct tissues. The cell and tissue specificities of different serotypes can be harnessed in the development of therapeutic vectors. For example, AAV8 efficiently transduces hepatocytes in mice and nonhuman primates upon systemic administration and is thus a preferred vehicle for gene delivery to the liver.38 AAVIn addition to the natural serotypes, a number of designer capsids with enhanced tissue tropism have been generated by means of rational and evolutionary approaches  or growth factor receptors (fibroblast growth factor receptor (FGFR) and hepatocyte growth factor receptor (HGFR) for AAV2; epidermal growth factor receptor for AAV6; or platelet‐derived growth factor receptor (PDGFR) for AAV5). Although these receptors have been described as co‐receptors for AAV infection, recent knockout experiments suggest they may have a rather modest role. In contrast, KIAA0319L  is widely expressed in many tissues and has been identified as an essential entry factor for multiple AAV serotypes.The differences in tropism are caused by variations in the capsid surface, which affects the preference for distinct cellular receptors. The initial cellular attachment is mediated by primary receptors, which are mainly glycan structures that are expressed by many cell types Figure2. Subs2.3.2 Acidification of late endosomes results in conformational changes in VP1 and VP2, leading to exposure of the unique N‐terminus of VP1 (VP1u), which contains a phospholipase A2 domain that supports the escape of AAV virions from the endosomal system, and a nuclear localization signal for transport into the nucleus. The low pH environment in late endosomes has been associated with autocleavage of the AAV capsid. Moreover, endosomal proteases play a pivotal role in priming the subsequent nuclear uncoating, and thus infectivity, of many viruses. Cathepsin B and cathepsin L are important for efficient transduction by AAV2 and AAV8. These proteases bind to and cleave the capsids of both serotypes, which leaves the virions intact, but presumably promotes the conformational change of the capsid and the exposure of VP1u, priming AAV for the downstream infection steps. In addition, VP1u exerts proteolytic activity against disordered proteins at neutral pH. However, the role of this function during the viral life cycle remains to be clarified.Following internalization, AAV is transported by microtubules within endosomal compartments to the perinuclear region Figure .55]5588cap s Figure .[88]3.4 Several rounds of transduction, cap recovery, and re‐packaging enriched AAVs capable of reaching the outer retina from the vitreous. Interestingly, isolated AAVs were almost exclusively derived from the peptide insertion library. The resulting AAV2.7m8 efficiently transduced the outer retina of mice and the fovea of nonhuman primates. In another study, Isgrig et al. used AAV2.7m8 as a gene delivery vehicle to cochlear inner and outer hair cells. The potential of this synthetically evolved capsid variant is currently being assessed in phase I and phase II clinical trials for the treatment of age‐related macular degeneration and diabetic retinopathy, respectively  and in nonhuman primates. Overcoming this limitation of evolutionary approaches performed in vivo will require more sophisticated enrichment strategies, such as combinations of in vitro and in vivo selection using different cell lines and species, and underlines the importance of preclinical studies with animal model surrogates for human patients.Regardless of the type of library used, one important requirement in screening is the coupling of genotype and phenotype. In the case of AAV, this is mainly achieved by transfecting the library plasmid at low concentrations. Previous studies suggest a so‐far poorly understood correlation between the identity of the capsid and the packaged genome.117 Sc3.7 Davidsson et al. extended this approach to enable barcoded rational AAV vector evolution screening, termed BRAVE. They designed an AAV production plasmid containing an ITR‐flanked GFP expression cassette with a barcode in the 3’ UTR and cis‐acting Rep‐Cap. The insertion of loxP sites enabled Cre recombination‐mediated linkage of the capsid variant to the barcode, which could be evaluated by next‐generation sequencing. The decoupling of the cap gene from the vector genome and the sequencing of expressed barcode mRNA add layers of flexibility, including the possibility to screen for AAV variants that undergo the full transduction cycle.In recent years, the construction of evolved AAV variants has been facilitated by the development of new techniques and high‐throughput methodologies. Especially, DNA barcoding of AAV coupled with next‐generation sequencing has facilitated the characterization of individual AAV variants with respect to, e.g., virion formation, transduction efficiency, blood clearance, and neutralizing epitopes.121 Dacap gene. More specifically, they tested the effects of all possible amino acid substitutions, insertions, deletions, and stop codons on viral production and in vivo biodistribution. The resulting capsid fitness landscape illustrates tolerated and deleterious mutations across the cap gene, and will be extremely helpful for future rational capsid engineering and library design. Moreover, the effects on neutralization with the A20 antibody and on thermal stability were investigated. In addition, stop codon screening in alternative reading frames among synonymous capsid sequences revealed three notable findings: first, nonsense mutations in the open reading frame (ORF) of the aap gene impair virion production, which underlines the role of the gene product in capsid assembly. Second, screening of the previously proposed gene X revealed no effect on the fitness of AAV. Third, a new gene was identified in a +1‐shifted ORF in VP1. The product of this gene, the so‐called membrane‐associated accessory protein (MAAP), seems to be involved in the so‐far‐inexplicable genome–phenotype coupling of AAV capsid libraries. The screening of additional serotypes might uncover mutual relationships as well as unique characteristics influencing viral fitness, and could help in generating optimized AAV variants.Recently, Ogden et al. presented an intriguing study that characterized all possible mutations in the AAV2 cap gene.33 Mor4Figuretrans to the rep‐cap plasmid. The third strategy transfers capsid properties from other serotypes by domain swapping. The following sections describe these strategies in more detail and give an overview of the latest developments.Compared to evolutionary approaches resulting in altered or enhanced tissue specificity of AAV, rational approaches based on fundamental research combined with insights from evolutionary engineering have opened the door to truly redirected viral vectors that are specific for distinct cellular receptors. Three main strategies are applied for equipping capsids with new functions Figure5. The 4.1) of the AAV2 capsid as a suitable site for the insertion of an integrin binding peptide, which efficiently retargeted AAV2 to integrin‐expressing cells. Since then, modification of this site has enabled, e.g., the targeting of distinct cell types, the affinity purification of viral vectors, the display of enzymes, and the insertion of peptide libraries for directed evolution  o Notably, the insertion of an integrin‐binding peptide at position 453 was influenced by residues R585 and R588 and the product was only functional in combination with arginine‐to‐alanine mutations. An alternative approach inserted leucine zippers at VP position 453 of AAV9 to noncovalently attach peptides to the capsid. Although the high molar excess of complementary peptide required for capsid functionalization calls for optimization, the system might offer a universal and modular tool for modifying the AAV surface.Position 453 lies in the variable region IV (VR IV) located in the highest protrusion of AAV2 (the GH2/GH3 loop) and has been identified as an alternative to the 587 insertion site.130 No Similarly, this site has been exploited for the insertion of a T7 gene 2 protein , a nanobody (13 kDa), and a HUH tag (21 kDa) into the capsid of AAV‐DJ. HUH tags are derived from bacterial or viral endonucleases and form covalent phosphotyrosine linkages with ssDNA in a sequence‐specific, orthogonal manner. Displaying the Escherichia coli‐derived mobilization protein A (mMobA) HUH tag on AAV‐DJ enabled the covalent attachment of commercial antibodies that had been conjugated to ssDNA by copper‐free click chemistry . Nevertheless, the study pioneered the use of the N‐terminus of VP2 as an anchor point for protein domains intended for capsid display and retargeting. In addition, VP2 turned out to be dispensable for viral production and infectivity, and it could tolerate the addition of larger protein domains such as 30‐kDa GFP. The use of disulfide‐free affibody molecules and DARPins (designed ankyrin repeat proteins) directed against cellular receptors opened the door to the targeting of distinct cell types in a highly specific and effective manner. This approach was extended to disulfide‐containing scFvs by employing an intein‐based trans‐splicing approach  DnaE intein was exploited for a generic protein‐to‐AAV coupling approach. NpuC‐displaying AAV particles readily coupled to NpuN‐fused DARPins and scFvs, showed comparable targeting efficiency to the genetically fused counterparts, and exhibited reduced transduction of off‐target cells. Therefore, this method could be a universal approach for the coupling of targeting domains to the surface of AAV, thus broadening the spectrum of proteins that can be attached to the capsid.An alternative strategy for displaying larger peptides and proteins on AAV is to fuse them to the N‐terminus of VP2, which leads to their exposure through the pore of the fivefold symmetry axis of the viral capsid Figure . This ap135 Imh Figure .[142 have been combined with VP2‐mediated antigen display on the viral capsid. The combined display and vector‐based expression of the Mycobacterium tuberculosis antigen AG85A resulted in a faster humoral immune response and did not require a booster dose for the induction of a memory response in mice.In addition to the retargeting of AAV, the possibility of modifying the capsid surface has led to the emergence of new strategies for the development of vaccines. Previous concepts of delivering DNA vaccines via AAV21 hav4.3 Chimeric virions generated in this way revealed capsid positions responsible for the differential tissue tropism and transduction kinetics of AAV serotypes. In addition to determining the structural properties crucial for transduction specificity and efficiency, these studies identified new positions that tolerate alterations and peptide insertions. For example, domain swapping and single amino acid exchanges between AAV2 and AAV8 revealed positions in the variable region VIII (VR VIII) of AAV8 that tolerate peptide insertions suitable for retargeting or the display of randomized peptide sequences. Moreover, receptor footprints on AAV serotypes were exploited for the directed development of chimeric AAV variants with altered tropism. Substituting a hexapeptide sequence located in the GH loop of AAV2 and containing residues R585 and R588 involved in heparan sulfate binding with those of other serotypes resulted in an AAV2/AAV8 chimera (AAV2i8) with a systemic transduction profile. Of note, AAV2i8 transduced cardiac and skeletal muscle with similar efficiency to AAV8, while showing markedly reduced targeting of the liver and a prolonged circulation half‐life in the blood. A similar approach was used to graft the galactose‐binding footprint from AAV9 onto AAV2 and AAV2i8. The resulting variants had unaltered tropism, but showed enhanced transduction and faster gene expression. In addition to receptor footprints, other capsid‐specific properties can also be exchanged between serotypes. AAV9, AAVrh.8, and AAVrh.10 are able to traverse the blood–brain barrier. This property was transferred to AAV1 by grafting the footprint from AAVrh.10 onto AAV1.Another approach for generating AAV variants with modified tropism is based on domain, peptide, and amino acid swapping between AAV serotypes Figure .[144,4.4 accompanied with improved intracellular trafficking to the nucleus and increased transgene expression. It has also been shown that the neddylation and SUMOylation pathways are upregulated during intracellular AAV trafficking. Mutation of predicted target sites for these ubiquitin‐like modifiers led to AAV2 variants with enhanced transduction and transgene expression.So far, the majority of rational capsid modifications have aimed at tailoring the specificity toward cellular receptors and/or protecting AAVs from NAbs. However, intracellular processes greatly affect the transduction efficiency of AAV‐based vectors. For example, the mutation of distinct serine, threonine, lysine, and tyrosine residues on the AAV capsid led to reduced ubiquitination and proteasomal degradation,151 ac55.1 The most prominent switches include protein–DNA, protein–protein, and protein–small molecule interactions that can be formed or dissociated in response to small molecules, enzymes, or light. This approach allows for the control of cellular processes such as transgene expression, receptor activation, or protein trafficking.Synthetic biology uses molecular switches to control biological functions in response to specific internal or external stimuli.154 Th5.1.1Figure This chemically induced dimerization pair has been used for the pharmacologic control of gene expression, receptor activation, kinase activity, protein trans‐splicing, and epigenome editing. The toolbox of synthetic‐biological switches could be similarly applied for the design of AAV variants that can be controlled by external stimuli. For example, the fusion of FKBP to VP2 of receptor‐blinded  AAV2 enabled controlled interaction with an FRB‐tagged DARPin directed against the EGF receptor , which forms heterodimers with the FKBP‐rapamycin binding (FRB) domain of mTOR in the presence of rapamycin or analogs  thereof Figure7a.[155.1.2 Moreover, proteases have been exploited to control extracellular processes and to develop cell‐free sensor circuits and smart drug delivery vehicles. The latter use environmental cues to determine their site and/or time of action, such as the decreased pH or elevated levels of matrix metalloproteases (MMPs) that often characterize the microenvironment of tumor tissues. pH‐ and MMP‐responsive delivery vehicles have the potential to specifically target such sites. A similar strategy has been developed for controlling AAV‐mediated gene transfer. The integration of negatively charged peptides into the heparan sulfate‐binding loop of AAV (between R585 and R588) effectively impaired the binding to heparin, which correlated with reduced cellular uptake. Insertion of MMP cleavage sites enabled the removal of the negatively charged peptide lock and the recovery of heparin binding and cell transduction  and phytochrome B (PhyB), both derived from the wall cress Arabidopsis thaliana. Cry2 homooligomerizes or forms heterodimers with CIB1 (cryptochrome‐interacting basic‐helix‐loop‐helix) upon blue‐light illumination and both interactions are reversed in darkness. In contrast, PhyB is a red‐light‐responsive photoswitch that associates with its interaction partner PIF (phytochrome interacting factor) upon red‐light illumination (660 nm) and dissociates in response to far‐red light (740 nm). The number of optogenetic switches is rapidly expanding, and spans the whole spectrum of visible light. A comprehensive overview of existing optogenetic tools including search engines for photoswitches and publications can be found at https://www.optobase.org/.Over the past decade, the steering of cellular functions with genetically encoded photoswitches has attracted considerable research interest. This field—termed optogenetics—exploits photoreceptors derived from, e.g., plants or bacteria as light switches to control intracellular and extracellular biological processes with unmatched temporal and spatial resolution.183 Op Gomez et al. exploited the nuclear transport of AAV, which is a rate‐limiting step during the transduction process, to develop red‐light‐tunable gene delivery. To this end, they displayed the phytochrome interaction partner PIF6 on AAV2 by fusing it to the N‐terminus of VP2. Expression of a nuclear localization signal (NLS) fused to PhyB enabled enhanced nuclear trafficking and thus gene delivery in response to red‐light illumination  are often chosen in gene replacement therapies to achieve maximal transgene expression. This was the case for the approved drugs Glybera, Luxturna, and Zolgensma. Enhancer elements and post‐transcriptional regulatory elements (PRE) can further increase the efficiency of gene expression. For example, the commonly used woodchuck hepatitis virus PRE (WPRE) or intron sequences are thought to mediate interaction with the spliceosome, increasing the stability and/or nuclear export of the mRNA.The expression of a transgene is controlled not only by the cellular uptake of the delivery vehicle and the trafficking of the genetic material to the nucleus, but also by coding and noncoding regulatory elements that act at the transcriptional or translational level Figure8a. The 5.2.1 and was recently exploited to restrict CRISPR/Cas9‐mediated genome editing to the liver of mice. Combinational incorporation of different miRNA targets can further enhance cell and tissue specificity. For example, compared to healthy hepatocytes, miRNA‐122 and miRNA‐199a expression is downregulated in hepatocellular carcinoma (HCC), and the incorporation of the respective miRNA binding sites into AAV8 efficiently restricted the expression of a suicide gene to HCC cell lines. Other approaches use computational vector design to identify cis‐acting regulatory modules (CRMs) that enhance the gene expression in target tissues and thereby allow the use of lower vector doses, reducing the risk of T cell‐mediated immune responses against AAV. In a genome‐wide in silico analysis, Chuah et al. identified hepatocyte‐specific CRMs that contain signatures of evolutionarily conserved binding sites for transcription factors associated with high expression in the liver. Intriguingly, one CRM candidate increased the expression of the human clotting factor IX (hFIX) up to 100‐fold, depending on the strength of the promoter used. Treatment of nonhuman primates with a self‐complementary AAV9 coding for hFIX under the control of a potent liver‐specific promoter and the liver‐specific CRM led to high  and sustained circulating levels of hFIX. A similar approach was used to enhance cardiac‐specific expression of genes delivered by AAV9.In cases where a specific tissue or cell type needs to be targeted, more sophisticated fine‐tuning of the gene expression could be advantageous and could be combined with the above‐described engineering of the viral capsid. Tissue‐specific promoters are usually weaker than constitutive promoters, but they add a layer of specificity to the gene expression profile and may help to reduce the effects of off‐target transduction. Additional strategies to increase the tissue specificity involve the exploitation of tissue‐specific expression profiles of miRNAs Figure ,c. miRNA191 an5.2.2 For example, bacterial repressor proteins, yeast transcription factors, and human hormone receptors have been exploited to develop eukaryotic gene switches that respond to clinically licensed antibiotics, flavonoids, amino acids, hormones, and other small molecules. The spectrum of transcriptional control elements has been further broadened by the introduction of chemically and optogenetically controlled protein–protein switches  in mice and primates, respectively. Their system comprises three components of a rapamycin‐inducible gene expression system, packaged into two AAVs. One vector constitutively expresses a DNA‐binding zinc finger homeodomain (ZFHD) fused to three repeats of FKBP, and FRB fused to the activation domain of the p65 subunit of human NFκB  limits the generic applicability in the design of vector genomes. Different strategies have been developed to enable the delivery of larger gene constructs, including overlapping dual vectors or trans‐splicing dual vectors.208 Hon Figure . The sysStrategies to spatially and/or temporally control the expression of a packaged transgene have great potential to be combined with engineered capsid variants. Developments in both capsid design and synthetic gene switches can be expected to advance gene delivery strategies.5.3The search for optimal gene delivery vehicles has focused not only on the engineering of independent virus species, but also on combining advantageous features of different virus family members or even virus orders. In the case of AAV, different features such as the low genotoxicity of its recombinant vector genome, or the cellular uptake and endosomal escape mechanisms have been exploited for the development of hybrid vectors.5.3.1 With the aim of developing parvoviral vectors for the treatment of hemoglobinopathies, Ponnazhagan et al. exploited B19's efficient tropism for human erythroid hematopoietic progenitor cells and packaged rAAV genomes into B19 capsids.The successful cross‐packaging of rAAV genomes into the parvorvirus B19 was reported more than 20 years ago.212 Wi Interestingly, bocaparvoviruses have an ssDNA packaging capacity of ≈5.5 kb, which permits the introduction of larger AAV expression cassettes , which resulted in efficient transduction of polarized human airway epithelial (pHAE) cells.213 In5.3.2 Insertion of rAAV expression cassettes into the intergenomic region of M13 phages displaying the RGD‐4C peptide enabled ligand‐targeted gene delivery to mammalian cells  was used for both PET imaging with a radiolabeled nucleoside analog and tumor killing by the administration of the prodrug ganciclovir. Recently, this AAVP platform was further developed to display additional peptide sequences on the major phage coat protein pVIII. A short positively charged Ala‐Lys‐Ala‐Ser peptide, e.g., reduced nonspecific protein adsorption and neutralization by antiphage antibodies. The pVIII‐mediated display of an endosomal escape peptide derived from influenza hemagglutinin markedly enhanced transduction efficiency of RGD‐4C‐AAVP.rAAV genomes can be packaged not only into capsids of related parvoviruses, but also into bacteriophages. Phages typically do not have any tropism for human cells but this capability can be introduced by the display of targeting ligands. To enable subsequent gene expression, eukaryotic regulatory elements are required. This was achieved by harnessing the compatibility of the ssDNA genomes of AAV and the M13‐derived phage dt‐tet.215 Ins Figure . The AAV To this end, AAV particles were attached to T4 heads via biotin‐avidin bridges. These T4‐AAV hybrids indeed mediated the uptake of the genetic material packaged in the T4 moiety. In addition to the genes packaged in AAV and T4, the T4 head could co‐deliver attached proteins into mammalian cells. The ability to simultaneously package DNA and to display protein cargos was exploited for the proof‐of‐concept development of combined DNA and protein vaccines against influenza and Yersina pestis. Interestingly, delivery of the genetic material via the T4 head led to a TH1‐biased immune response, presumably due to the prokaryotic origin of the DNA exhibiting nonmethylated CpG islands and thus provoking the activation of toll‐like receptors. In contrast, AAV‐mediated DNA delivery evoked a more balanced TH1 and TH2 response. This work describes a creative and interesting approach to the generation of new vaccines. The potential of the system, such as loading capacity for different genes, the maximal size of gene constructs that can be packaged, and the applicability to other therapeutic applications, still needs to be investigated.Another approach combined the packaging capacity of T4 phage heads (≈170 kbp of dsDNA) with the ability of AAV to be taken up by mammalian cells and to mediate efficient endosomal escape.217 To5.3.3 or antibody reduction by plasmapheresis and hemapheresis have shown some degree of success. Further methods for lowering the functional NAb titers include the administration of receptor‐blinded, empty capsid decoys capable of capturing anti‐AAV NAbs and the less specific approach of administering IgG‐cleaving proteases. For a detailed overview of immune responses to AAV and possible solutions, we refer the reader to the recent reviews by Nidetz et al. and Colella et al.As mentioned above, pre‐existing humoral immunity is one of the greatest challenges for AAV‐mediated gene therapy and has been addressed from various angles. Although forward‐ and backward‐directed evolution  to the transmembrane domain of the platelet‐derived growth factor leads to its exposure on the surface of cells and exosomes, and was used for the construction of brain‐targeted vexosomes.An alternative strategy to protect AAV from NAbs is their encapsulation in a protective polymer shield227 ors Figure .[228In general, vexosomes show enhanced transduction and broad tropism for many tissues. These features might be advantageous for the treatment of monogenetic diseases affecting many cell types in the body. However, for therapies that require the specific targeting of distinct tissues or cell types, e.g., in the case of tumor targeting, additional strategies will be needed to minimize off‐target transduction.6 although it was withdrawn from the market in 2010 due to the availability of alternative and safer therapies. Nevertheless, like Glybera, it pioneered a new therapeutic strategy. The murine origin of OKT3 could cause HAMA (human antimouse antibody) responses associated with mild to life‐threatening symptoms. Consequently, enormous efforts have been made to develop and manufacture humanized or fully human therapeutic antibodies. Today, these antibodies are successfully used for the treatment of different types of cancer, cardiovascular diseases, infectious diseases, or autoimmune diseases. Similarly, AAV‐based gene delivery offers huge potential for the development of new therapeutic approaches, such as gene replacement therapies, targeted virus‐directed enzyme prodrug therapy, or novel vaccination strategies. The approval of AAV‐based therapies has triggered an increased interest in the fundamental biology of this virus and in the engineering of new AAVs with improved functions. Both of these research topics go hand in hand and complement each other well. A deeper understanding of, e.g., cellular uptake mechanisms and intracellular trafficking will help to generate AAVs with enhanced transduction properties.The development of reliable and safe tools for the delivery of genes is a pressing goal, e.g., for the treatment of monogenetic diseases and the development of new therapeutic approaches. In recent years, only a limited number of delivery vehicles qualifying for in vivo applications have been identified. Among them, AAV has attracted great interest because the safety and functionality of this vector are supported by the approvals of three AAV‐based therapies. However, despite these successes, we are still far from having a universally applicable and adaptable gene delivery vehicle. Major obstacles include the presence of neutralizing antibodies in the majority of people, the need to improve targeting of specific tissues and cell types, and the limited efficiency of gene transfer. The first issue plays a minor role in very young patients lacking a fully trained immune system and mostly being seronegative for anti‐AAV antibodies, or for the treatment of partially immune‐privileged sites such as the brain or the eye. Luxturna for retinal gene therapy and Zolgensma for the treatment of spinal muscular atrophy in children appear to be low‐hanging fruits in this respect. However, they may have opened the door to the development and approval of additional AAV‐based therapies. Therapeutic monoclonal antibodies were in a similar situation over 30 years ago. Orthoclone OKT3 was the first antibody approved for clinical use in humans,235 alThe future of AAV as therapeutic gene delivery vehicle strictly depends on the question of whether broad in vivo applicability of AAV can be achieved.Additional factors that have to be taken into account, and which are often underestimated in academic settings, are the feasibility and expense of AAV production and downstream processing. Strategies that integrate all three major aspects—the engineering of AAV, the general in vivo applicability, and feasible production—are the most challenging yet potentially the most rewarding for achieving clinical breakthroughs.Here, synthetic biology provides valuable tools and concepts to address major challenges. The incorporation of chemical and optogenetic switches into capsid designs opens the door to temporal and spatial precision targeting of tissues and cells. These designs could be further complemented by systems that restore the transduction capability in response to endogenous disease markers such as specific proteases or the low pH in the microenvironment of tumors. Such approaches might minimize off‐target transductions and thus side‐effects thereby increasing the efficiency of the viral vectors. Moreover, synthetic‐biological switches enable adjustable and time‐controlled gene expression, which could be a promising means to time, dose, and potentially minimize immune responses against the therapeutic gene. The challenge of preexisting immunity against AAV can be tackled by various approaches, which include the creation of novel or ancestral capsid variants devoid of current NAb epitopes, the engineering of stealth shieldings, or the development of completely new viral vector hybrids. A notable advantage of synthetic‐biological approaches is the modularity and flexibility of the design concepts, which facilitates the customization and tailoring of AAV variants. microsystems engineering, and materials science. A similar trend can be anticipated in the engineering of AAV variants to tackle current clinical challenges. Developments in synthetic biology could enable the engineering of AAV vehicles that go beyond the standard capsid modifications and expression cassette designs. Although it is questionable whether the current early designs will ever enter the clinic, they could be considered as a first step toward next‐generation gene‐delivery vehicles. The hope awakened by this small virus is big: synergies among basic research, rational and evolutionary designs, and synthetic‐biological engineering could foster the emergence of new approaches both to improve current therapies and to address thus‐far untreatable illnesses.The current clinical trials with bioengineered capsids are promising and will likely trigger a new era of AAV‐based research and development. An important factor that will impact on or even define future investigations is the integration of multiple disciplines for the design or evolution of next‐generation AAV vehicles. The value of interdisciplinary developments is clear in other scientific fields. The combination of molecular biology and cell biology techniques with (bio‐)chemical, mathematical, physical, or technical methods has resulted in the emergence of new disciplines in, e.g., synthetic biology,238 miThe authors declare no conflict of interest.


One of the highlights of green chemistry is the development of techniques and procedures with low environmental impact. In the last years, deep eutectic solvents (DES) have become an important alternative to conventional organic solvents. For a period ionic liquids have provoked remarkable interest, but they have been displaced by DES because they show easier preparation methods, lower prices, many of them are biodegradable and compatible with biological systems. In addition, they show adjustable physicochemical properties, high thermal stability, low volatility and are compatible with water. In this paper is reviewed the state of the art of the use of DES paying special attention to the role of reaction media in organic synthesis. Deep eutectic solvents are an interesting alternative to conventional solvents, both as a reaction medium and for many processes and technologies used in chemistry and related sciences. They are easily prepared, versatile, many of them can be obtained from renewable sources and can be designed and adapted for specific processes. For all these reasons, they have been studied intensively in recent years and their interest is constantly growing. This paper reviews some of the most prominent applications and which are the fields of interest that may be a focus of applications in the near future. Criteria such as atom economy,2Conventional organic solvents are carbon‐based compounds. Most of them show high vapor pressure values and most of them are water immiscible. They constitute a heterogeneous group of volatile compounds chemically diverse and mainly obtained from the petrochemical industry. Organic solvents are widely used as reaction media, but in many other industrial branches and household products.Most conventional organic solvents show serious risks and hazards for handling, storing or using them because they use to be flammable, explosive, and they may form compounds with high toxicity when they break down at high temperatures. They produce harmful health effects when they come into direct contact with mucous, skin or eyes,From the point of view of environmental chemistry, most of organic solvents are considered as volatile organic compounds,To avoid the inherent risks and hazards related to the massive use of solvents in laboratories and industry in the last years, a lot of work has been done in two main directions:The publication of several guides and recommendations for the substitution of solvents with a high impact on human health and the environment, or potentially dangerous because of their flammability, or explosion risks2) based DES. The DES plays the role of solvent and Lewis acid and can be reused directly without any activation process five cycles, and the yield is in all cases upper than 89 %The classic Friedel‐Crafts reaction has been carried out for the synthesis of triarylmethanes (TRAMs) and diarylalkanes (DIAAs) choline chloride‐zinc chloride  chloride hexahydrate when mixed with alcohols at room temperature with good yieldsOther example of esterification reaction on carboxylic acids has been carried out with a quaternary ammonium salt of deep eutectic solvent (DES) which opens an access to esters using a family of DES as a solvent alkylating agent, and catalyst at the same timeCandida rugosa lipase (CRL)reached up to 44 % after 3 h of reaction and an enantiomeric excess of the ester of 62 %Because of the biocompatibility of many DESs, some experiments have been developed using enzymes in esterification and trans‐esterification reactions. Enzymatic esterification of racemic menthol with lauric acid in the presence of Candida antarctica lipase with vinyl decanoateTransesterification reactions can be performed with DES. As an example of such reactions are the synthesis of glycolipids. These products have paid attention in recent years as a way to prepare biodegradable surfactants. The enzymatic synthesis of glucose monodecanoate with two hydrophilic DES (choline:urea and choline:glucose) has been described in the presence of Transesterification reaction in DES has been used for the enzymatic biodiesel production from waste oils considering the procedure as an alternative to conventional production methods[6.7C−C bond formation is a key strategy in organic synthesis because it lets us build complex molecules from simpler structures. DESs has also been used in these reaction types and a lot of work has been done about.porcine pancreas lipase (PPL) to give the aldol product high yields. DES shows excellent compatibility with PPLThe well known aldol condensation reaction has been carried out on substituted benzaldehydes with aliphatic ketones in choline chloride/glycerol mixtures catalysed with S)‐(+)‐10‐camphorsulfonic acid (SB3‐Cy/CSA) as a reaction media and catalyst for carbon–carbon bond formation reaction via Claisen–Schmidt condensation.DES was obtained from 3‐(cyclohexyldimethylammonio)propane‐1‐sulfonate and  for the well known Knoevenagel condensation between aromatic aldehydes and active methylene compounds at room temperature6.82 in a molar ratio 1 : 2 that as in other cases play as dual role of solvent and catalyst to obtain the final products with excellent isolated yields of 95 %  and acetic acid or ethylene glycol as hydrogen bond donors.6.11DES have been used as reaction media for the synthesis of alkyl glycosides catalyzed by thermostable α‐amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen‐bond donors (HBD) performed best.6.12DESs have proved to be a good alternative to traditional organic solvents and in many biocatalytic processes,6.13DES may be used in polymer chemistry with three roles.As a reaction media in polymerization reactions.As monomers in the synthesis of copolymers.As media for modification a preexisting polymer.4F‐HF.An early example of polymerization using DES as reaction media was published in the synthesis of polyaniline polymer by electrochemical polymerization in an inorganic DES mixture formed by NH6.14A domino reaction is a process involving two or more bond‐forming transformations  which take place under the same reaction conditions without adding additional reagents and catalysts, and in which the subsequent reactions result as a consequence of the functionality formed in the previous step.Multicomponent Reactions (MCRs) are considered as a subclass of domino reactions, one in which three or more molecules react to form a single product in a one pot reaction.[The classic Biginelli, Ugi and Passerini multicomponent reactions have been carried out using DES and some examples can be found in literature.Biginelli is a three‐component reaction between an aldehyde, a ß‐ketoester and urea with acid catalysis. This reaction has been performed with several mixtures of HBD and HBA as reaction media in the synthesis of 3,4‐dihydropyrimidin‐2(1H)‐one and thiones derivatives[Ugi is a four‐component reaction that has been extensively investigated for the preparation of compounds with pharmaceutical interest. Such reaction can be carried out for example with Choline chloride/Urea showing higher yields than conventional solventsThe synthesis of α‐acyloxyamides may be performed with Passerini reaction. The α‐acyloxycarboxamide scaffold is present in many natural products as well as in pharmacologically active substances. This reaction can be accomplished in various choline chloride‐based deep eutectic solvents, mixing benzaldehyde, benzoic acid and cyclohexyl isocyanide in 1 : 1 : 1 proportions, without using any catalysts afforded products in excellent yieldThe multicomponent domino reactions of ketones, aldehydes and malononitrile in a deep eutectic solvent (DES) were developed. Urea‐choline chloride based DES is effective dual solvents/organocatalysts and affording a series of multisubstituted cyclohexa‐1,3‐dienamines7Due to demands for greener technologies, the substitution of conventional solvents for others less toxic and with lower environmental impact has become a priority objective for scientific and manufacturing communities. Deep eutectic solvents may be considered as a valuable alternative because they can be easily prepared from affordable starting materials, reusable in most cases and many of them are completely biodegradables. They can be used in many fields as a valuable substitutes of conventional organic solvents to avoid common inconveniences for the separation or extraction of high value natural products, and as reaction media for many types of chemical transformations. Chemists can replace satisfactory, organic solvents produced by the petrochemical industry by deep eutectic solvents in conventional reaction conditions, or when required with ultrasound and microwave irradiation. A remarkable group of deep eutectic solvents are prepared from renewable sources, therefore they are biocompatible so they may be used in enzymatic transformations and show a promising future in pharmacology and medicine applications. Literature shows the unstoppable growth of publications that use deep eutectic solvents, so they can be taken into account as a valuable tool in green chemistry. In this review are described some of the leading fields when deep eutectic solvents may be applied in the present and with promising perspectives for the next yearsThe authors declare no conflict of interest.Dr. Francisco G. Calvo‐Flores is Associate Professor of Chemistry in the Department of Organic Chemistry . He has developed its career at the University of Granada and during the last years he has been involved in several project focused on Green and Environmental Chemistry.Cristina Mingorance‐Sánchez studied chemsitry at the University of Granada, finishing the Degree in Chemistry in 2020. After her graduation, she started working in a fruit and vegetable company as a Quality Control Assistant, on sampling control, in accordance with the principles of the Hazard Analysis Critical Control Point (HACCP) of different food safety standards, developing effective self‐control systems.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Protein coronae formed with nanoparticles confer several useful properties. However, the non‐specific nature of protein corona formation makes it difficult to deliver specific proteins for therapeutic applications. Herein, we report on the construction of a new type of protein corona, termed binding‐mediated protein corona. This new corona enables the efficient and controllable delivery of functional proteins, which is otherwise challenging for conventional protein coronae. We show the design and delivery of the ribonucleoprotein corona for the CRISPR/Cas9 system. Successful gene editing in human cell lines (Hela and HEK293) demonstrates the efficient delivery, high stability, low cytotoxicity, and well‐controlled activity of the Cas9‐guide RNA ribonucleoprotein. The binding‐mediated protein corona strategy opens up new opportunities for therapeutic protein delivery. We report on the construction of a new type of protein corona, termed binding‐mediated protein corona. This new corona enables the efficient and controllable delivery of functional proteins, which is otherwise challenging for conventional protein coronae. Nanoparticles (NPs) are known to adsorb proteins non‐specifically, forming “protein coronae”.However, protein coronae have only been applied to the delivery of functional small molecules and oligonucleotides based on their electrostatic or hydrophobic interactions with the proteins. Protein coronae have not yet been used for targeted delivery of therapeutic proteins, because of the following challenges. Proteins can irreversibly bind to the nanoparticle surface, leading to changes of protein conformation and loss of biological functions, when the proteins are encapsulated in the inner layer of the corona.Here, we describe an affinity binding strategy to construct a new type of protein corona, termed binding‐mediated protein corona (BMPC) Scheme . The BMPWe chose to deliver the Cas9 ribonucleoprotein (RNP) as an example, consisting of the Cas9 protein and a specific single‐guide RNA (sgRNA), because direct delivery of RNP is an efficient approach for CRISPR genome editing to lower off‐target effects and immunogenicity. The commonly used liposome‐based methods for RNP delivery have high transfection efficiency,Construction of a BMPC comprises two main steps Scheme : conjugaWe applied the BMPC technique to the delivery of RNP of the CRISPR‐Cas9 system, using AuNPs and magnetic nanoparticles (MNPs) as the scaffolds. We designed a DNA linker that hybridizes with the targeting region of the sgRNA and a short thiol‐labeled oligonucleotide (SH‐DNA), forming a ternary complex . To prepare BMPC, we first conjugated the ternary complex of oligonucleotides to AuNPs through gold‐thiol bonds,We designed the DNA linker to hybridize with the targeting region of the sgRNA, silencing the enzymatic activity of the RNP without affecting the binding affinity of sgRNA to Cas9. To achieve controllable release of the RNP from the BMPC, we introduced two photocleavable sites within the domain of the DNA linker hybridizing to the sgRNA. Therefore, spatio‐temporally controlled release of RNP can be achieved through the use of UV illumination to break the photo‐cleavable linker (PC linker).To examine the success of constructing BMPC with desired structures, we prepared two binding‐mediated RNP coronae by incubating Cas9 with sgRNA‐functionalized AuNPs (sgRNA‐AuNPs) at 10:1 and 25:1 molar ratios. We named them RNP corona (10:1) and RNP corona (25:1). We characterized them using transmission electron microscopy (TEM), dynamic light scattering (DLS), and ultraviolet‐visible (UV/Vis) absorption spectra. TEM images of the sgRNA‐AuNPs showed an additional faint shell, approximately 11.5 nm thick, around the AuNPs, suggesting the formation of the ligand inner layer Figure . The sheTo confirm that all added Cas9 molecules were fully assembled in the form of binding‐mediated RNP corona, we centrifuged the mixture and separately analyzed the supernatant and the precipitate . The gel electrophoresis results show that there was no leakage of Cas9 into the solution (Lanes A–C) and the Cas9 was completely assembled to form the corona and only detected in the AuNP precipitates (Lanes F–G).To investigate the assembly efficiency and photoreactivity of the PC linker, we annealed the sgRNA, PC linker and SH‐DNA in an equal stoichiometric ratio. The appearance of one distinct band of expected mobility indicates successful assembly of the three oligonucleotide strands . With irradiation , the sgRNA was completely dissociated from the assembly , suggesting the photoreactivity of the PC linker.We evaluated the light‐activated release and control of the RNP by monitoring the enzymatic cleavage of a 702 bp dsDNA fragment of the EGFP gene (enhanced green fluorescence protein). Our results  confirmed the light‐activated release of RNP and the controllable activation of the Cas9‐sgRNA activity. The released RNP retained its nuclease activity to cleave the target DNA, with a comparable nuclease activity to that of the free RNP .To test whether cellular uptake of the binding‐mediated RNP corona can be rationally modulated through control of density of the RNP outer layer, we prepared a set of the RNP coronae using different Cas9 to sgRNA‐AuNP ratios, 1:1, 3:1, 5:1, 10:1, 15:1, 20:1, and 25:1, and determined their uptake in Hela cells. We observed that the cellular uptake of the RNP coronae increased with increasing Cas9 to sgRNA‐AuNP ratio Figure . BecauseWe reasoned that the efficient cellular uptake of the RNP corona was because of the enhanced interaction between the RNP corona outer layer and the cell membrane. We tested this hypothesis by tracking the cellular uptake of the RNP corona using TEM. TEM images show that the RNP coronae were localized on the membrane of Hela cells after 15 min of incubation Figure . With prTo demonstrate that the enhanced cellular uptake of RNP corona is not due to the AuNP core, we used magnetic nanoparticles (MNPs) as the scaffold to construct another binding‐mediated RNP corona and determined its cellular uptake. Similarly, enhanced cellular uptake was observed with increasing Cas9 to MNP ratio . Therefore, efficient cellular uptake of the RNP corona is attributed to the RNP outer layer that enhanced the interaction between the RNP corona and the cell membrane.3), a chemical reagent to disrupt the ATP production in cells and inhibit endocytosis,We then investigated a possible mechanism of cellular uptake of the binding‐mediated RNP corona. Treatment with sodium azide  and tracked its intracellular localization using confocal microscopy. The RNP coronae were mostly co‐localized with endosomes/lysosomes within 1 h of incubation, and then escaped from endosomes/lysosomes after incubation for 5 h . The stability of the RNP corona in the RNase A solution was also improved compared to the stability of a RNA reporter, and the stability of the RNP corona increased with increasing Cas9 to sgRNA‐AuNP ratios . These results suggest that the corona protected sgRNA from degradation, a benefit for controlled gene editing over an extended period of time. We also examined the stability of RNP corona in the presence of 1–10 mM glutathione (GSH), representing the higher end of intracellular GSH concentrations. Our results show that RNP corona remained stable in the presence of 1–10 mM GSH .Encouraged by the responsive release property and enhanced uptake efficiency of the RNP corona, we evaluated the gene editing potential of the activated RNP in Hela‐GFP cells. Successful editing of the EGFP transgene resulted in a loss of fluorescence, which was monitored using confocal microscopy and flow cytometry Figure . We incuWe also used the T7 endonuclease I (T7E1) mismatch cleavage assay to determine the editing efficiency of two genes in two cell lines Figure , which sWe extended application of the RNP corona system to gene editing in the HEK293 cells Figure , demonstIn summary, we have developed a binding‐mediated protein corona (BMPC) system for efficient delivery of functional CRISPR machinery and light‐activated genome editing. The RNP corona, which was formed with AuNPs, oligonucleotide ligand, and the sgRNA‐Cas9 ribonucleoprotein, protected the sgRNA from degradation and enhanced the delivery of the RNP into the cells. The RNP corona system exhibited low cytotoxicity to the Hela cells and maintained the nuclease activity of the RNP for gene editing. Incorporation of photocleavable linkers into the system enabled controlled activation of the sgRNA‐Cas9 RNP, resulting in photo‐responsive gene editing in human cells. In addition, because the Cas9 protein is not directly adsorbed onto the NPs, there is flexibility for encapsulating functional molecules, such as drugs and imaging probes, for therapeutic and biosensing applications. The BMPC strategy in combination with photo‐activation improves the delivery of RNP, offers optical control, and has potential for decreasing off‐target effects of the CRISPR‐Cas9 technology.The BMPC technique has three appealing features: tunable outer layer density and property, minimized irreversible interaction of the payload proteins with the core of NPs, and reduced leakage of the payload proteins. These features make BMPC distinct from other techniques using NPs as carriers for delivering the RNP. Others used nonspecific interactions to load RNP onto AuNPs and/or included coating of an additional polymer layer, and the resulting RNP carriers could not afford uniform assembly of oriented Cas9 or tunable cellular uptake or controllable release. BMPC is advantageous for efficient and controllable delivery of the CRISPR‐Cas9 RNP system and is promising for delivery of nucleic acids and proteins with a high efficiency, stability, and activity.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Medical schools are increasingly introducing technical clinical skills training from year 1. However, little research has determined students’ views of such training. This study compared the perceptions of student groups which received different levels of technical skills training during the early years of their undergraduate medical degree.n = 184) receiving 48 h of technical skills teaching and Phase curriculum (n = 94), receiving 12 h, voluntarily participated. A mixed methods design using a questionnaire and focus groups explored students’ views. Stage and Phase student questionnaire responses were compared using Mann Whitney U tests. Focus group transcripts underwent thematic analysis.Medical students from King’s College London’s Stage curriculum (n = 169) and Phase (n = 68) students identified year 1 as the best time to commence technical skills training. For the majority of the technical skills taught, Stage compared to Phase students reported feeling more prepared to perform them. Thematic analysis identified three main themes: Role of technical skills teaching in the early stages of medical education, impact on students’ learning and factors to consider when designing a medical undergraduate technical clinical skills programme.The majority of Stage  contains supplementary material, which is available to authorized users. Many undergraduate medical programmes introduce students to the clinical environment and the requisite skills during the early stages of training, moving away from the traditional model of distinct pre-clinical and clinical years. The term ‘clinical skills’ encapsulates a variety of abilities including history taking, physical examination, practical and technical skills. In the absence of clear definitions, a number of skills could be categorised as more than one of these terms . For exaResearch in relation to clinical skills training in the pre-clinical years has predominantly focused on human factors and the practicalities of physical examination through the use of patient contact. These experiences are well received by students –4 and haIt may be postulated that initiating technical skills teaching in the first year of medical training provides students a longer time frame during which they can practise and develop these skills which, in turn, may be expected to result in increased confidence and preparedness to perform them in clinical practise. However, given the limited physiological and clinical knowledge of students at the start of their medical education, commencing training at this stage may have little benefit or relevance to the students. Research regarding students’ perceptions of early technical skills training and the potential impact on their perceived ability to perform the skills is currently lacking.. The Stage programme comprises of 48 h of this teaching during year 1 compared to the 12 h provided to students over the first 2 years of the Phase programme.The undergraduate medical curriculum at King’s College London (KCL), UK, has undergone significant development culminating in the formation of the Stage Curriculum to replace the original Phase programme. One significant change has been the introduction of a technical clinical skills module in the first year of medical school. Based upon guidance from the GMC , studentThe present study aimed to explore student perceptions of technical skills training in the early stages of their medical education by comparing the views of two student groups with different levels of such training.A total of 184 year 1 students undertaking the Stage curriculum and 94 year 2 students undertaking the Phase curriculum voluntarily participated in the study. Students were recruited using e-mails to their student accounts inviting them to participate in the online survey and/or a focus group. A total of three recruitment e-mails were sent over a 2-week period.The study was conducted on the KCL medical school campus during April 2017 prior to student summative examinations. Year 1 students on the Stage curriculum had received 48 h of predominantly face-to-face teaching on technical skills considered appropriate for their level of training by the GMC Only two Phase students explicitly reported that a desire to learn the skills regardless of examination purposes was a motivating factor.n = 2) and Phase (n = 6) students raised concerns regarding the ability to retain the technical skills knowledge acquired during their first year over the long term:A minority of Stage  and Phase (n = 14) students cited that opportunities to practise the technical skills, both in sessions and throughout the medical curriculum, were an important component of such skills training, primarily to prevent skills being forgotten.The majority of Stage (n = 13) and Phase (n = 7) students as important. Stage students predominantly reported feedback to be beneficial in preventing the development of ‘bad habits’ prior to conducting the skills in the clinical environment. Phase students identified feedback as relevant with regard to aiding their preparation for upcoming examinations.Knowledgeable, engaging tutors and standardised teaching with clear instructions were considered by both groups as key factors when delivering clinical skills sessions. The opportunity to have constructive informal feedback on their performance during sessions was specifically highlighted by Stage


Bacterial colonization of the rhizosphere is critical for the establishment of plant–bacteria interactions that represent a key determinant of plant health and productivity. Plants influence bacterial colonization primarily through modulating the composition of their root exudates and mounting an innate immune response. The outcome is a horizontal filtering of bacteria from the surrounding soil, resulting in a gradient of reduced bacterial diversity coupled with a higher degree of bacterial specialization towards the root. Bacteria–bacteria interactions (BBIs) are also prevalent in the rhizosphere, influencing bacterial persistence and root colonization through metabolic exchanges, secretion of antimicrobial compounds and other processes. Traditionally, bacterial colonization has been examined under sterile laboratory conditions that mitigate the influence of BBIs. Using simplified synthetic bacterial communities combined with microfluidic imaging platforms and transposon mutagenesis screening approaches, we are now able to begin unravelling the molecular mechanisms at play during the early stages of root colonization. This review explores the current state of knowledge regarding bacterial root colonization and identifies key tools for future exploration. In contrast, P. putida lapA mutants show reduced attachment to abiotic surfaces and corn seeds and are defective for biofilm formation at 1‐hpi implying that LapA plays a role in the initial adhesion of P. putida  or capsular polysaccharide  play a role in both the primary and secondary attachment of diverse bacterial species. EPS is a major cell surface component composed of carbohydrate polymers, which promote cellular aggregation and irreversible binding to the root surface by forming bridges between bacterial cells (Burdman 2+ and Mg2+) and water availability  glucomannan mediates localized polar attachment of R. leguminosarum to pea and vetch root hairs through binding to plant lectins  mediated defence displayed distinct microbiome composition relative to wild‐type plants  sites located downstream of the highly conserved chromosomal glmS gene  operon, permitting crude quantification of N2 fixation. The ability to screen for elite competitiveness in large libraries of bacterial strains while simultaneously monitoring the expression of a biochemical marker will be revolutionary for the high‐throughput identification of agricultural inoculants.Competition between bacteria during colonization can alternatively be monitored by barcoding with oligonucleotides. In a recent example, 84 barcoded strains of et al., et al., et al., et al., et al., B. subtilis towards the root elongation zone (REZ) of Arabidopsis preceded colonization over the entire root length. This indicates that the REZ is a hotspot for initial bacterial root interactions, likely due to high concentrations of root exudates and that bacterial chemotaxis and motility towards these exudates is a prerequisite for root colonization  chip, which facilitates the growth of roots up to 800 μm wide and was successfully used to study bacterial root interactions with Aspen and Rice seedlings  is one such approach. TIS is a powerful technique whereby libraries of single‐insertion transposon mutants, which collectively saturate an organism's genome, are exposed to a specific condition and then analysed with next‐generation sequencing to simultaneously estimate the essentiality and/or fitness contribution of each gene in a bacterial genome. Comparison of gene mutation frequency in an ‘input pool’ relative to an ‘output pool’ following a challenge such as root colonization reveals whether a gene is essential, non‐essential, advantageous, or disadvantageous for growth and survival Fig. . There aRhizobium‐legume symbiosis at multiple stages of its development  free‐living growth in the rhizosphere, (ii) colonization of the root, (iii) nodule infection before differentiation into N2 fixing bacteroides and (iv) terminal differentiation into N2 fixing bacteroides  was recently used to identify bacterial genes important in the et al., en masse, resulting in the annotation of over 11 000 previously undefined protein‐coding genes  is an extension of TIS techniques in which the transposable element contains a unique, but random, 20 nucleotide DNA ‘barcode’, so that each individual transposon mutant within a pool is identifiable by sequencing (Wetmore Bacterial colonization of plant roots is a sequential, multi‐step process that begins in the rhizosphere with chemotaxis towards the root, followed by attachment and subsequent biofilm formation. To date, most genetic determinants involved in bacterial colonization have been evaluated in single‐strain studies under sterile conditions. Here we have highlighted that both PBIs and BBIs can influence bacterial colonization but characterizing their molecular mechanisms has been hindered due to the enormous scale of complexity and diversity of root‐associated microbiota. With the advent of new DNA‐integration systems and fluorescent reporters, marked seven‐member bacterial SynComs have been successfully cultivated offering unprecedented advancement towards deciphering the underlying principles of bacterial assembly in root niches. Moving forward, genetic exploration of SynComs using transposon mutagenesis‐based screening approaches will be instrumental in unveiling the novel genetics at play. Overall, a more thorough understanding of PBIs and BBIs will advance our understanding of bacterial ecological processes and be invaluable to optimize the future development of biofertilisers for sustainable agriculture, whether this be through targeted selection or engineering of elite plant growth‐promoting bacteria.


Accuracy of growth monitoring did not improve. Prevalence of stunting, underweight and inadequate interval growth at 6‐months corrected age decreased significantly more in the intervention clinics than in control clinics. Results suggest that integrating mHealth nutrition interventions is feasible and can improve child nutrition outcomes. Improved tool design may better promote accuracy.Infants born preterm, low birthweight or with other perinatal complications require frequent and accurate growth monitoring for optimal nutrition and growth. We implemented an mHealth tool to improve growth monitoring and nutritional status assessment of high risk infants. We conducted a pre–post quasi‐experimental study with a concurrent control group among infants enrolled in paediatric development clinics in two rural Rwandan districts. During the pre‐intervention period (August 2017–January 2018), all clinics used standard paper‐based World Health Organization (WHO) growth charts. During the intervention period (August 2018–January 2019), Kirehe district adopted an mHealth tool for child growth monitoring and nutritional status assessment. Data on length/height; weight; length/height‐for‐age (L/HFA), weight‐for‐length/height (WFL/H) and weight‐for‐age (WFA) z‐scores; and interval growth were tracked at each visit. We conducted a ‘difference‐in‐difference’ analysis to assess whether the mHealth tool was associated with greater improvements in completion and accuracy of nutritional assessments and nutritional status at 2 and 6 months of age. We observed 3529 visits. mHealth intervention clinics showed significantly greater improvements on completeness for corrected age (endline: 65% vs. 55%;  We evaluated whether using an mHealth tool can improve completeness and accuracy of growth monitoring and nutritional status assessment among at‐risk infants enrolled in a paediatric development clinic in rural Rwanda.Integrating the mHealth tool for growth monitoring and nutritional interventions was feasible and was associated with significantly greater improvements in completeness of growth monitoring but not improvements in growth monitoring accuracy.The mHealth tool was associated with reductions of malnutrition at 6 months suggesting that mHealth tools can help improve nutritional outcomes among high‐risk infants; however, additional design changes may be required to promote accuracy.1Globally, tremendous improvement has been made during the past decades in newborn care and reduced neonatal mortality and morbidity, especially in low‐ and middle‐income countries  growth charts are often used to assess child growth through calculation of z‐scores, including weight‐for‐length/height (WFL/H), weight‐for‐age (WFA), length/height‐for‐age (L/HFA) and head circumference‐for‐age among children under five  to provide structured follow up for high‐risk infants including infants born premature, LBW, or with other perinatal complications. The PDC programme is integrated into district hospitals and primary‐level community health centres to help high‐risk infants survive and achieve their full potential by providing comprehensive health, nutritional and developmental care with prevention, early detection of risks or complications and provision of specific interventions to identified problems , cleft lip/palate and trisomy 21. Other reasons for admission include developmental delay, hydrocephalus and infants aged less than 12 months following hospitalization for severe acute malnutrition. These high‐risk infants are followed up until age five.Details on PDC programme and protocol have been described elsewhere  and post‐intervention periods (August 2018–January 2019), regardless of the child's age. We assessed nutritional status at age 2 and 6 months (corrected for prematurity) among infants less than 6 months who enrolled in PDCs in Kirehe and Kayonza districts during the study period (August 2017–January 2019). Although is it possible for children to have their growth assessed at other ages, for example, if they present for a nonemergency acute illness, these ages were chosen purposively because they presented early growth (2 months) and the oldest age possible with our dataset (6 months) as a measure of longer term follow‐up. In addition, PDC programme systematically conducts growth assessments at ages 2 and 6 months such that we could expect to have complete data for most infants enrolled in the programme for at least 2 or 6 months at these ages. We included infants who have been enrolled before 2 months of age (corrected for prematurity) and had at least one PDC visit before or after 1 month of the target age for nutritional assessment (2 and 6 months). For all analyses, we excluded any post‐intervention visits from infants who initially enrolled during pre‐intervention period, but whose treatment continued during intervention period. This exclusion criterion was selected to avoid contamination of study groups. Our retrospective analysis included data on all eligible infants during the study period.2.4An mHealth tool was developed by Partners In Health/Inshuti Mu Buzima in collaboration with DTree International and ThingsPrime to support PDC health providers in conducting growth and nutritional assessments of PDC patients with the goal of improving early identification of growth failure and supporting provider decision making and counselling aligned with the protocol for the PDC programme. The mHealth tool is an android tablet/smartphone‐based application built using MangoLogic software with algorithms for growth monitoring and decision support that are based on the 2006 WHO Child Growth Standards and additional clinical information, including gestational age, based on PDC protocol for nutritional management of high risk infants. The tool is automated to plot child's anthropometric z‐scores and to calculate interval weight gain and automatically provides quick growth classification represented using colour‐coded visual pictures . The tool provides final decision on nutritional status and guiding messages for the health provider to plan nutritional interventions for the child. The function for mHealth tool to record entries was deactivated, and the tool does not track patient identifiers and entered measurements.To use the tool, the health care provider enters the patient's anthropometric data (length/height and weight), gestational age at birth and birthdate into the mHealth tool. The mHealth tool is programmed to calculate chronological age and corrected age for patients born premature (<37 weeks), as well as WFL/H, WFA and L/HFA z‐scores, and interval growth (average daily weight gain since last measurement). To aid interpretation of the calculations, we programmed the tool to determine the nutritional status of the patient according to the PDC protocol and provide a counselling message related to the patient's nutritional status, such as encouraging praise and counselling on play and communication when a child is growing well. The PDC provider uses the results and guiding messages provided by the mHealth tool to develop individualized nutrition care plans for the child and provides face to face nutritional counselling to the parents to address any specific concerns using additional counselling support in the PDC job aid.Prior to disseminating the tool to PDC health care workers, the mHealth tool went through several rounds of testing by Partners In Health/Inshuti Mu Buzima programme staff to validate the recommendations from the tool against the PDC protocol and standard WHO growth charts. The Android‐supported mHealth tool was installed onto tablets. Seven PDC health providers  from three health facilities with PDC in Kirehe district received a 2‐day training to learn how to use the tablet and tool through several case study‐based practical exercises. All health providers had experience in using personal android smartphones. Following the training, one tablet per health facility was distributed to each Kirehe district PDC and was primarily used by four trained nurses during clinical consultation. Health providers used the mHealth tool to conduct nutritional assessments and documented results from the tool  on hard copy clinic visit forms that were later entered into an electronic medical record (EMR). Health providers received on‐going mentorship on the use of the tool in the clinics by trained nutritionist and PDC programme manager from Partners In Health/Inshuti Mu Buzima as part of routine clinic mentorship. Nurses in the Kayonza district PDCs continued to use paper‐based growth monitoring. In our study sites, there was no additional formal training on the use of paper growth charts and interpretation, beyond overall PDC protocol training, which was attended by all PDC health care providers in in September 2017.2.5In both intervention and control sites, data were extracted from the EMR, which uses an OpenMRS system to record patients' information. Nurses routinely documented the clients' clinical information in patients' paper files, and that information was entered into the EMR by trained data officers within 1 week of the visit. Data on sociodemographic characteristics, anthropometric measurements, nurse‐reported child growth z‐scores , and nutritional status for the infants meeting inclusion criteria were extracted from the EMR. Data were deidentified prior to analysis. Monthly routine data quality assessments and validations are conducted regularly by district EMR coordinators with immediate correction of errors identified, and data were assessed for implausible values during the analysis process.2.6Data from the EMR records were assessed for plausibility and extreme values were flagged as data entry errors, investigated, and corrected, whenever possible. Our primary unit of analysis was PDC visits, which were cross‐classified into four exposure groups based on whether they occurred at either control vs. intervention sites during either the pre‐intervention or post‐intervention periods. The four exposure groups were (1) control pre‐intervention, (2) control post‐intervention, (3) intervention pre‐intervention and (4) intervention post‐intervention. We described the characteristics of PDC visits across exposure groups by calculating proportions for categorical variables and medians and interquartile ranges (IQRs) for continuous variables. A child was defined as preterm if their gestational age at birth was less than 37 weeks or gestational age was missing but the child was referred to the PDC clinic as preterm. Corrected age was calculated for any preterm infant with a documented gestational age as the infant's chronological age minus the number of weeks born premature, where weeks born early is defined as 40 weeks minus gestational age in weeks. LBW was defined as weight at birth less than 2500 g, though criteria for enrolment in PDCs match the criteria for referral of an infant born LBW to a hospital inpatient neonatal unit, which is less than 2000 g. Small for gestational age (SGA) was defined as birthweight less than the 10th percentile for gestational age and was calculated using INTERGROWTH‐21st standards . Stunting was defined as L/HFA z‐score of < −2; wasting was defined as WFL/H z‐score < −2; and underweight was defined as WFA z‐score < −2. Interval growth was categorized based on PDC protocol as adequate or in adequate depending on the child's corrected age Table . This anTo test whether intervention sites experienced greater improvements in growth monitoring completion, growth classifications accuracy and infants' nutritional status over time relative to control sites, we used a linear regression to conduct a ‘difference‐in‐difference’ analysis , L/HFA z‐score assessment , the WFA z‐score assessment  as well as the WFL/H z‐score assessment  had improved significantly more at intervention sites compared with control sites.Table The concordance of classification of the child's nutritional status and interval growth by health care providers using either mHealth tool or paper‐based WHO growth charts and gold standard calculations with Stata is described in Table p = 0.041), in pre‐ to post‐intervention, respectively. Underweight decreased from 47% to 33% in intervention clinics and increased from 30% to 53% in comparison clinics (p = 0.014). The proportion of infants with inadequate interval growth fell from 59% to 26% in intervention clinics, whereas it increased from 52% to 56% in comparison clinics (p < 0.001). In addition, the prevalence of wasting declined from 19% to 10% in intervention clinics, whereas it increased from 18% to 21% in comparison clinics, though the difference was not statistically significant (p = 0.366).Table 4The results of this study demonstrate a strong association between the introduction of an mHealth tool and improvements in the completeness in growth monitoring among care providers. The use of mHealth tool was also associated with significantly greater reductions in malnutrition among 6‐month old infants at sites receiving the mHealth intervention compared with those at sites not receiving the intervention. However, we did not find any improvements in accuracy associated with the introduction of the mHealth tool.In our study, there was a significant improvement of level of completeness at intervention clinics. The mHealth tool provides automatic calculation of corrected age, anthropometric z‐scores, and interval growth with a quick representation of classification of child nutritional status and guiding messages for clinical decisions. These features may have motivated the nurses to complete all aspects of the nutritional assessment, especially for harder‐to‐calculate growth classifications. The nurses in the PDCs reported that they appreciated the use of the mHealth tool, suggesting that the implementation of this tool may be feasible elsewhere. A similar finding is found in a study done in New Zealand where introduction of use of electronic growth charts at hospital was found to increase the rates of recording growth measurements and body mass index z‐scores (Dainty et al., Even though the accuracy of the nurses' documented growth classifications did not improve, the improvement in the completeness of nutritional assessments at the same level of accuracy may have been sufficient to generate meaningful improvements in quality of care. More complete growth monitoring may have increased the number of parents who received targeted nutritional counselling. We did not observe any improvements in the prevalence of malnutrition among infants at age 2 months, perhaps because it can take several months of intervention to improve nutritional status. However, we did observe significantly larger reductions in the prevalence of malnutrition among infants at age 6 months in clinics with the mHealth intervention compared with clinics without the intervention. This finding suggests that, even in the absence of improved accuracy, the increased completion associated with the mHealth intervention may have been sufficient to improve nutritional outcomes. This finding also suggests that quality improvements in nutritional interventions such as the PDC program may take several months to result in observable changes in nutritional status.Our intervention was not associated with an improvement in growth monitoring accuracy. Improving growth monitoring interpretation accuracy has been found to be difficult in other studies (Mutoro & Wright, This study has some limitations. The two pre‐ and post‐intervention groups are unmatched; however, the population and care providers in both comparison and intervention clinics serve similar populations and had generally the same characteristics. The key difference in the PDC programmes in these two districts during this study period was the extensive decentralization that occurred between the pre‐ and post‐intervention period in the intervention district. Although we cannot rule out that the significantly greater improvements associated with the introduction of an mHealth intervention reflect processes related to this decentralization rather than a true effect of the mHealth intervention, in general, we expect the quality of care delivered at health facilities that are developing new PDC clinics to be not as good as the quality of care delivered at hospitals. Therefore, we do not believe our results can be fully explained by bias due to the concurrent decentralization in the intervention district. We were not able to control other possible interventions from the Rwandan Government such as trainings provided to care providers, which may have differed between the intervention and control districts. However, as we collaborate closely with the facilities, we are not aware of any specific growth monitoring trainings for staff. In addition, we are unable to know whether the mHealth tool was used during PDC visits at the intervention sites as the tool does not track uses or record measurements; however, through qualitative feedback, the tool is very valued by providers, and we assume that it is used most of the time. Additional formal qualitative evaluation feedback may shed more light on this. Lastly, we used data that are routinely documented on patient charts by care providers and then entered into the EMR system, which led to some errors in recording of measurements and missing data. Despite regular data quality assessments by district EMR coordinators and identification and correction of implausible values during the analysis process, we were unable to verify the accuracy of raw measurements of weight, length/height and age that were recorded by the nurses and used to calculate the WHO standard growth classifications in our accuracy analyses.5The introduction of an mHealth tool was associated with significantly greater improvements in the completeness of nutritional assessments and nutrition status of infants at 6 months of age in intervention clinics. Although the accuracy of growth monitoring did not change, these findings suggest that integrating mHealth in providing nutrition interventions is feasible and may help in improving child nutrition outcomes. Further work is needed in improving the intervention with integrated digital systems with mHealth tool connected with EMR system and in improving the design of the tool with precise visibility and determination of z‐score. To reduce the level of inaccuracy, more research is needed to develop good training programmes to improve interpretation of growth charts as well as providing mentorship.The authors declare that they have no conflicts of interest.MN, ACK, SU and VB designed the study, interpreted results and drafted the manuscript. DAB provided methodologic assistance on study design, conducted statistical analysis and critically reviewed the manuscript. KB and CMK designed the study, supported interpretation of results, analysis and critical reviews of the manuscript. AN, KW, JB, SH, AU and EN contributed to results interpretation and manuscript revision. All authors have read and approved the final manuscript.ClinicalTrials.gov Identifier: NCT03665012.Data S1. Supporting informationClick here for additional data file.Figure S1. Selected screenshots of the PDC mHealth Tool after ImprovementClick here for additional data file.