Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism.

Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure-activity relationship study of Z22, a d-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, d-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of Z22 proved non-essential and was removed to yield the more active analog Z34. This dendrimer shows >83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade.

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended ß-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation.

In the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.

Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays.

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.

Deep learning guided image-based droplet sorting for on-demand selection and analysis of single cells and 3D cell cultures.

Uncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures. Here we demonstrate that deep neural networks can accurately classify single droplet images in real-time based on the presence and number of micro-objects including single mammalian cells and multicellular spheroids. This approach also enables the identification of specific objects within mixtures of objects of different types and sizes. The training sets for the neural networks consisted of a few hundred images manually picked and augmented to up to thousands of images per training class. Training required less than 10 minutes using a single GPU, and yielded accuracies of over 90% for single mammalian cell identification. Crucially, the same model could be used to classify different types of objects such as polystyrene spheres, polyacrylamide beads and MCF-7 cells. We applied the developed method for the selection of 3D cell cultures generated with Hek293FT cells encapsulated in agarose gel beads, highlighting the potential of the technology for the selection of objects with a high diversity of visual appearances. The real-time sorting of single droplets was in-line with droplet generation and occurred at rates up to 40 per second independently of image size up to 480 × 480 pixels. The presented microfluidic device also enabled storage of sorted droplets to allow for downstream analyses.

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics.

Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 °C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

Peptide Dendrimer-Lipid Conjugates as DNA and siRNA Transfection Reagents: Role of Charge Distribution Across Generations.

Transfection reagents are used to deliver DNA and siRNA into cells to achieve genetic manipulations, and may ultimately enable nonviral gene therapy. Progress in transfection reagents is limited by the fact that such reagents cannot be easily optimized due to their polymeric nature and/or difficult synthesis. We have developed a new class of well-defined and easily modifiable transfection reagents in the form of peptide dendrimers. These dendrimers self-assemble with DNA or siRNA and lipofectin to form nanoparticles which efficiently enter mammalian cells and liberate their nucleic acid cargo. By systematically modifying the amino acid sequence of our dendrimers we have found that their transfection efficiency depends on the distribution of positive charges and hydrophobic residues across the dendrimer branches. Positive charges present in all three generations lead to efficient DNA delivery, whereas siRNA delivery requires charges in the outer two generations combined with a hydrophobic dendrimer core.

Efficient Transfection of siRNA by Peptide Dendrimer-Lipid Conjugates.

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure-activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure-activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future.

Combinatorial Synthesis of Structurally Diverse Triazole-Bridged Flavonoid Dimers and Trimers.

Flavonoids are a large family of compounds associated with a broad range of biologically useful properties. In recent years, synthetic compounds that contain two flavonoid units linked together have attracted attention in drug discovery and development projects. Numerous flavonoid dimer systems, incorporating a range of monomers attached via different linkers, have been reported to exhibit interesting bioactivities. From a medicinal chemistry perspective, the 1,2,3-triazole ring system has been identified as a particularly attractive linker moiety in dimeric derivatives (owing to several favourable attributes including proven biological relevance and metabolic stability) and triazole-bridged flavonoid dimers possessing anticancer and antimalarial activities have recently been reported. However, there are relatively few examples of libraries of triazole-bridged flavonoid dimers and the diversity of flavonoid subunits present within these is typically limited. Thus, this compound type arguably remains underexplored within drug discovery. Herein, we report a modular strategy for the synthesis of novel and biologically interesting triazole-bridged flavonoid heterodimers and also very rare heterotrimers from readily available starting materials. Application of this strategy has enabled step-efficient and systematic access to a library of structurally diverse compounds of this sort, with a variety of monomer units belonging to six different structural subclasses of flavonoid successfully incorporated.

Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13.

The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.

Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.

Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease.

Mutation of the tobacco etch virus (TEV) protease nucleophile from cysteine to serine causes an approximately ∼104 -fold loss in activity. Ten rounds of directed evolution of the mutant, TEVSer , overcame the detrimental effects of nucleophile exchange to recover near-wild-type activity in the mutant TEVSer X. Rather than respecialising TEV to the new nucleophile, all the enzymes along the evolutionary trajectory also retained the ability to use the original cysteine nucleophile. Therefore the adaptive evolution of TEVSer is paralleled by a neutral trajectory for TEVCys , in which mutations that increase serine nucleophile reactivity hardly affect the reactivity of cysteine. This apparent nucleophile permissiveness explains how nucleophile switches can occur in the phylogeny of the chymotrypsin-like protease PA superfamily. Despite the changed key component of their chemical mechanisms, the evolved variants TEVSer X and TEVCys X have similar activities; this could potentially facilitate escape from adaptive conflict to enable active-site evolution.

In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

Ultrarapid generation of femtoliter microfluidic droplets for single-molecule-counting immunoassays.

We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10(6) per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

Peptide dendrimer/lipid hybrid systems are efficient DNA transfection reagents: structure--activity relationships highlight the role of charge distribution across dendrimer generations.

Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure-activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6-10-fold over commercial reagents under their respective optimal conditions. Emerging structure-activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity.

A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets.

The enormous reduction of assay volume afforded by compartmentalization into picolitre water-in-oil droplets is an exciting prospect for high-throughput biology. Maintaining the activity of encapsulated proteins is critical for experimental success, for example in in vitro directed evolution, where protein variants are expressed in droplets to identify mutants with improved properties. Here, we present a simple and rapid method to quantitatively compare concentrations of fluorescent molecules in microdroplets. This approach allows an assessment of different emulsification procedures and several oil/surfactant mixtures for biochemical compatibility, in particular in vitro protein expression. Based on determining droplet fluorescence vs. droplet diameter, the method uses the gradient of such curves as a 'concentration correlation coefficient' (CCC) that is directly proportional to fluorophore concentration. Our findings suggest that generation of droplets using a microfluidic flow-focusing device gave no more protein expression than droplet production by the bulk methods of vortexing and homogenizing. The choice of oil/surfactant, however, was found to be critical for protein expression and even encapsulation of purified protein, highlighting the importance of careful selection of these components when carrying out biochemical experiments in droplets. This methodology will serve as a quantitative test for the rapid optimization of droplet-based experiments such as in vitro protein expression or enzymatic assays.

SNAP display: in vitro protein evolution in microdroplets.

SNAP display is based on the covalent reaction of the DNA repair protein AGT (O(6)-alkylguanine DNA alkyltransferase, the "SNAP-tag") with its substrate benzylguanine (BG). Linear, BG-labelled template DNA is encapsulated in water-in-oil emulsion droplets with a diameter of a few micrometres (i.e. 1 mL of emulsion contains ∼10(10) compartments). Each droplet contains only a single DNA copy, which is transcribed and translated in vitro. The expressed AGT fusion proteins attach to their coding DNA via the BG label inside the droplet, which ensures that a specific genotype-phenotype linkage is established. Subsequently, the emulsion is broken and protein-DNA conjugates, which constitute a DNA-tagged protein library, selected via affinity panning. This method will prove a useful addition to the array of in vitro display systems, distinguished by the stability of DNA as the coding nucleic acid and the covalent link between gene and protein.