Biology subjects

Fernandez, L. A.

Publications and source records attributed to Fernandez, L. A..

6 recordsLinked to original sources

bioRxiv2024

Synthetic bacteria with programmed cell targeting and protein injection suppress tumor growth in vivo

Bacterial living therapeutics (BLTs) hold promise for treating cancer and other human diseases because they can be engineered and transported into the microbiota (e.g., of tumors, gastrointestinal tract) to deliver therapeutic payloads. Current approaches rely on the natural tropism of the bacterial chassis used and trigger the local release of protein cargoes, typically through active extracellular secretion or bacterial lysis. BLTs capable of targeting specific cellular subsets and delivering payloads intracellularly might provide new therapeutic opportunities and improve efficacy while reducing off-target effects. We used synthetic biology to develop BLTs that can deliver defined cargo proteins into the cytoplasm of target cells. We designed a modular synthetic bacterium with programmed adhesion to cells by targeting defined cell surface antigen and armed with an inducible type III secretion system (T3SS) for injection of a protein cargo of interest. As a proof of principle, we programmed synthetic bacteria to recognize the epidermal growth factor receptor (EGFR) and inject the catalytic fragments of the potent adenosine diphosphate-ribosyltransferase toxins ExoA and TccC3. These BLTs demonstrated the ability to trigger robust tumor cell death in vitro. Intratumoral administration of these synthetic bacteria suppressed tumor growth in vivo and prolonged the survival of treated animals when the tumor cells were recognized by the engineered bacteria. These results demonstrate the potential of programming cell targeting and controlled protein injection for the development of effective and specific BLTs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/590337v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eb769dorg.highwire.dtl.DTLVardef@b84b39org.highwire.dtl.DTLVardef@152b8b0org.highwire.dtl.DTLVardef@1db4108_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

microbiology↗

bioRxiv2021

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

In vivo evolution of antibodies facilitates emergence of novel target specificities from pre-existing clones. In this work we show how mutagenic ssDNA recombineering of camel-derived nanobodies encoded in a bacterial genome enables clonal hyper-diversification and the rise of new properties. As a proof-of-principle we used a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) and evolved it towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying on their surface this nanobody fused to the intimin outer membrane anchor domain were subjected to multiple rounds of mutagenic ssDNA recombineering targeted to the CDR1, CDR2 and CDR3 regions of its genomically encoded VHH sequence. Binders to the new antigen (EPEC TirM) were then selected upon immunomagnetic capture of bacteria bearing the corresponding nanobody variants. As a result, several modified nanobodies were identified which maintained recognition of EHEC TirM but acquired the ability to bind the new antigen with high affinity (Kd ~20 nM). The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Our experimental workflow empowers the evolution of nanobodies displayed on the surface of bacterial cells for a large number of potential applications in medical and industrial biotechnology.

synthetic biology↗

bioRxiv2020

Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

molecular biology↗

bioRxiv2019

In vivo diversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

Diversification of specific DNA segments typically involve in vitro generation of large sequence libraries and their introduction in cells for selection. Alternative in vivo mutagenesis systems on cells often show deleterious offsite mutations and restricted capabilities. To overcome these limitations, we have developed an in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. The transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP elongation. While the efficiency of this platform is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.

bioengineering↗

bioRxiv2018

A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

synthetic biology↗