Biology subjects

Portell, A.

Publications and source records attributed to Portell, A..

5 recordsLinked to original sources

bioRxiv2026

Receptor-guided AAV Tropism Engineering via MATCH

Precise control over viral tropism remains a major challenge in the development of gene delivery technologies. We present MATCH (Modulation of AAV Tropism through Conjugation to Homing proteins), a modular biochemical method that enables programmable, receptor-guided retargeting of adeno-associated viruses (AAVs) through site-specific covalent protein conjugation. By incorporating a SpyTag peptide motif into selected AAV capsid loops, MATCH allows one-step, stoichiometrically defined attachment of recombinant SpyCatcher-linked targeting proteins to the viral surface. Using mosaic AAV-DJ and AAV9 capsids with controlled SpyTag incorporation, we achieve efficient assembly and tunable ligand display. MATCH-AAVs conjugated to an anti-CD3 single-chain antibody efficiently activate and transduce resting human T cells within mixed PBMC populations in vitro, achieving transduction levels of up to [~]58% of total PBMCs. Conjugation to transferrin receptor (TfR1)-binding proteins yielded enhanced brain transduction in vivo, with murine TfR1-targeted MATCH-AAV9 exhibiting up to an 84-fold increase in brain expression relative to wild-type AAV9. Human TfR1-targeted vectors similarly enabled robust, receptor-dependent transduction both in vitro and in humanized mouse models. Both TfR-targeted vectors enabled widespread transduction of the parenchyma, consistent with TfR1-mediated crossing of the blood-brain barrier. Finally, we establish a streamlined one-pot "Mix-and-MATCH" production strategy in which capsid and targeting ligands are co-expressed during vector generation, yielding functional, targeted AAVs at titers comparable to conventional production. This simple and generalizable synthetic-biology approach provides a versatile toolkit for rational AAV tropism engineering, offering a scalable route to custom vector design for research and therapeutic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/715691v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1935815org.highwire.dtl.DTLVardef@7c05faorg.highwire.dtl.DTLVardef@6c8857org.highwire.dtl.DTLVardef@16b2097_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Regulatable In Vivo Gene Expression via Adaptamers

Precise, reversible control of transgene expression is essential for safe and durable gene therapy, yet current inducible systems remain difficult to translate in vivo due to large size, limited induction duration, and dependence on immunogenic regulators. Here we present the adaptamer (ADAR modulatable aptamer), a compact (<120 bp) RNA switch that couples an FDA-approved small-molecule-responsive aptamer with endogenous ADAR-mediated RNA editing to regulate expression. Ligand binding stabilizes a double-stranded RNA structure that recruits ADAR to convert a stop codon into a sense codon, restoring downstream protein translation. This cleavage-free, post-transcriptional mechanism enables precise, small-molecule-dependent modulation without exogenous protein machinery. We demonstrate that adaptamers are highly functional across multiple cell lines, including human T-cells. In mice, AAV-delivered adaptamer-controlled FGF21 expression induced metabolic remodeling, significantly increasing energy expenditure and reversing obesity. This minimal, programmable system offers a clinically compatible approach for tunable genetic medicines and safe deployment of pleiotropic and dose-limited proteins.

synthetic biology↗

bioRxiv2024

A Novel Engineered U7 Small Nuclear RNA Scaffold Greatly Increases in vitro and in vivo ADAR-Mediated Programmable RNA Base Editing

Custom RNA base editing using the endogenous human Adenosine Deaminase Acting on RNA (ADAR) enzyme presents a promising approach for precision therapeutics, alleviating concerns of permanent DNA damage or immunogenicity from1 foreign bacterial proteins such as CRISPR/Cas. ADAR can be directed to act on therapeutic RNA targets by antisense guide RNAs (gRNAs) that create a substrate for ADARs adenosine-to-inosine (effectively A-to-G) deamination activity. Delivery of gRNAs via a DNA expression construct provided by Adeno-Associated Virus (AAV) might allow life-long duration of the therapy. However, a major challenge for RNA editing using gene-encoded gRNAs and endogenous levels of ADAR is achieving sufficient gRNA activity inside cells, especially in therapeutic situations where AAV delivery may provide as low as one viral genome per cell. Here we show that embedding antisense gRNAs into a U7 small nuclear RNA (snRNA) framework and adding hnRNP A1 binding domains greatly increases the efficiency of custom RNA editing. This increased editing efficiency allows for detectable RNA editing from a single genomic insertion of gRNA construct per cell, which enabled a pooled library screen of 750+ gRNA variations to further improve the SmOPT U7 hairpin system. The screen revealed critical residues responsible for RNA editing and generated new SmOPT and U7 hairpin variants that further boosted RNA editing. The final design, combined with an improved synthetic U7 promoter, resulted in up to 76% targeted editing with a single integrated copy of construct per cell, representing a 10- to 100-fold increase over existing circular gRNA approaches. Using systemic in vivo AAV delivery, we achieved an unprecedented 75% RNA editing in the total brain of a mouse model of Hurler syndrome. Our novel SmOPT U7 system also improved published antisense oligos for DMD exon skipping, currently in clinical trials, by up to 25-fold in differentiated myoblasts, and therefore represents a universal scaffold for ADAR-based RNA editing as well as other antisense RNA therapies.

bioengineering↗

bioRxiv2022

Reprogramming Adeno-Associated Virus Tropism Via Displayed Peptides Tiling Receptor-Ligands

Adeno-associated viruses (AAVs) are common gene therapy vectors, however, their effectiveness is hindered by poor target tissue transduction and off-target delivery. Hypothesizing that naturally occurring receptor-ligand interactions could be repurposed to engineer tropism, we fragmented all annotated protein ligands known to bind human receptors into tiling 20-mer peptides and displayed these onto the surface loops of AAV5 and AAV9 capsids at two sites. The resulting four capsid libraries, comprising >1 million AAV variants, were screened across 9 tissues in C57BL/6 mice. Tracking variant abundance, we identified >250,000 variants which packaged into capsids, and >15,000 variants which efficiently transduced at least one mouse organ. We individually validated 21 AAV variants with 74.3% of the organ tropism predictions accurately reproducing, confirming overall screen efficacy. Systematic ligand tiling enabled prediction of putative AAV-receptor interactions, which we successfully validated by targeted genetic perturbations. Comprehensive peptide tiling also enabled examination of homologous peptide activity. Interestingly, we observed functional peptides tended to be derived from specific domains on ligands. Notably, certain peptides also displayed consistent activity across mice strains, capsid insertion contexts, and capsid serotypes, including novel immune orthogonal serotypes. Further analyses of displayed peptides revealed that biophysical attributes were highly predictive of AAV variant packaging, and there was a machine learnable relationship between peptide sequence and tissue tropism. We anticipate this comprehensive ligand peptide tiling and display approach will enable engineering of tropism across diverse viral, viral-like, and non-viral delivery platforms, and shed light into basic receptor-ligand biology.

bioengineering↗

bioRxiv2022

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.

bioengineering↗