Biology subjects

Rufino-Ramos, D.

Publications and source records attributed to Rufino-Ramos, D..

3 recordsLinked to original sources

bioRxiv2023

Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

bioengineering↗

bioRxiv2023

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

molecular biology↗

bioRxiv2023

Extracellular communication between brain cells through functional transfer of Cre mRNA

1In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

cell biology↗