Biology subjects

Behlke, M. A.

Publications and source records attributed to Behlke, M. A..

9 recordsLinked to original sources

bioRxiv2026

Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

Chemical modifications such as 2'-O-methyl (2'-OMe) are central to the efficacy and tolerability of RNA therapeutics. We recently identified that 2'-OMe RNA fragments as short as three nucleotides can exert opposing effects on Toll-like receptor 8 (TLR8) sensing in a motif-dependent manner. This discovery raises important considerations for degradation products of chemically modified RNA therapeutics, which may generate such immunologically active fragments. Here, leveraging their short length, we systematically map how base and sugar modifications within 3-mer oligonucleotides regulate TLR7 and TLR8 responses, and we resolve the structural basis for both TLR8 potentiation and TLR7/8 antagonism by RNA fragments. Building on these insights, we report the development of a dual TLR7/8 inhibitory oligonucleotide with therapeutic potential in autoimmune disease. Together, these findings provide unprecedented resolution of the immunomodulatory properties of oligonucleotide modifications on TLR7/8 and establish 3-mer oligonucleotides as the shortest functional class of RNA therapeutics described to date.

immunology↗

bioRxiv2024

2'-O-Methyl-guanosine 3-base RNA fragments mediate essential natural TLR7/8 antagonism

Recognition of RNA fragments by Toll-like receptors (TLR) 7 and 8 is a key contributor to the initiation of a protective innate immune response against pathogens. A long-standing enigma is how degradation products of host RNAs, generated by the daily phagocytic clearance of billions of apoptotic cells, fail to activate TLR7 and TLR8 signalling1. Here, we report that select 2-O-methyl (2-Ome) guanosine RNA fragments as short as 3 bases, including those derived from host-RNAs, are potent TLR7 and TLR8 antagonists that reduce TLR7 sensing in vivo. Mechanistically, antagonistic fragments are directed towards a distinct binding site on these proteins by 5-end 2-Ome guanosine. Our results indicate that host-RNAs evade detection by TLR7/8 due to a pool of abundant host ribosomal 2-Ome-modified RNA fragments that naturally antagonize TLR7 and TLR8 sensing to avoid auto-immunity. Crucially, rare TLR7 and TLR8 mutations located at this antagonistic site decrease the inhibitory activity of 2-Ome guanosine RNA fragments and lead to auto-immunity in patients. Our findings also establish that select chemically synthesised 3-base oligonucleotides can harness the protective anti-inflammatory activity of this natural immune checkpoint for therapeutic targeting of TLR7-driven diseases. One Sentence SummaryShort 2-O-Methyl RNA fragments are natural TLR7/8 antagonists

immunology↗

bioRxiv2024

CCR2 silencing in sensory neurons blocks bone cancer progression

The peripheral nervous system contributes to cancer growth, in part by shaping the immunological niche of the tumor. How the nervous system influences bone cancer progression, and whether the underlying neuroimmune pathways can be targeted therapeutically, remain unclear. Here we demonstrate a profound influence of the peripheral nervous system on tumor progression that can be countered by silencing chemokine receptor signaling in sensory neurons. Axotomy of the tumor-innervating femoral nerve inhibits tumor progression in animals bearing bone cancer, whereas intrathecal delivery of the tumor-associated proinflammatory chemokine CCL2 promotes both tumor growth and allodynia. Silencing CCR2 in dorsal root ganglion (DRG) neurons with a newly developed lipid nanoparticle-formulated Dicer-substrate siRNA impedes tumor progression and pathological bone remodeling, and relieves bone cancer-associated pain. Mechanistically, bone cancer drives CCR2-dependent transport of substance P and CGRP along the tumor-innervating femoral nerve, and these neuropeptides expand the tumor-associated macrophage population; silencing CCR2 in DRG neurons normalizes the neuropeptide milieu and ameliorates altered bone remodeling. We thus define a targetable neuroimmune axis that contributes to cancer progression. HighlightsO_LICancer progression activates sensory neurons, driving pain hypersensitivity and neuropeptide release. C_LIO_LIAxotomy of the tumor-innervating femoral nerve impedes tumor progression. C_LIO_LICCL2-CCR2 signaling in DRG neurons promotes pain hypersensitivity and cancer growth. C_LIO_LISilencing CCR2 in the DRG reduces pain hypersensitivity, tumor-associated macrophage numbers and cancer growth. C_LI

neuroscience↗

bioRxiv2022

Comparative analysis of CRISPR off-target activity discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells

While CRISPR-based editing most often occurs at DNA sequences with perfect homology to the guide RNA (gRNA), unintended editing can occur at highly homologous regions (i.e., off-target (OT) sites). Due to the pace at which genome editing therapies are approaching clinical applications, there is an emerging need to define effective workflows for investigating OT editing effects. A number of homology-dependent, in silico-based prediction methods and wet lab-based empirical methods exist to investigate OT editing, but few have been subjected to analytical assessment or head-to-head comparison in human primary cells using an ex vivo editing process optimized for high-fidelity gene editing. Therefore, we sought to compare publicly available in silico tools (COSMID, CCTop, and Cas-OFFinder) as well as empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) in the context of ex vivo hematopoietic stem and progenitor cell (HSPC) editing. To do so, we edited CD34+ HSPCs using 11 different guide RNAs (gRNAs) complexed with HiFi Cas9, then performed targeted next-generation sequencing of ~200-site panels containing a range of nominated OT sites identified by in silico and empirical methods. We identified an average of 0.45 OT sites per gRNA at an indel detection limit of 0.5%. This study confirmed the marked improvement in specificity with HiFi Cas9 compared to wild-type Cas9 without compromising on-target activity when delivered as an RNP. Additionally, all HiFi Cas9 OT sites using a standard 20nt gRNA were identified by all OT detection methods with one exception (SITE-seq did not identify an OT generated by an AAVS1 gRNA). This resulted in high sensitivity for the majority of OT nomination tools, however due to the large number of false positives called by most methods, in silico-based COSMID and empirical methods DISCOVER-Seq and GUIDE-Seq attained the highest positive predictive value. We did not find the empirical methods identified off-target sites that were not also identified by bioinformatic methods when delivered as an RNP complex. Finally, this study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity as well as positive predictive value which would enable more efficient identification of potential off-target sites without compromising a thorough examination for any given gRNA.

genomics↗

bioRxiv2021

Clinically Relevant Gene Editing in Hematopoietic Stem Cells for the Treatment of Pyruvate Kinase Deficiency Hemolytic Anemia

Pyruvate Kinase Deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene, which constitutes the main cause of chronic non-spherocytic hemolytic anemia. PKD incidence is estimated in 1 in 20,000 people worldwide. The PKLR gene encodes for the erythroid pyruvate kinase protein (RPK) implicated in the last step of the anaerobic glycolysis in red blood cells. The defective enzyme fails to maintain normal erythrocyte ATP levels, producing severe hemolytic anemia, and can be fatal in severe patients. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cells (HSPC) transplantation, so far. However, HSPC transplant is associated with a significant morbidity and mortality, especially in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR/Cas9 system and rAAVs for donor matrix delivery to build an efficient and safe system to knock-in a therapeutic donor at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient recipient mice. Moreover, erythroid cells derived from edited PKD-HSPCs restored normal levels of ATP, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene editing strategy may represent a lifelong therapy to restore RPK functionality in RBCs of patients and correct PKD. Single Sentence SummaryClinically relevant gene editing in hematopoietic stem cells for the treatment of Pyruvate Kinase Deficiency.

genetics↗

bioRxiv2020

CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing

CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair footprints that result from non-homologous end joining (NHEJ) after double stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building our understanding into a novel analysis tool (CRISPAltRations) improved results quality. We validated our software using simulated rhAmpSeq amplicon sequencing data (11 gRNAs and 603 on- and off-target locations) and demonstrate that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user-interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a UI we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.

bioinformatics↗

bioRxiv2018

In vivo selection for corrected β-globin alleles after CRISPR/Cas9 editing in human sickle hematopoietic stem cells enhances therapeutic potential

Sickle Cell Disease (SCD), one of the worlds most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected {beta}-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.\n\nONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

genetics↗

bioRxiv2018

Engineered transfer RNAs for suppression of premature termination codons

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

molecular biology↗

bioRxiv2018

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of sixty different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

cell biology↗