Biology subjects

Novelli, G.

Publications and source records attributed to Novelli, G..

5 recordsLinked to original sources

bioRxiv2026

Long-read cross-platform validation reveals novel repeat features in myotonic dystrophy type 2

The broader application of long-read sequencing (LRS) for repeat expansion characterization in myotonic dystrophy type 2 (DM2) and other repeat expansion disorders (REDs) remains limited by the lack of systematic validation and benchmarking of sequencing results and bioinformatic workflows. Here, we performed an orthogonal cross-platform validation of previously generated Oxford Nanopore Technologies (ONT) data by sequencing the same DNA samples with Pacific Biosciences (PacBio) HiFi following amplification-free targeted enrichment in a cohort of 8 DM2 patients. Despite substantial differences in sequencing chemistry and coverage, the two platforms showed high concordance in repeat size estimation, somatic mosaicism, and repeat architecture. This validation confirmed the presence of the (TCTG)n motif and enabled the identification of a previously unreported (CCCG)n motif at the 3' end of expanded alleles, further highlighting the structural complexity of the CNBP expansion. Through this analysis, we also established a bioinformatic workflow that improved ONT-based repeat characterization, addressing limitations in motif resolution and enabling more accurate analysis of CNBP expansions. Overall, this study provides a validated framework for LRS-based CNBP repeat analysis, supporting the integration of these technologies into routine molecular investigation for DM2 and other REDs.

genomics↗

bioRxiv2022

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully-expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire [~]50-kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype- phenotype correlations for the better stratification of DM2 patients in clinical trials.

genomics↗

bioRxiv2021

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

bioengineering↗

bioRxiv2020

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

synthetic biology↗

bioRxiv2020

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

biochemistry↗