Biology subjects

Weinberger, L.

Publications and source records attributed to Weinberger, L..

7 recordsLinked to original sources

bioRxiv2026

Limitations of inferring antiviral efficacy of interfering particles from observational natural histories

Recently Hariharan et al.2 reported naturally arising defective HIV genomes capable of conditional replication and interference in humans. While this work makes an important contribution to the field of therapeutic interfering particles (TIPs), particularly with respect to safety and tolerability, it also raises fundamental issues regarding: (i) whether the presented data constitute a valid test of, or support conclusions about, the therapeutic potential of TIPs and (ii) technical issues pertaining to the reported basic reproductive numbers (R0). Hariharan et al. conclude that the findings "raise concerns about the effectiveness of TIPs." However, the data presented do not constitute a valid test of therapeutic efficacy. Here, we (i) clarify that post-hoc observational natural history cannot adjudicate the success or failure of an intervention, (ii) show new analysis highlighting how the reported R0 measurements are internally inconsistent with the within-host viral dynamics reported, and (iii) explain that alternative, well-established mechanisms remain sufficient to explain the reported observations.

microbiology↗

bioRxiv2023

Comparative analysis between RNA-seq and single-molecule RNA FISH indicates that the pyrimidine nucleobase idoxuridine (IdU) globally amplifies transcriptional noise

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability, but the physiological roles of noise have remained difficult to determine in the absence of generalized noise-modulation approaches. Previous single-cell RNA-sequencing (scRNA-seq) suggested that the pyrimidine-base analog (5-iodo-2-deoxyuridine, IdU) could generally amplify noise without substantially altering mean-expression levels but scRNA-seq technical drawbacks potentially obscured the penetrance of IdU-induced transcriptional noise amplification. Here we quantify global-vs.-partial penetrance of IdU-induced noise amplification by assessing scRNA-seq data using numerous normalization algorithms and directly quantifying noise using single-molecule RNA FISH (smFISH) for a panel of genes from across the transcriptome. Alternate scRNA-seq analyses indicate IdU-induced noise amplification for ~90% of genes, and smFISH data verified noise amplification for ~90% of tested genes. Collectively, this analysis indicates which scRNA-seq algorithms are appropriate for quantifying noise and argues that IdU is a globally penetrant noise-enhancer molecule that could enable investigations of the physiological impacts of transcriptional noise. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

systems biology↗

bioRxiv2022

A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters

The high transmissibility of SARS-CoV-2 is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, post-exposure TIP dose lowers SARS-CoV-2 nasal shedding and at 5 days post-infection infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Co-housed contact animals exposed to infected, TIP-treated, animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals co-housed with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission. SignificanceCOVID-19 vaccines are exceptionally effective in preventing severe disease and death, but they have mixed efficacy in preventing virus transmission, consistent with established literature that parenteral vaccines for other viruses fail to prevent mucosal virus shedding or transmission. Likewise, small-molecule antivirals, while effective in reducing viral-disease pathogenesis, also appear to have inconsistent efficacy in preventing respiratory virus transmission including for SARS-CoV-2. Recently, we reported the discovery of a single-administration antiviral Therapeutic Interfering Particle (TIP) against SARS-CoV-2 that prevents severe disease in hamsters and exhibits a high genetic barrier to the evolution of resistance. Here, we report that TIP intervention also reduces SARS-CoV-2 transmission between hamsters.

microbiology↗

bioRxiv2020

RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Random-Deletion Library sequencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1s cis and trans elements. The resulting landscape recapitulated HIV-1s known cis-acting elements (i.e., LTR, {Psi}, and RRE) and surprisingly indicated that HIV-1s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, {Psi}, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported [~]300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKVs cis-acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections. ImportanceRecent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)--a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral cis- and trans-acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

synthetic biology↗

bioRxiv2020

Tripartite Inhibition of SRC-WNT-PKC Signalling Consolidates Human Naive Pluripotency

Different conditions have been devised to isolate MEK/ERK signalling independent human naive pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naive conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NF{kappa}B, WNT/{beta}CATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naive human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naive pluripotency were found between mouse and human. Finally, we establish alternative naive conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naive PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naive pluripotency and may advance its utilization in future translational applications. Highlights of key findingsO_LICombined inhibition of SRC, WNT and PKC signaling consolidates human naive pluripotency C_LIO_LIStable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent human naive PSCs C_LIO_LIOpposing roles for ACTIVIN and WNT/{beta}CATENIN signaling on mouse vs. human naive pluripotency C_LIO_LI2i and MEK/ERKi independent alternative human naive PSC conditions via inhibiting NOTCH/RBPj signaling C_LI

developmental biology↗

bioRxiv2018

Disrupting Transcriptional Feedback Yields an Escape-Resistant Antiviral

From microbes to cancers, drug-resistant escape variants cause significant morbidity and mortality1-7. Here we present proof-of-concept that disruption of viral auto-regulatory (feedback) circuits strongly inhibits viral replication and confers an extremely high barrier to the evolution of resistance. Using DNA duplexes, we develop single-molecule feedback-circuit disruptors that interfere with transcriptional negative feedback in human herpesviruses (both Herpes Simplex Virus 1 and Cytomegalovirus) thereby increasing viral transcription factors to cytotoxic levels. Feedback disruptors exhibit low-nanomolar to picomolar IC-50s, reduce viral replication >100-fold in culture and in mice, and synergize with the standard-of-care antivirals. Strikingly, no feedback-disruptor escape mutants evolved over >60 days of culture, in contrast to approved antivirals to which resistance rapidly evolved. Overall, the results demonstrate that molecular targeting of feedback circuitry could yield escape-resistant antivirals, potentially enabling development of a new class of antimicrobials.

microbiology↗

bioRxiv2017

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.

developmental biology↗