bims-dresag Biomed News
on DNA damage and repair, cellular senescence and aging, gene therapy
Issue of 2021–07–18
twelve papers selected by
Pengyi Yan, Shanghai Jiao Tong University
  1. Stochastic initiation of DNA replication across the human genome.
  2. Targeting viruses in their phase.
  3. Molecular pathology of rare progeroid diseases.
  4. Senescence-Induced Chemoresistance in Triple Negative Breast Cancer and Evolution-Based Treatment Strategies.
  5. Guard proteins keep watch at epithelial walls.
  6. CRISPR-based diagnostics.
  7. Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.
  8. Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.
  9. Switching on prodrugs using radiotherapy.
  10. PARP inhibitors in prostate cancer: when to use them?
  11. Advancing a rapid, high throughput screening platform for optimization of lentivirus production.
  12. Oncogenic mutations drive intestinal cancer initiation through paracrine remodeling.
  1. Mol Cell. 2021 Jul 15. pii: S1097-2765(21)00502-5. [Epub ahead of print]81(14): 2873-2874
    Stochastic initiation of DNA replication across the human genome.
    David M MacAlpine.
      Wang et al. (2021) comprehensively map DNA replication initiation events across the human genome using single-molecule optical resolution mapping and find that initiation events are randomly distributed across broad initiation zones that are only utilized in a stochastic fashion across a population of cells.
    DOI:  https://doi.org/10.1016/j.molcel.2021.06.022
  2. Nat Rev Mol Cell Biol. 2021 Jul 14.
    Targeting viruses in their phase.
    Paulina Strzyz.
      
    DOI:  https://doi.org/10.1038/s41580-021-00400-7
  3. Trends Mol Med. 2021 Jul 13. pii: S1471-4914(21)00178-7. [Epub ahead of print]
    Molecular pathology of rare progeroid diseases.
    Matthias Rieckher, George A Garinis, Björn Schumacher.
      Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. Recent advances from studies in animal models of PSs have provided new insight into the role of DNA repair mechanisms in human disease and the physiological adaptations to accumulating DNA damage during aging. The molecular pathology of PSs is reminiscent of the natural aging process, highlighting the relevance for a wide range of age-related diseases. Recent progress has led to the development of novel therapeutic strategies against age-related diseases that are relevant to rare diseases as well as the general aging population.
    Keywords:  DNA damage response; DNA repair; age-related diseases; aging; progeroid syndromes
    DOI:  https://doi.org/10.1016/j.molmed.2021.06.011
  4. Front Oncol. 2021 ;11 674354
    Senescence-Induced Chemoresistance in Triple Negative Breast Cancer and Evolution-Based Treatment Strategies.
    Anindita Chakrabarty, Shayantani Chakraborty, Ranjini Bhattacharya, Goutam Chowdhury.
      Triple negative breast cancer (TNBC) is classically treated with combination chemotherapies. Although, initially responsive to chemotherapies, TNBC patients frequently develop drug-resistant, metastatic disease. Chemotherapy resistance can develop through many mechanisms, including induction of a transient growth-arrested state, known as the therapy-induced senescence (TIS). In this paper, we will focus on chemoresistance in TNBC due to TIS. One of the key characteristics of senescent cells is a complex secretory phenotype, known as the senescence-associated secretory proteome (SASP), which by prompting immune-mediated clearance of senescent cells maintains tissue homeostasis and suppresses tumorigenesis. However, in cancer, particularly with TIS, senescent cells themselves as well as SASP promote cellular reprograming into a stem-like state responsible for the emergence of drug-resistant, aggressive clones. In addition to chemotherapies, outcomes of recently approved immune and DNA damage-response (DDR)-directed therapies are also affected by TIS, implying that this a common strategy used by cancer cells for evading treatment. Although there has been an explosion of scientific research for manipulating TIS for prevention of drug resistance, much of it is still at the pre-clinical stage. From an evolutionary perspective, cancer is driven by natural selection, wherein the fittest tumor cells survive and proliferate while the tumor microenvironment influences tumor cell fitness. As TIS seems to be preferred for increasing the fitness of drug-challenged cancer cells, we will propose a few tactics to control it by using the principles of evolutionary biology. We hope that with appropriate therapeutic intervention, this detrimental cellular fate could be diverted in favor of TNBC patients.
    Keywords:  adaptive therapy; chemotherapy; evolution; senescence; senescence-associated stemness; therapy-induced senescence; triple negative breast cancer
    DOI:  https://doi.org/10.3389/fonc.2021.674354
  5. Immunity. 2021 Jul 13. pii: S1074-7613(21)00248-X. [Epub ahead of print]54(7): 1366-1368
    Guard proteins keep watch at epithelial walls.
    Alejandra Zárate-Potes, Katja Dierking.
      Cells can detect pathogens through guard proteins that sense disturbances in core cellular processes, but the exact mechanisms often remain elusive. In this issue of Immunity, Orzalli et al. identify Bcl-2 family members as guard proteins that detect virus-induced translational inhibition and induce pyroptosis in human keratinocytes.
    DOI:  https://doi.org/10.1016/j.immuni.2021.06.004
  6. Nat Biomed Eng. 2021 Jul;5(7): 643-656
    CRISPR-based diagnostics.
    Michael M Kaminski, Omar O Abudayyeh, Jonathan S Gootenberg, Feng Zhang, James J Collins.
      The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.
    DOI:  https://doi.org/10.1038/s41551-021-00760-7
  7. J Gene Med. 2021 Jul 16. e3377
    Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.
    Shufeng Ma, Jie Lv, Zinan Feng, Zhili Rong, Ying Lin.
      The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, and has great potential for clinic applications. The system consists of two key molecules, a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, but new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the current review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific, and safer gene editing.
    Keywords:  CRISPR/Cas9; CRISPR/Cas9 delivery; gene editing; guide RNA; guide RNA engineering
    DOI:  https://doi.org/10.1002/jgm.3377
  8. J Vis Exp. 2021 Jun 28.
    Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.
    Rebeca Carballar-Lejarazú, Thai Binh Pham, Adam Kelsey, Taylor Tushar, Anthony A James.
      Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.
    DOI:  https://doi.org/10.3791/62607
  9. Nat Chem. 2021 Jun 10.
    Switching on prodrugs using radiotherapy.
    Jin Geng, Yichuan Zhang, Quan Gao, Kevin Neumann, Hua Dong, Hamish Porter, Mark Potter, Hua Ren, David Argyle, Mark Bradley.
      Chemotherapy is a powerful tool in the armoury against cancer, but it is fraught with problems due to its global systemic toxicity. Here we report the proof of concept of a chemistry-based strategy, whereby gamma/X-ray irradiation mediates the activation of a cancer prodrug, thereby enabling simultaneous chemo-radiotherapy with radiotherapy locally activating a prodrug. In an initial demonstration, we show the activation of a fluorescent probe using this approach. Expanding on this, we show how sulfonyl azide- and phenyl azide-caged prodrugs of pazopanib and doxorubicin can be liberated using clinically relevant doses of ionizing radiation. This strategy is different to conventional chemo-radiotherapy radiation, where chemo-sensitization of the cancer takes place so that subsequent radiotherapy is more effective. This approach could enable site-directed chemotherapy, rather than systemic chemotherapy, with 'real time' drug decaging at the tumour site. As such, it opens up a new era in targeted and directed chemotherapy.
    DOI:  https://doi.org/10.1038/s41557-021-00711-4
  10. Endocr Relat Cancer. 2021 Jun 01. pii: ERC-21-0133.R1. [Epub ahead of print]
    PARP inhibitors in prostate cancer: when to use them?
    Nely Díaz-Mejía, David García-Illescas, Rafael Morales-Barrera, Cristina Suarez, Jacques Planas, Xavier Maldonado, Joan Carles, Joaquin Mateo.
      Poly (ADP-ribose) polymerase (PARP) inhibitors have antitumor activity in advanced prostate cancer associated to loss of homologous recombination repair (HRR) function. About 20% of all patients with advanced prostate cancer present germline or tumor mutations in HRR-related genes, the commonest being BRCA2, mutated in approximately 10% of all advanced prostate cancers. Challenges related to sample availability, tumor heterogeneity and access to NGS technology need to be addressed for a successful implementation of genomic stratification in routine clinical practice. The recent regulatory approvals of PARP inhibitors olaparib and rucaparib represent the first molecular biomarker-guided drugs for men with prostate cancer. While these findings represent a significant advance in the field of precision medicine and prostate cancer, there are still many unsolved questions to deliver optimal use of PARP inhibitors in this disease. Several clinical trials have shown that different mutations in different genes associate with distinct magnitudes of sensitivity to PARP inhibitors, with BRCA2 mutations associating to more frequent and durable responses, questioning the benefit for subset of patients with mutations in other HRR-associated genes. In this review, we scrutinize the clinical development of different PARP inhibitors for the treatment of advance prostate cancer, and we discuss how the study of further biomarkers and the design of rational drug combinations can aid towards maximizing patient benefit from this drug class.
    DOI:  https://doi.org/10.1530/ERC-21-0133
  11. Biotechnol J. 2021 Jul 14. e2000621
    Advancing a rapid, high throughput screening platform for optimization of lentivirus production.
    Sneha Gopal, Adam E Osborne, Lindsay Hock, Jill Zemianek, Kun Fang, Gretchen Gee, Ronit Ghosh, David McNally, Steven M Cramer, Jonathan S Dordick.
       BACKGROUND: Lentiviral Vectors (LVVs) hold great promise as delivery tools for gene therapy and chimeric antigen receptor T cell (CAR-T) therapy. Their ability to target difficult to transfect cells and deliver genetic payloads that integrate into the host genome makes them ideal delivery candidates. However, several challenges remain to be addressed before LVVs are more widely used as therapeutics including low viral vector concentrations and the absence of suitable scale-up methods for large-scale production. To address these challenges, we have developed a high throughput microscale HEK293 suspension culture platform that enables rapid screening of conditions for improving LVV productivity.
    KEY RESULTS: High density culture (40 million cells/mL) of HEK293 suspension cells in commercially available media was achieved in microscale 96-deep well plate platform at liquid volumes of 200 μL. Comparable transfection and LVV production efficiencies were observed at the microscale and in conventional shake flasks and a 1-L bioreactor, indicating that significant scale-down does not affect LVV concentrations and predictivity of scale-up. Optimization of production step allowed for final yields of LVVs to reach 1.5 × 107  TU/mL.
    CONCLUSIONS: The ability to test a large number of conditions simultaneously with minimal reagent use allows for the rapid optimization of LVV production in HEK293 suspension cells. Therefore, such a system may serve as a valuable tool in early stage process development and can be used as a screening tool to improve LVV concentrations for both batch and perfusion based systems. This article is protected by copyright. All rights reserved.
    Keywords:  HEK293 suspension cells; High-throughput transfection; Lentiviral titers; Lentiviral vectors
    DOI:  https://doi.org/10.1002/biot.202000621
  12. Cancer Cell. 2021 Jul 12. pii: S1535-6108(21)00335-4. [Epub ahead of print]39(7): 913-915
    Oncogenic mutations drive intestinal cancer initiation through paracrine remodeling.
    Helen H N Yan, April S Chan, Suet Yi Leung.
      Three articles in Nature show that intestinal stem cells with cancer-promoting mutations could shape the surrounding normal tissue in their favor to promote clonal fixation and field expansion, raising the possibility of developing therapeutic strategies that maintain or enhance the health of normal cells to out-compete the mutant cells.
    DOI:  https://doi.org/10.1016/j.ccell.2021.06.013
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