bims-dresag Biomed News
on DNA damage and repair, cellular senescence and aging, gene therapy
Issue of 2021–07–11
six papers selected by
Pengyi Yan, Shanghai Jiao Tong University
  1. Targeting Serine in Cancer: Is Two Better Than One?
  2. Senescence and Senolytics in Cardiovascular disease: Promise and Potential Pitfalls.
  3. Multiplexed Genome Engineering with Cas12a.
  4. Genomic instability signals offer diagnostic possibility in early cancer detection.
  5. High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies.
  6. Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.
  1. Trends Cancer. 2021 Jul 01. pii: S2405-8033(21)00134-5. [Epub ahead of print]
    Targeting Serine in Cancer: Is Two Better Than One?
    Aitziber Buqué, Lorenzo Galluzzi, David C Montrose.
      Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.
    Keywords:  DNA damage; PHGDH; PSAT1; PSPH; colorectal cancer; glycine
    DOI:  https://doi.org/10.1016/j.trecan.2021.06.004
  2. Mech Ageing Dev. 2021 Jul 05. pii: S0047-6374(21)00112-3. [Epub ahead of print] 111540
    Senescence and Senolytics in Cardiovascular disease: Promise and Potential Pitfalls.
    W Andrew Owens, Anna Walaszczyk, Ioakim Spyridopoulos, Emily Dookun, Gavin D Richardson.
      Ageing is the biggest risk factor for impaired cardiovascular health, with cardiovascular disease being the cause of death in 40% of individuals over 65 years old. Ageing is associated with an increased prevalence of atherosclerosis, coronary artery stenosis and subsequent myocardial infarction, thoracic aortic aneurysm, valvular heart disease and heart failure. An accumulation of senescence and increased inflammation, caused by the senescence-associated secretory phenotype, have been implicated in the aetiology and progression of these age-associated diseases. Recently it has been demonstrated that compounds targeting components of anti-apoptotic pathways expressed by senescent cells can preferentially induce senescence cells to apoptosis and have been termed senolytics. In this review, we discuss the evidence demonstrating that senescence contributes to cardiovascular disease, with a particular focus on studies that indicate the promise of senotherapy. Based on these data we suggest novel indications for senolytics as a treatment of cardiovascular diseases which have yet to be studied in the context of senotherapy. Finally, while the potential benefits are encouraging, several complications may result from senolytic treatment. We, therefore, consider these challenges in the context of the cardiovascular system.
    Keywords:  Ageing; atherosclerosis; cardiovascular; heart failure; inflammation; remodelling; senescence; senolytic
    DOI:  https://doi.org/10.1016/j.mad.2021.111540
  3. Methods Mol Biol. 2021 ;2312 171-192
    Multiplexed Genome Engineering with Cas12a.
    Niels R Weisbach, Ab Meijs, Randall J Platt.
      Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.
    Keywords:  CRISPR; CRISPR array synthesis; CRISPR-Cas12a; Cas12a; Gene editing; Genome engineering; Multiplexed; Orthogonal genome engineering; Transcriptional regulation
    DOI:  https://doi.org/10.1007/978-1-0716-1441-9_11
  4. Trends Genet. 2021 Jul 01. pii: S0168-9525(21)00165-7. [Epub ahead of print]
    Genomic instability signals offer diagnostic possibility in early cancer detection.
    Sarah Killcoyne, Aisha Yusuf, Rebecca C Fitzgerald.
      Emerging evidence from the large numbers of cancer genomes analyzed in recent years indicates that chromosomal instability (CI), a well-established hallmark of cancer cells, is detectable in precancerous lesions. In this opinion, we discuss the association of this instability with tumor progression and cancer risk. We highlight the opportunity that early genomic instability presents for the diagnosis of esophageal adenocarcinoma (EAC) and its precancerous lesion, Barrett's esophagus (BE). With a growing body of evidence suggesting that only a small pool of cancer-related genes are involved in early tumor development, we argue that general genomic instability may hold greater diagnostic potential for early cancer detection as opposed to the identification of individual mutational biomarkers.
    Keywords:  Barrett’s esophagus; chromosomal instability; early detection
    DOI:  https://doi.org/10.1016/j.tig.2021.06.009
  5. Nat Commun. 2021 07 06. 12(1): 4138
    High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies.
    Jicong Cao, Eva Maria Novoa, Zhizhuo Zhang, William C W Chen, Dianbo Liu, Gigi C G Choi, Alan S L Wong, Claudia Wehrspaun, Manolis Kellis, Timothy K Lu.
      Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.
    DOI:  https://doi.org/10.1038/s41467-021-24436-7
  6. J Cell Biol. 2021 Sep 06. pii: e202101092. [Epub ahead of print]220(9):
    Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.
    Magdalena P Crossley, Joshua R Brickner, Chenlin Song, Su Mon Thin Zar, Su S Maw, Frédéric Chédin, Miaw-Sheue Tsai, Karlene A Cimprich.
      R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.
    DOI:  https://doi.org/10.1083/jcb.202101092
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