Nonclinical Safety Evaluation of Advanced Therapies
Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard in Toxicologic Pathology, 2018
Over the last several years, a new form of gene therapy has emerged that has, based on its ease of use, changed the face of therapeutic approaches in this area and has significant potential to expand rapidly. The concept of “genome editing” is now possible with the advent of engineering various DNA nucleases to target locations in the genome with high specificity and induce double-strand breaks with the intent of eliminating or restoring the function of an endogenous gene or target the insertion of an exogenous gene (Cox 2015). At this point in time, four major classes of nucleases with the intent of current or eventual therapeutic use exist: 1) meganucleases, 2) zinc finger nucleases (ZFNs), 3) transcription activator–like effector nucleases (TALENs) and 4) CRISPR-associated nuclease Cas9. While the meganuclease, ZFN and TALEN systems, recognize DNA via protein/DNA interactions, Cas9 utilizes what is referred to as a “guide RNA” to direct the nuclease to its target. The ZFN- and TALEN-based technologies have entered into clinical trials, and the meganuclease technology may soon follow (Tebas 2014, Qasim 2017). Given the ease of production of individual or even libraries of such targeting guide RNAs for CRISPR/Cas9, there is a substantial amount of focus on this platform. This field is rapidly evolving, with newer platforms emerging that avoid DNA cleavage and modify nucleotides directly (Komor 2016), thus, it will be important for the toxicologic pathologist to keep track of the progress in this area.
Genome Editing for Genetic Lung Diseases
Anthony J. Hickey, Sandro R.P. da Rocha in Pharmaceutical Inhalation Aerosol Technology, 2019
Clustered regularly interspaced short palindromic repeats/associated endonuclease (CRISPR/Cas) systems are essentially RNA-guided nucleases.4,5,29,30 Class 2 CRISPR/Cas systems use a single Cas endonuclease that cleaves DNA upon target recognition by CRISPR RNA (crRNA).4,5,29,30 A target specific crRNA and a trans-activating CRISPR RNA (tracrRNA) of Cas9 could be fused to form a single guide RNA (sgRNA).4,5,29,30 The Cas endonucleases bind a protospacer adjacent motif (PAM) sequence, and use a RNA guide sequence located in the crRNA to recognize the complementary DNA sequence.4,5,29,30 By changing the guide sequences, it is very simple to target most genomic sequences, making CRISPR/Cas system a powerful genome editing tool. Cas9 system, in particular Streptococcus pyrogenes Cas9 (SpCas9), has a simple PAM requirement (NGG, indicating GG or CC are required) and high potency and, thus, is now broadly used in biomedical research and drug development.4,5,29,30
Gene Therapy for Lung Cancer
Kenneth L. Brigham in Gene Therapy for Diseases of the Lung, 2020
Several antisense approaches have been investigated. One approach is through the use of antisense oligonucleotides, in which the phosphodiester backbone is modified to methylphosphonate or phosphorothioate to reduce degradation by nucleases. These modified antisense oligonucleotides can enter tumor cells by endocytosis and form DNA-RNA duplexes with endogenous sense mRNA, inhibiting translation. Another approach to inactivating mRNA is via ribozymes. Ribozymes are essentially antisense oligonucleotides that contain RNase active sites. A ribozyme possessing this activity allows catalytic gene ablation by sequence-specific cleavage of the target transcript. A third approach is to use plasmids or viral vectors for transferring an open reading frame fragment of the desired gene oriented backwards (3' to 5') behind a powerful promoter, resulting in the production of an antisense RNA. Antisense RNA transcribed from these constructs form a RNA duplex with sense mRNA inhibiting translation (89).
DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology
Published in Journal of Extracellular Vesicles, 2019
Elisa Lázaro-Ibáñez, Cecilia Lässer, Ganesh Vilas Shelke, Rossella Crescitelli, Su Chul Jang, Aleksander Cvjetkovic, Anaís García-Rodríguez, Jan Lötvall
The release of cellular DNA in many different structural forms such as apoptotic blebs, histone/DNA complexes or nucleosomes, DNA/RNA-lipoprotein complexes or virtosomes, DNA traps, etc., has been well documented [7–10]. Such extracellular structures, classified in umbrella terms such as circulating DNA or cell-free DNA, largely serve to protect the DNA from nucleases that are present in, for example, the circulation and to reduce the likelihood of DNA being seen as a danger signal by the immune system [11]. As nucleases are essential enzymes that control DNA repair and therefore, genomic stability, their defects or absence are associated with diseases in which the sensing of self-nucleic acids is critical [12]. For instance, the knockout of the DNase I and II family members are linked to severe autoimmune and metabolic diseases [13].
Stem cell therapies for wound healing
Published in Expert Opinion on Biological Therapy, 2019
Nina Kosaric, Harriet Kiwanuka, Geoffrey C Gurtner
Genetic engineering methods can further enhance the therapeutic potential of stem cells [91]. As previously discussed, several challenges must be overcome prior to clinical adoption of MSC therapy, including preserving cell viability and activity upon exposure to the wound microenvironment [92], and developing strategies to enhance cell migration. Several approaches to genetically modify cells exist, including retroviral/lentiviral and non-integrating viral vector, plasmid DNA, and mRNA transfection, and nanoparticle delivery transduction [91,93]. These approaches are limited in their clinical application as viral delivery poses a high risk of oncogenic insertional mutagenesis and non-viral delivery of plasmids is not preserved as cells propagate. In the last decade, nuclease-mediated gene targeting with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 have allowed investigators to modify, remove, or insert genes with high precision.
Characterization of DNA cleavage produced by seminal plasma using leukocytes as a cell target
Published in Systems Biology in Reproductive Medicine, 2019
Elva I. Cortés-Gutiérrez, Carlos García De La Vega, Javier Bartolomé-Nebreda, Jaime Gosálvez
In addition to these well-known sperm DNA damage effectors, several reports have suggested the existence of an endogenous sperm nuclease in mammalian species, which cleaved DNA into loop-sized fragments of about 50 kb. DNA-loop domains are condensed into a single protamine toroid and protamine toroids are linked by chromatin stretches that are more sensitive to nucleases than the DNA packed within the toroids. Thus, sperm chromatin structure allowing that sperm can be digested at the sites of attachment to the nuclear matrix (Sotolongo et al. 2003). This nucleolytic activity would be equivalent to that of other nucleases that have been found in somatic cells. Some of these nucleases become activated during apoptosis and are able to cleave DNA into similar-sized fragments (Li et al. 1999; Yakovlev et al. 2000; Solovyan et al. 2002; Boulares et al. 2002a, 2002b). In humans and other mammalian species, the true nature of ejaculated spermatozoa presenting nuclear DNA damage is still an open area of research. The multifactorial source of agents producing DNA damage yield a confounding environment where the final result (DNA damage) is observed but remaining cryptic the original source of the DNA damage (Barroso et al. 2000; Arnon et al. 2001). Within this scenario, the presence of DNase in the ejaculate can represent a potential inducer of DNA damage. The presence of proteins at the seminal plasma with full capacity to produce DNA cleavage, their identification, their specific conditions to become active or inactive and their clinical consequences, is still an open area of research.
Related Knowledge Centers
- Biochemistry
- DNA Repair
- Enzyme
- Genome Instability
- Molecular Cloning
- Nucleic Acid
- Nucleotide
- Phosphodiester Bond
- Nick
- Immunodeficiency