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.
Aptamers and Cancer Nanotechnology
Mansoor M. Amiji in Nanotechnology for Cancer Therapy, 2006
In the initial step, a library of random nucleotides flanked by fixed nucleotides is generated by solid phase oligonucleotide synthesis. The random nucleotides serve to add complexity to the pool while the fixed sequences are utilized for polymerase chain reaction (PCR) amplification. In the case of isolating DNA aptamers, the oligonucleotide pool is incubated with the target of interest, and the bound fragments are partitioned and directly amplified by PCR system. The resulting pool is used in a follow-up round of selection and amplification, and the process is repeated until the affinity for the target antigen plateaus. Typically, this will be achieved in six to ten rounds of SELEX. After the last round of SELEX, aptamers are cloned in plasmids, amplified, sequenced, and their binding constants are determined (Figure 16.1). These aptamers may be subject to additional modification such as size minimization to truncate the nucleotides not necessary for binding characteristics and nuclease stabilization by replacing naturally occurring nucleotides with modified nucleotides (i.e., 2′-F pyrimidines, 2′-OCH3 nucleotides) that are poor substrates for endo- and exonuclease degradation.
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].
Application in Gene Editing in Ovarian Cancer Therapy
Published in Cancer Investigation, 2022
Shuang Luo, Yujiao Wang, Yongyu Tao, Shuo Li, Zirui Wang, Wei He, Hangxing Wang, Nan Wang, Jianwei Xu, Hailiang Song
Gene editing, also known as genome editing or genome engineering, is a new and accurate genetic engineering technology that can be used to modify specific target genes. The technique relies on genetically engineered nucleases (also known as “molecular scissors”) that cut double-stranded DNA at specific loci in the genome and induce the repair of these breaks through non-homologous terminal connections or homologous recombination, finally leading to targeted mutations. Based on this characteristic, nucleases can efficiently carry out site-directed gene editing. At present, the technique is used in a variety of clinical fields, such as gender identification, and is also a promising strategy for OC therapy. Its ability to specifically knockout genes, promote or silence gene expression, can be used to alter the high expression of oncogenes and the low expression of tumor suppressor genes in OC, so it can be used as a new therapeutic approach. Although the status of gene editing has been greatly improved, gene editing still faces great challenges in ovarian cancer: first, how to accurately locate ovarian cancer-related genes; second, how to ensure the efficacy and safety of patients after gene therapy, and last but not least, how to promote the use of gene therapy for ovarian cancer in clinical practice. This review summarizes the application of gene editing in the treatment of OC and the underlying mechanisms, in order to stimulate its use in the clinic and improve the survival rate and quality of life of OC patients.
CRISPR and personalized Treg therapy: new insights into the treatment of rheumatoid arthritis
Published in Immunopharmacology and Immunotoxicology, 2018
Fatemeh Safari, Safar Farajnia, Maryam Arya, Habib Zarredar, Ava Nasrolahi
As regard, maintaining the stability of ex vivo expanded Tregs is difficult, so using genome editing technologies may improve this bottleneck for achieving powerful suppressive Tregs. In this end, site-specific nucleases as novel genome engineering technologies have been used for modification of a broad range of cells and organisms. Genome editing technologies such as zinc finger nucleases (ZFNs) as the first generation technique, TALENs array (Transcription activator-like effector nucleases) and recently RNA-based nuclease may successfully improve the adoptive Treg therapy. Clustered regularly interspaced short palindromic repeats in combination with Cas 9, provides the encouraging strategy for genome editing of eukaryotes108. High specificity of the CRISPR–Cas9 system is indebted to small RNAs that precisely guide the Cas9 nuclease to the target site of the genome through Watson–Crick base pairing109. Robust phenotypic effects and great validation rate of these sequence-specific programmable nucleases represent the promising window in the future applications. The combination of programmability with the permanently mutagenic or non-mutagenic manner110 of the Cas9 provides various strong genetic tools for gene knocking in, out or down and transcriptional activation. Wild-type Cas9 with two nuclease sites (HNH and Ruvc) mediates perturbation in the genome by indel (insert-deletion) mutations111 (Figure 3).
Related Knowledge Centers
- Biochemistry
- DNA Repair
- Enzyme
- Genome Instability
- Molecular Cloning
- Nucleic Acid
- Nucleotide
- Phosphodiester Bond
- Nick
- Immunodeficiency