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Gene Therapy in Oral Tissue Regeneration
Published in Vincenzo Guarino, Marco Antonio Alvarez-Pérez, Current Advances in Oral and Craniofacial Tissue Engineering, 2020
Fernando Suaste, Patricia González-Alva, Alejandro Luis, Osmar Alejandro
In bacteria, the delivery of genetic material has been done through the use of plasmids, which are circular DNA molecules that replicate and segregate independently on the host bacterial chromosome (episomes); this allows that the sequences cloned in these vectors are expressed stablely in the bacterial cytosol. Bacterial transformation emerged as one of the first approaches to introduce genetic material into a cell. In this strategy the permeability of the cell membrane is disturbed either by thermal shock or by the use of an electric field (electroporation). Additionally, cellular mechanisms such as conjugation and phage-mediated transduction were used to transfer genetic elements from one cell to another.
Current developments in human stem cell research and clinical translation
Published in Christine Hauskeller, Arne Manzeschke, Anja Pichl, The Matrix of Stem Cell Research, 2019
Stephanie Sontag, Martin Zenke
A more cost-effective alternative for generating footprint-free iPSCs are episomal plasmids, which are equipped with viral origins of replication (e.g. oriP/EBNA from Epstein-Barr virus) to ensure a sufficient expression period to reprogram somatic cells (Yu et al., 2009). While the initial reprogramming efficiencies were poor, in recent years progress has been made in plasmid design, and reprogramming efficiencies of episomal plasmids range from 0.001% to 0.02% (Rao and Malik, 2012). So far no study has reported on the integration of episomal plasmids in the host genome but as DNA could potentially integrate into the genome, whole genome sequencing should be performed on episomal reprogrammed iPSCs to ensure transgene-free iPSCs.
Gene Therapy
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Currently, in addition to efficient and safe gene delivery, the duration of expression, especially in the case of nonviral vectors, seems to be the largest unsolved problem. In principle, depending on the disease, a short-term transient expression or a long-term sustained expression can be designed by using appropriate DNA cassettes. DNA sequences can regulate rate, duration, and location of expression. While current nonviral delivery systems aim for episomal delivery (i.e., not integrating into the chromosome), the next generation of plasmids may contain sequences for adhesion to the nucleus and self-replication. They may also contain peptide stretches specifying nuclear localization (Boulikas, 1995; 1996), site-specific chromosome integration, and possibly homologous recombination.
Therapies in preclinical and clinical development for Angelman syndrome
Published in Expert Opinion on Investigational Drugs, 2021
Theodora Markati, Jessica Duis, Laurent Servais
A viral-mediated ex vivo gene therapy (cell therapy) using AAV is presently at late preclinical development, close to clinical testing. This will be the first time for a therapeutic approach of its kind to be tested for AS. A major challenge when translating results of viral-mediated gene therapies from mice and non-human primates to humans is dose scaling; this is particularly challenging for AS, as UBE3A is required throughout the brain and the threshold of expression needed for phenotypic rescue remains unknown. The most straightforward routes of administration for such therapies in the case of AS are those directed into the subarachnoid space, intrathecally via lumbar puncture or via intra-cisterna magna injection, and intracerebroventricularly. Certainly, improved bioavailability in the CNS can be achieved using these routes; however, they are highly interventional, especially considering the likelihood that redosing will be required. Nevertheless, AAVs have a good transduction capability with neurons. Additionally, their genome usually remains as extrachromosomal episomes in transduced cells and does not incorporate into the host genome [74]. This is reassuring from a safety perspective but raises concerns for the durability of expression. In contrast, the upcoming ex vivo gene therapy presents the advantage of being a permanent treatment: by using lentiviral programming, the UBE3A gene is integrated into the chromosomes of the MSCs and therefore is copied with cell divisions.
An update on gene therapy for lysosomal storage disorders
Published in Expert Opinion on Biological Therapy, 2019
Murtaza S. Nagree, Simone Scalia, William M. McKillop, Jeffrey A. Medin
An ideal gene therapy would be a life-long treatment, and, despite the long half-life of AAV episomes, they may not be able to provide decades of transgene persistence. As such, methods are being developed to deliver genome-editing components together with therapeutic constructs that may provide a more lasting response. This would combine the integrative benefit of LV with the tropism control exhibited by AAV (and certain serotypes). The principle is to encourage the integration of therapeutic DNA into known and ‘benign’ loci. For example, the AAVS1 locus is a natural integration site for wild-type AAV [84]. While recombinant AAV predominantly reside episomally, it is possible to use double-strand break-mediated homologous recombination to insert the desired therapeutic construct into the AAVS1 locus [84]. Alternatively, methods to achieve hepatocyte-specific editing of the albumin locus have been developed [85]. These integrations utilize the natural albumin promoter to attain high levels of liver-specific expression of a desired protein with reduced potential for genotoxicity due to heterologous transgenic promoters. The latter method is under clinical investigation for the treatment of MPS I and MPS II in phase I trials (NCT02702115 and NCT03041324). An analogous method is also under investigation for the treatment of Fabry disease [86,87].
Evolving role of human papillomavirus as a clinically significant biomarker in head and neck squamous cell carcinoma
Published in Expert Review of Molecular Diagnostics, 2019
Caitlin McMullen, Christine H. Chung, Juan C. Hernandez-Prera
Most HPV infections are cleared by immune response without causing cancer; however, HR viral subtypes may not clear as rapidly, and its persistence induced malignant transformation [21]. After transmission, the virus binds to different cell surface receptors and enters the cells located in the reticulated epithelium within in the tonsillar crypts of the host [22]. The viral DNA migrates to the nucleus and establishes itself as an episome at a low copy number or integrates to the host cell genome. After integration, the host cellular machinery coordinates viral replication, amplification, and transcription, which lead to the expression of oncoproteins [16]. HR viral subtypes produce oncoproteins that inactivate tumor suppressor genes and initiate the malignant transformation of the host cell. More specifically, the viral E6 oncoprotein promotes degradation of the p53 protein by ubiquitination, which disrupts the p53-mediated cellular response to DNA damage [23,24]. The E7 oncoprotein ubiquitinates the retinoblastoma protein (pRb), and its degradation causes in the release of E2F which then initiates the transcription of S phase genes [25,26]. In an attempt to inhibit the E7-mediated cell cycle progression to the S phase, feedback loops are activated and result in increased expression of host protein, p16INK4A [27].