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Recombinant DNA Technology and Gene Therapy Using Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
There are different gene therapy approaches. For example, a patient’s cells may be removed, modified using the viral vector with the gene insert (therapeutic transgene), and then reintroduced into the body. This is known as the ex vivoapproach. Alternatively, the viral vector with the gene insert could be introduced directly to an organ or systemically to modify the target cells within the patient’s body. This is known as the in vivo approach (Figure 7.2). (Mietzsch and Agbandje-McKenna 2017; Colavito 2007; Minkoff and Baker 2004; Kurreck and Stein 2016). It should be recognized that the ex vivo gene therapy approach is built on knowledge gained from successful bone marrow transplants using patient-matched hematopoietic (blood) stem cell donors. This is known as allogeneic bone marrow transplantation. With gene therapy, now the patient’s own cells can be modified and given back to them in an approach known as an autologous bone marrow transplantation (Dunbar et al. 2018).
Challenges in Delivering Gene Therapy
Published in Yashwant Pathak, Gene Delivery, 2022
Gene therapy usually has two main ways of delivery: in vivo and ex vivo. Another name for these delivery systems would be “direct delivery” for in vivo and “cell-based delivery” for ex-vivo delivery. In in vivo, the work performed, in this instant the delivery of gene therapy, is performed within the natural condition of the organism or quite literally within the organism. Ex-vivo refers to the opposite, which would be outside the living organism [6]. Ex-vivo can almost be compared to that of invitro. Invitro, refers to the work within a set environment, such as a test tube. Ex-vivo gene therapy would use the concept of invitro, as ex-vivo refers to cells being taken out of the body and then transduced with the gene in a test tube, invitro, and then simply readministered to the body, where the gene expression can start to occur. Below in the figure is a summary of direct and cell-based delivery (Figure 1.3).
Basic genetics and patterns of inheritance
Published in Hung N. Winn, Frank A. Chervenak, Roberto Romero, Clinical Maternal-Fetal Medicine Online, 2021
Gene therapy involves insertion of normal copies of genes into individuals who have genetic diseases. This can potentially be accomplished by either somatic cell or germ cell gene therapy. Most work thus far has focused on somatic cell gene therapy. There are two ways to approach somatic cell gene therapy. Ex vivo gene therapy involves removing a patient’s cells from the body, inserting the normal gene copy into the cells, and then returning the cells to the body. In in vivo gene therapy, cells are treated while inside the patient’s body. For successful gene therapy, the cell requiring treatment must be easily accessible and relatively long-lived. Some of the earliest human gene therapy trials were performed for severe combined immune deficiency due to adenosine deaminase deficiency, using bone marrow stem cells. Other cells under consideration for therapy have included lymphocytes, hepatocytes, muscle cells, and respiratory epithelial cells. More recently, gene therapy for Leber’s congenital amaurosis has been accomplished by replacement of the defective gene locally to the retina of the eye. Types of genetic diseases that are amenable to somatic cell gene therapy are primarily autosomal recessive or X-linked disorders that result in almost total lack of normal protein. Reconstitution of even 5% to 10% of normal protein levels appears to be sufficient to treat these diseases. Dominant disorders that are caused by heterozygosity for mutant and normal genes (dominant-negative mutations) are not likely to be treatable by gene replacement; methods to block production of the mutant protein will be required.
Generation of a novel ex-vivo model to study re-endothelialization
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Siti Sarah Azman, Muhammad Dain Yazid, Nur Azurah Abdul Ghani, Raja Zahratul Azma Raja Sabudin, Mohd Ramzisham Abdul Rahman, Nadiah Sulaiman
An ex-vivo model proves valuable as it enables research to be done in a controlled environment, ensuring reproducibility within the laboratory setting. Moreover, it offers the advantage of focusing on specific cellular processes or mechanisms that may be challenging to study in-vivo due to complexity of the organism. Many studies have utilised short to moderate culture periods for ex-vivo tissues [31]. Notably, in vascular tissue research, studies have demonstrated the viability of ex-vivo tissue culture for up to 14 days, facilitating real-time observations following various treatments [18,32]. Therefore, this endothelial-denuded artery model provides a controlled environment outside of a living organism, enabling researchers to investigate the process of EC regeneration and repair following endothelial injury. The ex-vivo model allows for a targeted study of specific cellular or molecular responses following endothelial injury, which is challenging to achieve in an in-vivo model. Moreover, as the denudation protocol specifically targets the EC layer without damaging the SMC, it becomes a valuable tool to evaluate tissue-engineered vascular grafts (TEVG).
IFN-γ activates the tumor cell-intrinsic STING pathway through the induction of DNA damage and cytosolic dsDNA formation
Published in OncoImmunology, 2022
Hui Xiong, Yu Xi, Zhiwei Yuan, Boyu Wang, Shaojie Hu, Can Fang, Yixin Cai, Xiangning Fu, Lequn Li
Fresh tissues were obtained from patients undergoing pulmonary resection before radiation or chemotherapy at the Department of Thoracic Surgery, Tongji Hospital. Ex vivo culture was performed as previously described.33 Briefly, the tumor tissues were dissected into 1 mm3 cubes and placed on a gelatin sponge (Hushida, Jiangxi, China) in RPMI-1640 media supplemented with 10% heat-inactivated FBS. Then, the indicated amounts of IFN-γ or anti-CD3 mAb, anti-PD-1 Ab, and anti-IFN-γ Ab were added to the cultures. The tissues were cultured at 37°C for 48 h and collected for RNA and protein extraction. This study was performed following the Declaration of Helsinki. The use of human tissue samples was approved by the Institutional Ethics Committee of Huazhong University of Science and Technology.
Comparison of two radiofrequency-based hemostatic devices: saline-linked bipolar vs. cooled-electrode monopolar
Published in International Journal of Hyperthermia, 2022
Xavier Moll, Dolors Fondevila, Félix García-Arnas, Fernando Burdio, Macarena Trujillo, Ramiro M. Irastorza, Enrique Berjano, Anna Andaluz
The comparison between ex vivo and in vivo results is not straightforward. While it would seem reasonable that the coagulation sizes were smaller in vivo due to the heat sink effect (absent in the ex vivo model), we found larger coagulations, at least with the 5-mm Coolingbis® and the Aquamantys® (see Figure 6), possibly due to the difference of the initial tissue temperature (room temperature in the ex vivo and 37 °C in the in vivo model), which directly affects electrical conductivity and therefore Joule effect heating. In the context of RF tumor ablation, we already observed computer results predicting larger coagulation sizes for the in vivo case at 37 °C than the ex vivo at room temperature [15]. In other words, the initial temperature of the tissue possibly has a much greater impact than the heat sink effect by blood perfusion.