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Retroviral Gene Transfer in Autologous Bone Marrow and Stem Cell Transplantation
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Rafat Abonour, Kenneth Cornetta
In addition to stable integration, retroviral vectors are attractive cell markers because very sensitive methods of vector detection, namely the polymerase chain reaction, are available. Also, if the marker gene confers drug resistance, it may be possible to select out marked cells in vitro. Lastly, retroviral gene transfer is a simple technical procedure that does not expose the marked cell to toxic compounds or radioactivity that might alter the function of the marked cells. Retroviral vectors have been used extensively in vitro to mark human leukemic cell lines with an efficiency of 10-60% using supermite transduction protocols (Hogge and Humphries, 1987; Smith and Benchimol, 1987; Cornetta et al., 1993).
Gene Transfer into Human Hematopoietic Stem Cells
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
Serguei Kisselev, Tatiana Seregina, Richard K. Burt, Charles J. Link
Similar trials on eleven adult patients with multiple myeloma or breast cancer were performed by Dunbar and colleagues.99 In these trials, NeoR retroviral gene transfer into bone marrow and peripheral blood-derived CD34 enriched cells was performed and followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. Out of 10 evaluable patients with the marker gene detected at the time of engraftment, 3 had persistence of the marker gene for more than 18 months post transplantation. Marker gene was detected in multiple cell lineages, including both myeloid and lymphoid cells. The data suggested that better long-term marking originated from peripheral blood derived cells than from marrow. However, the levels of marking were low, with between 0.1 to 0.01% cells being positive. The marker was detected both in lymphoid and myeloid cell lineages.99,100
Gene Delivery for Intervertebral Disc
Published in Raquel M. Gonçalves, Mário Adolfo Barbosa, Gene and Cell Delivery for Intervertebral Disc Degeneration, 2018
Gianluca Vadalà, Luca Ambrosio, Vincenzo Denaro
The latter was adopted in a study conducted by Sowa et al. (2011), who investigated the use of an AAV vector containing a novel control system, namely, RheoSwitch, which uses a specific promoter region that enables transgene expression only in the presence of an activator ligand. The vector carrying a GFP marker gene was transfected in rabbit NP cells in vitro; increasing concentrations of the activator ligand were added and fluorescence was then assessed during and after its removal. Results showed that fluorescence was time and dose dependent: it diminished 24 hours after removal of the ligand and was hardly detectable after 48 hours. Further, this regulation system has been tested in vivo using healthy rabbits that received an intradiscal injection of the virus. GFP expression was reported to be high in animals treated with both virus and activator ligand, while animals receiving either one of them did not show any appreciable fluorescence. In addition, no GFP expression was detected in surrounding tissues, thus possibly designating RheoSwitch as a reliable regulation system for IDD gene therapy (Sowa et al. 2011).
Brain Gene Expression Profiling of Individuals With Dual Diagnosis Who Died by Suicide
Published in Journal of Dual Diagnosis, 2020
Brenda Cabrera-Mendoza, Cristóbal Fresno, Nancy Monroy-Jaramillo, Gabriel Rodrigo Fries, Consuelo Walss-Bass, David C. Glahn, Patricia Ostrosky-Wegman, Alma Delia Genis-Mendoza, José Jaime Martínez-Magaña, Ana Luisa Romero-Pimentel, Carlos Enrique Díaz-Otañez, Fernando García-Dolores, Eli Elier González-Sáenz, Roberto Cuauhtemoc Mendoza-Morales, Gonzalo Flores, Rubén Vázquez-Roque, Humberto Nicolini
To identify whether the gene expression profiles could reflect differences in cell type compositions among the groups, we performed a cell type–specific marker enrichment with NeuroExpresso, which is a database of brain cell type–specific gene expression profiles (Mancarci et al., 2017). This tool was used through its implementation as R package known as markerGeneProfile (Mancarci et al., 2017). Briefly, the estimation of marker gene profiles was performed for each cellular type as described in Mancarci et al. (2017). This estimation is based on a PCA obtained from the expression of mouse cell type–specific markers provided by NeuroExpresso to estimate the relative abundance of a cell type. Mouse cell type–specific markers were transformed to their respective human gene orthologs with the homoloGene tool (NCBI Resource Coordinators, 2017). For comparing the proportion of cell type–specific markers between the groups, we used a two-side Wilcoxon rank sum test for each cellular type.
Gut microbiota in ALS: possible role in pathogenesis?
Published in Expert Review of Neurotherapeutics, 2019
Pamela A. McCombe, Robert D. Henderson, Aven Lee, John D. Lee, Trent M. Woodruff, Restuadi Restuadi, Allan McRae, Naomi R. Wray, Shyuan Ngo, Frederik J. Steyn
The marker-gene method, which uses Polymerase Chain Reaction (PCR) to amplify a genomic region selected for specific properties, provides an overview of the microbial composition in the samples [47,48]. The region of interest usually includes segments of sequence that are highly variable between species flanked by conserved segments. The highly variable regions serve as barcodes for specific microorganisms and the conserved regions serve as the binding sites for PCR primers, common across micro-organism classes. Common genes are used in marker-gene analyses. These are 16S rRNA for identifying bacteria and archaea and 18S rRNA and internal-transcribed spacer (ITS) for fungi [49]. A key variable in marker-gene data is the sequencing technology that is used and the length of DNA reads that are generated [50]. For example, Illumina NextSeq 500 generates 2 × 250 paired-end reads that effectively cover 400–500 bp (i.e. two variable regions of the 16s RNA gene). The marker-gene approach is well tested, fast, and has a relatively low cost [47–49] and the specific marker-gene amplification reduces the possibility of contaminations from host DNA [49]. However, the coverage is at the level of genus, and therefore can miss the true complexity of the microbial community in the sample. Moreover, PCR amplification bias can provide an inaccurate view of the species distribution [47,48,51].
Production of CAR T-cells by GMP-grade lentiviral vectors: latest advances and future prospects
Published in Critical Reviews in Clinical Laboratory Sciences, 2019
Mansour Poorebrahim, Solmaz Sadeghi, Elham Fakhr, Mohammad Foad Abazari, Vahdat Poortahmasebi, Asma Kheirollahi, Hassan Askari, Alireza Rajabzadeh, Malihe Rastegarpanah, Aija Linē, Angel Cid-Arregui
Two functional titration assays are usually used to quantify transduction events: (1) measuring vector DNA copies by q-PCR, and (2) counting transgene-expressing cells in the target cell population. Measurement of transduced cells by transgene expression is the most reliable method to determine infectious LV titers. This method can be used only to titer stocks of vectors that carry reliable marker genes, which can be easily monitored by microscopy or flow cytometry, or when specific antibodies against the LV encoded protein(s) are available. Marker genes that are used in these assays include neo resistance gene, green fluorescent protein, or other living colors. The marker gene used should be controlled by a promoter that is active in the transduced cells (e.g. HEK293T). However, functional analysis of transgene expression restricted to reporter proteins cannot discriminate cells with single or multiple integration events and may not be reliable to measure expression of transgenes under the control of tissue-specific promoters [117,118,123]. Detection of proviral DNA copy numbers in infected cells by q-PCR can be used to measure infectious vector copy numbers. This is the most accurate and straightforward method to measure the titer of the lentivirus. The q-PCR technique is independent of promoter functionality and allows absolute quantitation of transduction events [118,124]. However, the best assay for functionality of the vector is the technique that measures product in cells supporting the activity of the promoter driving the transgene.