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Adeno-Associated Virus-Based Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Successful gene transfer with integration and regulated gene expression in hematopoietic lines has been reported. Samulski and co-workers (149) used a recombinant AAV vector containing a marked human γ globin gene and DNAse I hypersensitivity sites from the locus control region (LCR) of the β-globin gene cluster to transduce K562 erythroleukemia cells. High-level, hemin-inducible expression of the marked γ-globin gene was detectable in clonal populations. This same gene was regulatable in BFU-E grown from CD34+ human cells (150). About 20% to 40% of BFU-E carried levels at 4% to 71% of endogenous γ-globin mRNA. When pooled BFU-E from the peripheral blood of a patient with sickle cell anemia was used, the hemoglobin F as measured with HPLC increased from 26% to 40%. AAV has been used to transfer the NeoR gene to T and B lymphocyte clones (151). Transduction with antisense for the HIV LTR in an HIV-infected cell line inhibited HIV production and replication (152). Suppression of human α globin gene mRNA by 91% in K562 which express high levels in culture was achieved using the α globin promoter driving the antisense sequence (153).
Transgenic Experiments with Interleukin-5
Published in Gerald J. Gleich, A. Barry Kay, Eosinophils in Allergy and Inflammation, 2019
Colin J. Sanderson, Malcolm Strath, Ian Mudway, Lindsay A. Dent
As IL-5 is normally expressed only after antigen stimulation, in a subpopulation of T cells, the production of transgenic mice in which IL-5 is produced constitutively should provide a constant source of IL-5 in a physiologically relevant manner. Two different approaches have been used to produce transgenic mice expressing IL-5. First, the IL-5 gene was ligated to the human CD2 locus control region (LCR) to give constitutive expression of IL-5 by all T cells (9). Second, the IL-5 cDNA was ligated to the mouse metallothionein promoter to give a low-level constitutive production of IL-5 from spleen, liver, kidney, and bone marrow. Treatment with heavy-metal ions induced expression of the metallothionein promoter and increased the serum IL-5 levels by about fivefold (25).
Identification of HPV types 6 and 11 in skin tags using PCR
Published in Cut Adeya Adella, Stem Cell Oncology, 2018
J. Karayana, N.K. Jusuf, I.B. Putra
All of the samples were subjected to PCR, using primers specific to the HPV genome. The presence of HPV type 6 was investigated by PCR amplification of 258-361 bp (base pairs) target from the locus control region (LCR) using the sequence forward primer 5’-TAG- GGGACGG TCCTCTATTC-3’ and reverse primer 5’-GCAACAGCCTCTGA GTCACA-3’. The presence of HPV type 11 was investigated by PCR amplification of 356 bp target from the L1 region using the sequence forward primer 5’-GAATACATGCGCCATGTGGA-3’ and reverse primer 5’-AGCAG ACGTCCGTCCTC GAT-3’. The reaction was carried out in a volume of 20 pl containing 12 ul PCR fast ready mix (KAPA 2G®), 2.5 pl forward primer, 2.5 pl reverse primer, and 5 ul DNA isolate. The amplification cycles comprised 1 min at 95°C for initial denaturation, 15 seconds at 95°C for denaturation, 15 seconds at 55-65°C for annealing, 5 seconds/kb at 72°C for extension, and 10 mins at 72°C for the final extension using a thermocycler (Applied BiosynthesisTm Veriti 384®).
Novel Insights into the Pathophysiology and Treatment of Sickle Cell Disease
Published in Hemoglobin, 2023
Aderson da Silva Araújo, Ana Cristina Silva Pinto, Clarisse Lopes de Castro Lobo, Maria Stella Figueiredo, Sandra Fátima Menosi Gualandro, Sara Teresinha Olalla Saad, Rodolfo Delfini Cancado
The control of the expression of HbF in SCD patients is another avenue of research. The globin genes are under the control of distal regulatory elements, a cluster of enhancers called the locus control region, located approximately 60 kb upstream of the genes. Chromatin looping takes place between distal enhancers and globin gene promoters to regulate transcription; the resulting three-dimensional chromatin organization is dynamic and correlates with the transcriptional activity of globin genes [21]. One of the major regulators of the switch from HbF to HbA (or HbS in SCD patients) is BCL11a, a zinc finger protein that acts as a chromatin modifier and transcriptional repressor of HbF [22]. Several genetic approaches to interfere with BCL11a expression and binding sites are ongoing (see below) [8].
Targeting fetal hemoglobin expression to treat β hemoglobinopathies
Published in Expert Opinion on Therapeutic Targets, 2022
Human β-like globin genes are in a cluster on the short arm of chromosome 11 at p15.4. Their expression is controlled by a super-enhancer upstream of the ε-globin gene (HBE) called the locus control region or LCR. (Figure 2) The LCR loops to globin gene promoters, and along with many recruited protein complexes, governs developmental and high-level globin-gene expression. HbF is encoded by two closely linked and structurally similar genes, HBG2 and HBG1 (referred to herein as HBG). A switch from HbF to HbA (α2βA2) expression starts midway through gestation and by age 6 mos. Is complete with HbF constituting <1% of total hemoglobin. Levels of HbF in sickle cell anemia are much higher than those of normal people and take 5 to 10 years to stabilize depending on the HBB haplotype. In β0 thalassemia, even though HbF is the only hemoglobin present except for a small amount of HbA2 its levels are almost always insufficient for normal life.
Effect of deletions in the α-globin gene on the phenotype severity of β-thalassemia
Published in Hemoglobin, 2022
Dipankar Saha, Prosanto Kr. Chowdhury, Amrita Panja, Debashis Pal, Kaustav Nayek, Gispati Chakraborty, Prashant Sharma, Reena Das, Surupa Basu, Raghunath Chatterjee, Anupam Basu
Thalassemia syndromes are inherited monogenic disorders, primarily associated with anemia. Patients suffering from thalassemia display a wide variation in clinical presentations such as the age of presentation, transfusion requirements, hepatosplenomegaly and others. This ailment is caused by inherited mutations primarily in the β-globin gene (HBB) that limit the synthesis of hemoglobin (Hb) [1]. In the HBB gene, about 400 different aberrant loci have been identified that are responsible for β hemoglobinopathies or β-thalassemia (β-thal) [2]. Based on their position and nature, they result in β-thal major (β-TM) or β-thal intermedia (β-TI) types of β hemoglobinopathies. Mutations on the HBB gene include defective splicing or early frameshifts, resulting in complete disruption of mRNA synthesis, thus categorized as β0 mutations. On the other hand, certain substitution mutations may lead to alternative amino acids being placed or mutations in the promoter or locus control region (LCR), which may lead to retarded synthesis of the β chain, categorized as β+ mutations [3]. However, it has been observed in different populations, that the phenotypic heterogeneity and variations in clinical presentation and manifestations are not always explainable by the mutations solely on the HBB gene [4,5]. This leads to a diagnostic and therapeutic dilemma.