Nancy E. Seidel

Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis

Defects of the ankyrin-1 gene are the most common cause in humans of hereditary spherocytosis, an inherited anemia that affects patients of all ethnic groups. In some kindreds, linked -108/-153 nucleotide substitutions have been found in the upstream region of the ankyrin gene promoter that is active in erythroid cells. In vivo, the ankyrin erythroid promoter and its upstream region direct position-independent, uniform expression, a property of barrier insulators. Using human erythroid cell lines and primary cells and transgenic mice, here we have demonstrated that a region upstream of the erythroid promoter is a barrier insulator in vivo in erythroid cells. The region exhibited both functional and structural characteristics of a barrier, including prevention of gene silencing in an in vivo functional assay, appropriate chromatin configuration, and occupancy by barrier-associated proteins. Fragments with the -108/-153 spherocytosis-associated mutations failed to function as barrier insulators in vivo and demonstrated perturbations in barrier-associated chromatin configuration. In transgenic mice, flanking a mutant -108/-153 ankyrin gene promoter with the well-characterized chicken HS4 barrier insulator restored position-independent, uniform expression at levels comparable to wild-type. These data indicate that an upstream region of the ankyrin-1 erythroid promoter acts as a barrier insulator and identify disruption of the barrier element as a potential pathogenetic mechanism of human disease.

Improved Amphotropic Retrovirus‐Mediated Gene Transfer into Hematopoietic Stem Cells

Abstract: The efficiency of amphotropic retrovirus‐mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. 1–3 In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7‐fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30‐fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G‐CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10‐fold increase in bone marrow HSC 14 days after treatment. 4 In this report we show that when rhesus monkey bone marrow cells collected 14 days after G‐CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

Exploiting Pre-rRNA Processing in Diamond Blackfan Anemia Gene Discovery and Diagnosis

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single‐copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre‐rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre‐rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre‐60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene. Am. J. Hematol. 89:985–991, 2014.

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.

Development of a Stable Retrovirus Vector Capable of Long‐Term Expression of γ‐Globin mRNA in Mouse Erythrocytes

Abstract: Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human γ‐globin gene showed position‐independent, copy number‐dependent expression of a linked γ‐globin mRNA. We generated a “double‐copy” Ank/Aγ‐globin retrovirus vector that transferred two copies of the Ank/Aγ‐globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long‐term repopulating hematopoietic stem cells. Expression of Ank/Aγ‐globin mRNA in mature red blood cells was approximately 8% of the level of mouse α‐globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Development of a High‐Titer Retrovirus Producer Cell Line and Strategies for Retrovirus‐mediated Gene Transfer into Rhesus Monkey Hematopoietic Stem Cells

Retroviral-mediated gene transfer into pluripotent hematopoietic stem cells has been difficult to achieve in large animal models. We have compared several infection protocols in a murine model system and concluded that bone marrow can be maintained and infected in vitro for 2-6 days. We have also developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (CFU) per milliliter of culture medium. Autologous rhesus monkey bone marrow cells were co-cultured with either high- (2 x 10(10) CFU/ml) or low- (5 x 10(6) CFU/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone marrow cells from all three animals reconstituted with cells co-cultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow co-cultured with the low-titer producer clone exhibited no evidence of gene transfer.