Small nuclear ribonucleoproteins (snRNPs) based gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Antisense Oligonucleotides ◽  
Mammalian Cells ◽  
Globin Gene ◽  
Premature Stop Codon ◽  
Critical Element ◽  
Globin Mrna ◽  
Exon 2 ◽  
U7 Snrnp ◽  
Hiv 1

In 1968, Weinberg and Penman initially coined the term snRNA. Splicing is the process of eliminating introns from pre-RNA and combining exons The two forms of splicing are constitutive and alternative. U7 snRNP is a critical element in the unique 3′ end processing of replication-dependent histone (RDH) premRNAs. U7 Sm OPT uses antisense oligonucleotides to control pre-mRNA splicing. U7 snRNP has shown potential in preclinical studies and human clinical trials. DMD, ALS, thalassemia, HIV-1 infection, and spinal muscular atrophy are excellent illustrations of the majority of the problems (SMA) SmD1 and SmD2, which are found in other U snRNPs, are replaced by Lsm10 and Lsm11, respectively. There are just 500 molecules of U7SnRNP in a cell. Interesting because of its size, great stability, and tendency to collect in the nucleus of U 7 snRNA. The snRNP particle may hybridize to practically any RNA sequence in the nucleoplasm by altering the motif.U7 snRNA gene therapy is often employed to repair splicing abnormalities. The utilization of U7 Sm OPT-implanted antisense oligonucleotides has a multitude of potential therapeutic applications. More effective are the locations that bind U1 and U2 snRNPs to suppress splicing. useful for addressing splicing mistakes in muscular dystrophy, DMD, ALS, thalassemia, HIV-1 infection, and SMA PTCH1, BRCA1, and CYP11A have all been fixed with it. X-linked recessive muscular wasting illness, DMD. Individuals with exon 2 deletions either have asymptomatic or mildly symptomatic dystrophin levels. ANTISENSE oligonucleotides were introduced into the U7 Sm OPT and given via AAV to treat patients. These cells missed exon 2, resulting in an alternative translation starting at exon 6. (through an internal ribosome entrance region) The NIH's next clinical study will commence in January of 2020. U7 Sm OPT bifunctional gene therapy was successful in treating muscular dystrophy.Superoxide dismutase 1 (SOD1) gene mutations cause amyotrophic lateral sclerosis (ALS). SOD1 function was restored in a single study using U7Sm OPT to help rats with ALS. When given at birth, this medicine postponed sickness onset and enhanced life expectancy by 92% and 58%, respectively. Mutations in intron 2 produce a premature stop codon and hinder translation of full-length globin. Antisense oligonucleotides that span this region target nucleotides 102 to 130 of globin mRNA exon 1.5′ or 3′ splice site oligonucleotides in mammalian cells have been found to fix globin mRNA. The 654T > G mutation causes severe thalassemia symptoms. Combining U7 Sm OPT with induced pluripotent stem cells (iPSCs) led to a successful decrease of the globin gene

Development of Viral Vectors for Gene Therapy of β-Chain Hemoglobinopathies: Optimization of a γ-Globin Gene Expression Cassette

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2208-2216 ◽  
Author(s):  
Qiliang Li ◽  
David W. Emery ◽  
Magali Fernandez ◽  
Hemei Han ◽  
George Stamatoyannopoulos
Keyword(s):  
Gene Therapy ◽  
Globin Gene ◽  
Expression Cassette ◽  
Globin Genes ◽  
Internal Deletion ◽  
Globin Mrna ◽  
Retrovirus Vector ◽  
Globin Promoter ◽  
Intron 2 ◽  
Β Chain

Progress toward gene therapy of β-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of theAγ- and β-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that theAγ-globin promoter could be deleted to −159 without affecting expression, while deleting the β-globin promoter to −127 actually increased expression compared with longer fragments. Expression from the optimal β-globin gene promoter was consistently higher than that from the optimal Aγ-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the β-globin locus control region (termed a μLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the -globin locus. All three enhancers increased expression from the β-globin gene to roughly the same extent, while the HS-40 element was notably less effective with theAγ-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Aγ-globin gene linked to the β-globin promoter. Inclusion of extended 3′ sequences from either the β-globin or the Aγ-globin genes had no significant effect on expression. A 714-bp internal deletion ofAγ-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a −127 β-globin promoter, anAγ-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, γ-globin expression averaged 166% of murine -globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine -globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for γ-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

scholarly journals The Human ARF Cell Cycle Regulatory Gene Promoter Is a CpG Island Which Can Be Silenced by DNA Methylation and Down-Regulated by Wild-Type p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6457-6473 ◽  
Author(s):  
Keith D. Robertson ◽  
Peter A. Jones
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Mammalian Cells ◽  
Cpg Island ◽  
Regulatory Gene ◽  
Critical Element ◽  
Wild Type ◽  
Exon 2 ◽  
P53 Expression ◽  
Wild Type P53

ABSTRACT The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check.

scholarly journals Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

1997 ◽  
Vol 17 (5) ◽  
pp. 2698-2707 ◽  
Author(s):  
N M Yeilding ◽  
W M Lee
Keyword(s):  
Myogenic Differentiation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
C2c12 Cells ◽  
Regulatory Function ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Exon 2 ◽  
Coding Sequences

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

Development of Viral Vectors for Gene Therapy of β-Chain Hemoglobinopathies: Optimization of a γ-Globin Gene Expression Cassette

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2208-2216 ◽  
Author(s):  
Qiliang Li ◽  
David W. Emery ◽  
Magali Fernandez ◽  
Hemei Han ◽  
George Stamatoyannopoulos
Keyword(s):  
Gene Therapy ◽  
Globin Gene ◽  
Expression Cassette ◽  
Globin Genes ◽  
Internal Deletion ◽  
Globin Mrna ◽  
Retrovirus Vector ◽  
Globin Promoter ◽  
Intron 2 ◽  
Β Chain

Abstract Progress toward gene therapy of β-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of theAγ- and β-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that theAγ-globin promoter could be deleted to −159 without affecting expression, while deleting the β-globin promoter to −127 actually increased expression compared with longer fragments. Expression from the optimal β-globin gene promoter was consistently higher than that from the optimal Aγ-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the β-globin locus control region (termed a μLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the -globin locus. All three enhancers increased expression from the β-globin gene to roughly the same extent, while the HS-40 element was notably less effective with theAγ-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Aγ-globin gene linked to the β-globin promoter. Inclusion of extended 3′ sequences from either the β-globin or the Aγ-globin genes had no significant effect on expression. A 714-bp internal deletion ofAγ-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a −127 β-globin promoter, anAγ-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, γ-globin expression averaged 166% of murine -globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine -globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for γ-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

scholarly journals Repair of thalassemic human  -globin mRNA in mammalian cells by antisense oligonucleotides

1996 ◽  
Vol 93 (23) ◽  
pp. 12840-12844 ◽  
Author(s):  
H. Sierakowska ◽  
M. J. Sambade ◽  
S. Agrawal ◽  
R. Kole
Keyword(s):  
Antisense Oligonucleotides ◽  
Mammalian Cells ◽  
Globin Mrna

scholarly journals Regulated expression of amplified human beta globin genes

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 733-739 ◽  
Author(s):  
D Rund ◽  
C Dobkin ◽  
A Bank
Keyword(s):  
Gene Therapy ◽  
Mrna Expression ◽  
Globin Gene ◽  
Serial Passage ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Neomycin Resistance

Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.

Functional requirements for phenotypic correction of murine β-thalassemia: implications for human gene therapy

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3275-3282 ◽  
Author(s):  
Derek A. Persons ◽  
Esther R. Allay ◽  
Denise E. Sabatino ◽  
Patrick Kelly ◽  
David M. Bodine ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mrna Expression ◽  
Cell Morphology ◽  
Human Gene ◽  
Globin Gene ◽  
Therapeutic Benefit ◽  
Red Cell ◽  
Globin Mrna ◽  
Human Gene Therapy ◽  
Extramedullary Erythropoiesis

Abstract As initial human gene therapy trials for β-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve β-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe β-thalassemia. Generation of β-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, β-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human γ-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous α-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, γ-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total α-globin mRNA in patients with severe β-thalassemia in whom 20% of erythroid precursors express the vector genome.

Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3261-3261
Author(s):  
Karina Laflamme ◽  
Laura Elnitski ◽  
Ashley N. Owen ◽  
Patrick G. Gallagher ◽  
David M. Bodine
Keyword(s):  
Gene Therapy ◽  
Globin Gene ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Wild Type ◽  
Binding Region ◽  
Globin Mrna ◽  
Primary Mouse ◽  
Double Copy

Abstract Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are >20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of >4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Following Beta-Globin Gene Transfer, the Production of Hemoglobin Depends Upon the Beta-Thalassemia Genotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 978-978 ◽  
Author(s):  
Laura Breda ◽  
Carla Casu ◽  
Laura Casula ◽  
Dorothy A. Kleinert ◽  
Nicoletta Bianchi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Alternative Splicing ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Globin Gene ◽  
Dependent Manner ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Splicing Mutations ◽  
Murine Erythroleukemia

Abstract Abstract 978 Preclinical studies showed that β-thalassemia can be cured in mice by lentiviral-mediated transfer of the human β-globin gene. Based on these studies, clinical trials have been proposed or are underway. However, to date no study has addressed the efficacy of gene therapy in relationship to the different nature of the β-globin mutations. To address this question, we developed a new pre-clinical approach to predict the potential outcome of gene transfer in vivo, testing erythroid progenitor cells (ErPC) of a large group of β-thalassemic patients. The β0-39 and β+ IVS1-110 are two of the mutations represented in the majority of the patients enrolled in our study. Specifically, the β0-39 mutation modifies codon 39 into a premature termination codon, which, in the mutated mRNA, is easily identified and degraded by the non-sense mediated mRNA decay machinery. This and similar mutations leading to absent β-globin synthesis are defined as β0. The β+ IVS1-110 mutation, instead, activates an aberrant 3' cryptic splice site without completely abolishing normal splicing. For this reason some normal mRNA and protein is made and mutations such as this are classified as β+. We divided all patients, homozygous or compound heterozygous for β0 and β+ mutations, into three groups: β0/0, β+/+ or β+/0. Cells from patients (N=33) were infected with T9W, a lentiviral vector carrying the human β-globin gene and large elements from the human LCR, which was previously shown to cure thalassemic mice. Differentiated ErPCs of β0/0 patients (N=10), infected with T9W, increased their HbA levels in a vector-copy-number (VCN) dependent manner, reaching values similar to the expected normal ones. Conversely, ErPCs of β+/0 and β+/+ patients (N:23) responded only slightly or not at all, even though the transgenic β-globin mRNA was highly expressed and α-globin aggregates were observed. We observed that the β+ IVS1-110 mRNA appears more stable, compared to the β0-39 mRNA, representing, on average, 49% of the total β-globin mRNA. Similar results were observed with other alternative β+ splicing mutations analyzed. This led us to hypothesize that the aberrant mRNAs, generated by the alternative splicing, prevent the transgenic β-globin mRNA from being translated. We generated a second lentiviral vector in which a human ankyrin regulatory element flanks the transgenic human β-globin cassette (AnkT9W), after chromosomal integration. We compared T9W versus AnkT9 in murine erythroleukemia (MEL) cells with comparable VCN. AnkT9W expressed up to 3.5 times more human β-globin mRNA and produced nearly two times more absolute chimeric Hb (α-mouse:β-human). Furthermore, AnkT9W-RNA fractions in the cytoplasm had a net shift toward the highest multi-polysomal component, implying a translational advantage. In mice, compared to T9W, AnkT9W markedly improved the phenotype of thalassemic animals engrafted with lentiviral transduced thalassemic bone marrow cells. And finally, AnkT9W completely rescued HbA synthesis in cells of patients (N=5) carrying alternative splicing mutations, with HbA increasing according to VCN and simultaneously reducing the amount of α-globin aggregates. No major differences in HbA synthesis were observed in β0/0 cells infected with T9W or AnkT9W. We believe that the ankyrin element is responsible for increasing transcription of the β-globin transgenic mRNA during the early phases of erythroid differentiation, efficiently competing with stable aberrant mRNAs for translation. We are performing chromatin analyses and generating new vectors to further characterize the function of the ankyrin element. These new findings may have profound implications in designing gene therapy trials and in understanding the genotype/phenotype variability observed in β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1022-1022 ◽  
Author(s):  
Raechel Peralta ◽  
Audrey Low ◽  
Aneeza Kim ◽  
Sue Murray ◽  
Shuling Guo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mrna Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Antisense Oligonucleotides ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Mrna ◽  
Dose Dependent

Abstract Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis. B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression. To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures: Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

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