Rosalba Di Marzo

A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference

The application of RNA interference (RNAi) to stem cell–based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant γ-globin gene. Expression of both γ-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage–specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34+ cells from patients with sickle cell anemia led to erythroid-specific expression of the γ-globin transgene and concomitant reduction of endogenous βS transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

The spectrum of β-thalassaemia mutations in Sicily

To characterize β‐thalassaemia genes among the Sicilian population we have previously determined the DNA haplotypes in the β‐globin gene cluster of 99 β‐thal chromosomes. We found seven haplotypes, although 95 of 99 β‐thal chromosomes contained framework 1 and framework 3 β genes. We have now determined the mutation in all 99 of these β‐thal genes by the use of oligonucleotide hybridization, PCR‐amplification and direct genomic sequencing, and direct restriction analysis. Our results indicate that (1) the β0‐39 mutation is most frequent (35%): (2) β0‐39, IVS‐1 nt 110 and IVS‐1 nt 6 mutations account for 90% of β‐thal genes: (3) the IVS‐1 nt 6 mutation is more frequent in thalassaemia intermedia (77%) than in Cooleys disease (34%): (4) the association between haplotypes and specific mutations is imperfect, but mutation spread has occurred within haplotypes containing the same β‐gene framework: (5) the β0‐39 and the IVS‐1 nt 6 mutations, with a mutation spread to two major haplotypes, may be older than the IVS‐1 nt 110 mutation: (6) these data make possible first‐trimester prenatal diagnosis in many families (85%) in Sicily using only three pairs of oligonucleotides. In addition, a new mutation, a frameshift at codon 76 due to loss of a C residue, was found in a single β‐thal chromosome.

Age at diagnosis as an indicator of eligibility for BRCA1 DNA testing in familial breast cancer

This paper reports the results of 1428 β-thalassemia chromosomes studied in Sicily during a hemoglobinopathy control program starting in 1983. Molecular screening was performed by direct restriction enzyme analysis, allele specific oligonucleotide (ASO) hybridization, reverse dot blot analysis (RDB) and, for the rare or new mutations, by direct sequencing of polymerase chain reaction (PCR) products. Using these approaches 1410 (98.7%) out of 1428 β-globin gene defects were characterized, involving 22 different β-thalassemia mutations. Three of these were present at high frequency (β∘39, IVS1, 110 and IVS1,6); the other β-globin gene defects were found at low frequency. In the latter, we found a smaller group of mutations at a frequency lower than 10% (IVS1, 1, IVS2, 745, β S) and a larger one at a frequency lower than 2% [-87, IVS1,2, IVS2,1, fr 6, fr 8 (-AA), fr 44, fr 76, -101, IVS1, 116, IVS1, 3′end G-C, IVS1,5 G-A, IVS1,5 G-C, cod 30, Lepore, deltaβ, β C]. The possible origin of this very large number of mutations is discussed, taking into account the historical point of view. Moreover, this approach has made a first trimester prenatal diagnosis program possible in our region in practically all cases, with a great improvement in general thalassemia management.

Desensitization to hydroxycarbamide following long-term treatment of thalassaemia intermedia as observed in vivo and in primary erythroid cultures from treated patients.

Hydroxycarbamide (HC) is a pharmacological agent capable of stimulating fetal haemoglobin (HbF) production during adult life. High levels of HbF may ameliorate the clinical course of β‐thalassaemia and sickle cell disease. The efficacy of HC for the treatment of thalassaemia major and thalassaemia intermedia is variable. Although an increase of HbF has been observed in most patients, only some patients experience significant improvement in total haemoglobin levels. This study aimed to determine the effectiveness and safety of short‐ (1 year) and long‐term (mean follow‐up 68 months) HC treatment in 24 thalassaemia intermedia patients. Additionally, we evaluated if primary erythroid progenitor cells cultured from treated patients responded to HC treatment in a manner similar to that observed in vivo. Our results confirm a good response to HC after a short‐term follow‐up in 70% of thalassaemia intermedia patients and a reduction of clinical response in patients with a long follow‐up. Erythroid cultures obtained from patients during treatment reproduced the observed in vivo response. Interestingly, haematopoietic stem cells from long‐term treated patients showed reduced ability to develop into primary erythroid cultures some months before the reduction of the ‘in vivo’ response. The mechanism of this loss of response to HC remains to be determined.

Treatment with hydroxycarbamide for intermedia thalassaemia: decrease of efficacy in some patients during long-term follow up

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The Sea Urchin sns5 Insulator Protects Retroviral Vectors From Chromosomal Position Effects by Maintaining Active Chromatin Structure

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

Induction of gamma‐globin gene transcription by hydroxycarbamide in primary erythroid cell cultures from Lepore patients

Increased expression of fetal haemoglobin (HbF) may ameliorate the clinical course of beta‐thalassemia and sickle cell disease. Some pharmacological agents, such as hydroxycarbamide (HC), can increase fetal haemoglobin synthesis during adult life. Cellular selection and/or molecular mechanisms have been proposed to account for this increase. To explore the mechanism of action of HC we focused on homozygous Hb‐Lepore patients that presented with high fetal haemoglobin levels and were good responders to HC treatment “in vivo”. We performed primary erythroid cultures from peripheral blood of four homozygous Lepore patients. The increase in HBG (γ‐globin) transcription levels and HbF content in these cultures, after HC treatment, were detected by quantitative real time polymerase chain reaction analysis and flow cytometric analysis. Primary transcript “in‐situ” hybridization analysis showed a 2‐fold increase in the number of cells expressing both HBG alleles in HC‐treated erythroid cultures. These studies, demonstrating the larger number of biallelic HBG expressing cells, suggest that HC is able to stimulate the activation of HBG transcription. These observations provide evidences that the molecular mechanism of action is involved in the increase of fetal haemoglobin production by HC.

Quantification of HBG mRNA in primary erythroid cultures: prediction of the response to hydroxyurea in sickle cell and beta‐thalassemia

Increased expression of fetal hemoglobin (HbF) may ameliorate the clinical course of hemoglobinopathies like sickle cell disease (SCD) and β‐thalassemia. Hydroxyurea (HU) can stimulate HbF production in these diseases but the response is highly variable indicating the utility of developing an in vitro test to predict the patients response to HU. We assessed whether the HbF response of patients with SCD and thalassemia intermedia (TI) to HU correlates with HBG (both γ‐globin genes) expression in their cultured erythroid progenitors following exposure to HU.

Efficacy of Rapamycin as Inducer of Hb F in Primary Erythroid Cultures from Sickle Cell Disease and β-Thalassemia Patients.

Abstract Phenotypic improvement of hemoglobinopathies such as sickle cell disease and β-thalassemia (β-thal) has been shown in patients with high levels of Hb F. Among the drugs proposed to increase Hb F production, hydroxyurea (HU) is currently the only one proven to improve the clinical course of these diseases. However, Hb F increase and patient’s response are highly variable, indicating that new pharmacological agents could be useful for patients not responding to HU or showing a reduction of response during long-term therapy. In this study we evaluated the efficacy of rapamycin, a lypophilic macrolide used for the prevention of acute rejection in renal transplant recipients, as an inducer of Hb F production. The analyses were performed in cultured erythroid progenitors from 25 sickle cell disease and 25 β-thal intermedia (β-TI) patients. The use of a quantitative Real-Time-polymerase chain reaction ReTi-PCR technique and high performance liquid chromatography (HPLC) allowed us to determine the increase in γ-globin mRNA expression and Hb F production in human erythroid cells treated with rapamycin. The results of our study demonstrated an increase in vitro of γ-globin mRNA expression in 15 sickle cell disease and 14 β-TI patients and a corresponding Hb F increase. The induction by rapamycin, even if lower or similar in most of samples analyzed, in some cases was higher than HU. These data suggest that rapamycin could be a good candidate to be used in vivo for the treatment of hemoglobinopathies.