Dorothy A. Kleinert

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Toward optimizing the use of deferasirox: potential benefits of combined use with deferoxamine

Patients with β-thalassemia require iron chelation therapy to protect against progressive iron overload and non-transferrin-bound iron. Some patients fail to respond adequately to deferoxamine and deferasirox monotherapy while others have side effects which limit their use of these drugs. Since combining deferiprone and deferoxamine has an additive effect, placing all patients into net negative iron balance, we investigated the possibility that combining deferasirox and deferoxamine would lead to similar results. We conducted 34-day metabolic iron balance studies in six patients in whom the relative effectiveness of deferasirox (30 mg/kg/day) and deferoxamine (40 mg/kg/day) was compared, alone and in combination. Patients consumed fixed low-iron diets; daily urinary and stool iron excretion were determined by atomic absorption. Red blood cell transfusions were given prior to each drug treatment to minimize the effects of ineffective erythropoiesis. Serial safety measures, hematologic parameters, serum chemistries, ferritin levels and urinalyses were determined. All patients were in negative iron balance when treated with deferoxamine alone while four of six patients remained in positive balance when deferasirox monotherapy was evaluated. Daily use of both drugs had a synergistic effect in two patients and an additive effect in three others. Five of six patients would be in negative iron balance if they used the combination of drugs just 3 days a week. No significant or drug-related changes were observed in the blood work-ups or urinalyses performed. We conclude that supplementing the daily use of deferasirox with 2 – 3 days of deferoxamine therapy would place all patients into net negative iron balance thereby providing a convenient way to tailor chelation therapy to the individual needs of each patient.

Quality of life in thalassemia: A comparison of SF‐36 results from the thalassemia longitudinal cohort to reported literature and the US norms

Thalassemia is a chronic, inherited blood disorder, which, in its most severe form, causes life-threatening anemia. Advances in treatment have led to increased life expectancy however the need for chronic blood transfusions and chelation therapy remains a significant burden for patients. Our study compared health related quality of life (HRQOL) from the Thalassemia Clinical Research Networks (TCRNs) Thalassemia Longitudinal Cohort (TLC) study to US norms and assessed association with clinical variables. There were 264 patients over age 14 who completed the Medical Outcomes Study 36-Item Short Form Health Survey version 2 (SF36v2) baseline assessment. When compared to US norms, TLC patients had statistically significant (P < 0.05) worse HRQOL on five of the eight subscales (physical functioning, role-physical, general health, social functioning, and role-emotional) and on both summary scales (physical component summary and mental component summary). Women, older patients, and those with more disease complications and side effects from chelation reported lower HRQOL. In general, adolescents and adults with thalassemia report worse HRQOL than the US population, despite contemporary therapy. The SF-36 should become a standard instrument for assessing HRQOL in thalassemia to determine predictors of low HRQOL which may be better addressed by a multidisciplinary team.

Symptoms of depression and anxiety in patients with thalassemia: Prevalence and correlates in the thalassemia longitudinal cohort

Thalassemia is an inherited blood disorder that requires lifelong adherence to a complicated and burdensome medical regimen which could potentially impact emotional functioning of patients. The importance of understanding and promoting healthy emotional functioning is crucial not only to psychological well‐being, but also to physical health as it has been shown to impact adherence to medical regimens [ 1–4 ]. The current study aimed to [ 1 ] determine the prevalence of depressive and anxiety symptoms in adolescent and adult patients with thalassemia; and [ 2 ] explore possible demographic, medical, and psychosocial correlates of these symptoms in 276 patients (14–58 years old, M age = 27.83; 52% female). Overall, most patients did not report experiencing significant symptoms of anxiety and depression (33% of participants indicated experiencing symptoms of anxiety and 11% symptoms of depression). Females and older patients were more likely to experience these symptoms than males and younger patients. Symptoms of anxiety and depression were positively associated with self‐report of difficulty with adherence and negatively associated with quality of life. Given these findings, regular screening for anxiety and depression symptoms could help to identify at‐risk individuals to provide them with appropriate psychological support with the goal of improving both emotional and physical health. Am. J. Hematol., 2010.

Pain as an emergent issue in thalassemia

Thalassemia is a congenital blood disorder often requiring chronic blood transfusions and iron chelation therapy [1,2]. While advances in treatment have resulted in increased life expectancy [3], extended life spans have exposed previously unidentified issues, including bodily pain. The aim of this study was to examine the prevalence, severity, predictors, and effects of pain in 265 adultstadolescents and 103 children with thalassemia. Overall, 69% of adults/adolescents reported bodily pain on the SF-36v2 health survey, with 28% reporting at least moderate pain. Parents reported pain in 56% of children using the PF-28 child health questionnaire, with only 11% reporting pain fairly often. There were no significant differences in pain in children with thalassemia compared with the general population. In adults/ adolescents, pain increased significantly with age (P = 0.005), more so than in the general population. This study highlights the fact that children and young adults with thalassemia experience pain comparable to the general population, whereas older adults (aged 35+) experience greater pain. Our findings show that increased pain is associated with decreased quality of life and increased anxiety and depression.

Red blood cell transfusions for thalassemia: results of a survey assessing current practice and proposal of evidence-based guidelines.

In the absence of curative treatment, such as stem cell transplant, regular transfusions remain the mainstay of therapy for individuals with thalassemia major, a syndrome that results from marked ineffective erythropoiesis and the resultant anemia. The primary objectives of transfusion therapy are twofold: to suppress ineffective erythropoiesis and to ensure appropriate growth and development through childhood. In practice, a number of different transfusion protocols are in use across the developed world, with on‐demand transfusion still being the paradigm in most of the developing world with limited resources.

A preclinical approach for gene therapy of β-thalassemia

Lentiviral‐mediated β‐globin gene transfer successfully treated β‐thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different β‐globin mutations found in patients. Most mutations can be classified as β0 or β+, based on the amount of β‐globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two‐phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human β‐globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooleys anemia patients.

Longitudinal monitoring of cardiac siderosis using cardiovascular magnetic resonance T2* in patients with thalassemia major on various chelation regimens: a 6-year study.

Cardiovascular magnetic resonance (CMR) and hepatic magnetic resonance imaging (MRI) have become reliable noninvasive tools to monitor iron excess in thalassemia major (TM) patients. However, long‐term studies are lacking. We reviewed CMR and hepatic MRI T2* imaging on 54 TM patients who had three or more annual measurements. They were managed on various chelation regimens. Patients were grouped according to their degree of cardiac siderosis: severe (T2*, <10 msec), mild to moderate (T2* = 10–20 msec), and no cardiac siderosis (T2*, >20 msec). We looked at the change in cardiac T2*, liver iron concentration (LIC) and left ventricular ejection fraction (LVEF) at years 3 and 5. In patients with severe cardiac siderosis, cardiac T2* (mean ± SD) improved from 6.9 ± 1.6 at baseline to 13.6 ± 10.0 by year 5, mean ΔT2* = 6.7 (P = 0.04). Change in cardiac T2* at year 3 was not significant in the severe group. Patients with mild to moderate cardiac siderosis had mean cardiac T2* of 14.6 ± 2.9 at baseline which improved to 26.3 ± 9.5 by year 3, mean ΔT2* = 11.7 (P = 0.01). At baseline, median LICs (mg/g dry weight) in patients with severe, mild–moderate, and no cardiac siderosis were 3.6, 2.8, and 3.3, whereas LVEFs (mean ± SD) (%) were 56.3 ± 10.1, 60 ± 5, and 66 ± 7.6, respectively. No significant correlation was noted between Δ cardiac T2* and Δ LIC, Δ cardiac T2*, and Δ LVEF at years 3 and 5. Throughout the observation period, patients with no cardiac siderosis maintained their cardiac T2* above 20 msec. The majority of patients with cardiac siderosis improve cardiac T2* over time with optimal chelation. Am. J. Hematol. 88:652–656, 2013.

Parvovirus B19 Quiescence during the Course of Human Immunodeficiency Virus Infection in Persons with Hemophilia

To detect and characterize parvovirus B19 infection during the course of progressive immune deficiency from human immunodeficiency virus (HIV), ten subjects enrolled in the Multicenter Hemophilia Cohort Study were followed for 6.4 to 15 years from HIV seroconversion through extreme immune deficiency. Four to five sera or plasma samples from each subject, collected at predetermined CD4+ lymphocyte levels, were tested for immunoglobulin G (IgG) and M (IgM) B19 antibodies and DNA. All 42 samples were positive for B19 IgG antibodies, and three were weakly positive for IgM antibodies. Only one sample, collected coincident with HIV seroconversion, was unequivocally positive for B19 DNA. No persistent hematologic adverse effects of B19 infection were observed. Thus, although B19 IgG antibodies are highly prevalent among HIV‐infected persons with hemophilia or related disorders, B19 viremia and its hematologic consequences were not detected, even with severe depletion of CD4+ lymphocytes. If primary B19 infection occurs after immune deficiency, however, the consequences may be more adverse. Am. J. Hematol. 56:248–251, 1997.

Contextually appropriate emotional word use predicts adaptive health behavior: Emotion context sensitivity and treatment adherence.

Emotion context sensitivity is the ability to respond emotionally in a manner that is functionally appropriate for the context in which the emotion arises. This study examined the relationship between emotion context sensitivity and treatment adherence in adults with the chronic illness Thalassemia. Emotional responses were measured by examining the frequency of positive and negative emotional words used to answer two interview questions that created two different emotional contexts. Consistent with previous research on adaptive and contextually appropriate emotions, negative emotion words were related to adherence in the context of the disease itself, while positive emotion words were related to adherence in the context of coping.