Maria Ester Bernardo

The role of conditioning in hematopoietic stem cell gene therapy.

Gene therapy (GT) approaches based on autologous hematopoietic stem cells (HSC) corrected ex vivo have shown therapeutic benefit in a number of inherited disorders. GT bares the advantage of allowing each patient to be her/his own donor while reducing the risks of immune-mediated complications as compared with allogeneic hematopoietic stem-cell transplantation (HSCT). In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. With regard to HSC-GT for inherited genetic disorders, the ideal conditioning regimen should aim to contain toxicity by reducing the dosage and/or the number of chemotherapeutic agents administered, in comparison to fully myeloablative preparations employed in conventional allogeneic HSCT. To meet this aim, a profound knowledge of the disease-specific biological background and of the therapeutic transgene levels, as well as of the key principles of transplantation, are required. While low-dose conditioning is sufficient to create a mixed chimerism when gene-corrected cells are endowed with a natural selective advantage, such as in the case of immune deficiencies, myeloablative doses are necessary when high levels of engraftment are required in disease such as lysosomal storage disorders and beta thalassemia. Therefore, the intensity and type of conditioning regimen administered to patients undergoing HSC-GT should be tailored to reach a minimal efficacious therapeutic target level while sparing toxicity. Novel strategies based on monoclonal antibodies selectively depleting blood cells and associated with limited extramedullary toxicity might be successfully employed in the context of HSC-GT in the near future. This review focuses on the role of the conditioning regimen in HSC-GT, and in particular, it highlights the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome.

A new enzyme-linked immunosorbent assay for a total anti-T lymphocyte globulin determination: Development, analytical validation, and clinical applications

Background: Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versus-host disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with &bgr;-Thalassemia, receiving allogeneic HSCT. Methods: Diluted serum samples were incubated with Goat-anti-Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200–0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with &bgr;-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days −5, −4, and −3 before HSCT (day 0). Blood sampling for PK evaluation was performed on days −5, −4, and −3 before and after drug infusion; and then from day −2 to +56. Results: The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day −5; 85.9 and 199.2 mcg/mL on day −4; 153 and 270.9 mcg/mL on day −3, respectively. The median PK values of CL was 0.0029 (range: 0.0028–0.0057) L·kg−1·d−1, Vd was 0.088 (range: 0.025–0.448) L/kg and t1/2 was 20.2 (range: 5.8–50.2) days. Conclusions: These data suggest that given the marked interindividual variability of total ATLG disposition, the development of a validated ELISA method and the possibility to measure PK parameters in paediatric populations are essential steps to optimize drug therapeutic regimens.

Phenotypic and functional characterization of mesenchymal stromal cells isolated from pediatric patients with severe idiopathic nephrotic syndrome

BACKGROUNDnIdiopathic nephrotic syndrome (INS) is one of the most common renal diseases in the pediatric population; considering the role of the immune system in its pathogenesis, corticosteroids are used as first-line immunosuppressive treatment. Due to its chronic nature and tendency to relapse, a significant proportion of children experience co-morbidity due to prolonged exposure to corticosteroids and concomitant immunosuppression with second-line, steroid-sparing agents. Mesenchymal stromal cells (MSCs) are multipotent cells that represent a key component of the bone marrow (BM) microenvironment; given their unique immunoregulatory properties, their clinical use may be exploited as an alternative therapeutic approach in INS treatment.nnnMETHODSnIn view of the possibility of exploiting their immunoregulatory properties, we performed a phenotypical and functional characterization of MSCs isolated from BM of five INS patients (INS-MSCs; median age, 13 years; range, 11-16 years) in comparison with MSCs isolated from eight healthy donors (HD-MSCs). MSCs were expanded ex vivo and then analyzed for their properties.nnnRESULTSnMorphology, proliferative capacity, immunophenotype and differentiation potential did not differ between INS-MSCs and HD-MSCs. In an allogeneic setting, INS-MSCs were able to prevent both T- and B-cell proliferation and plasma-cell differentiation. In an in-vitro model of experimental damage to podocytes, co-culture with INS-MSCs appeared to be protective.nnnDISCUSSIONnOur results demonstrate that INS-MSCs maintain the main biological and functional properties typical of HD-MSCs; these data suggest that MSCs may be used in autologous cellular therapy approaches for INS treatment.

MSCs for Gastrointestinal Disorders

Recent experimental findings and clinical trials have shown the ability of mesenchymal stromal cells (MSCs) to home to damaged tissues and to produce paracrine factors with anti-inflammatory properties, resulting in reduction of inflammation and functional recovery of the damaged tissues. These properties, in the context of regenerative medicine, are being tested in chronic inflammatory disorders of the gastrointestinal (GI) tract. Phase I–II clinical trials indicate that intravenous and local injection of ex vivo-expanded MSCs are feasible and safe in luminal and fistulizing Crohn’s Disease (CD) and in end-stage liver diseases. Large randomized clinical trials are warranted to properly establish the role of MSC therapy for these diseases, in comparison with conventional treatment. This chapter focuses on recent research on the anti-inflammatory/reparative properties of MSCs and discusses the potential clinical applications of MSC-based cellular therapy in GI disorders.

Immunomodulatory Properties of MSCs

Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from several human tissues and expanded ex vivo for clinical use. They comprise a heterogeneous population of cells, which, through production of growth factors, cell-to-cell interactions and secretion of matrix proteins, play a key role in the regulation of haematopoiesis. In recent years, several experimental studies have shown that MSCs are endowed with potent immunomodulatory properties directed in vitro at all cells involved in immune responses. Due to their immunomodulatory and engraftment-promoting properties, MSCs have been tested in the clinical setting both to facilitate haematopoietic engraftment and to treat steroid-resistant acute graft-versus-host disease (GvHD). More recently, experimental findings and clinical trials have focused on the ability of MSCs to home to injured tissues and to produce paracrine factors with anti-inflammatory properties, resulting in functional recovery of damaged tissues. The mechanisms through which MSCs exert this pleomorphic therapeutic potential rely on some key properties of these cells: the capacity to home to sites of injury, the ability to blunt exaggerated immune responses and the ability to secrete soluble factors capable of stimulating both the survival and recovery of injured cells. This chapter focuses on recent advances in MSC biology and summarises the clinical studies on their immunomodulatory and anti-inflammatory properties, particularly in the setting of allo- and autoimmune disorders.

MSCs in Pediatric Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is an accepted treatment for some children with certain life-threatening conditions such as malignant diseases, immune deficiency, inborn errors of metabolism, bone marrow failure syndromes, and hemoglobinopathies. The introduction of alternative donor population such as cord blood and haploidentical transplantations has resulted in this treatment becoming more readily available. Inherent risks with alternative donor sources are mainly immune-mediated complications, with graft rejection or graft failure and graft-versus-host disease (GVHD) as main obstacles. Mesenchymal stromal cells (MSCs) through cell-to-cell interactions, production of growth factors, and secretion of matrix proteins play a vital role in the regulation of hematopoiesis and exhibit a wide range of immunomodulatory and anti-inflammatory properties in vitro and in vivo. Use of MSCs in phase I/II clinical studies with HSCT in children indicates that their infusion is safe and effective in preventing graft failure after T-cell-depleted allogeneic HSCT from an HLA-disparate relative as well as in rescuing patients with severe, steroid-refractory GVHD.