Biomedical subjects

Marta Serafini

Publications and source records attributed to Marta Serafini.

5 recordsLinked to original sources

PubMed2025

Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability.

BACKGROUND: Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality. METHODS: We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness. RESULTS: Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use. CONCLUSIONS: Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

Humans↗

PubMed2007

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells.

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Animals↗

PubMed2006

Pluripotency in adult stem cells: state of the art.

For many years, it has long been known that stem cells derived from adult tissues maintain the capacity for self-renewal and differentiation into multiple cell types that are characteristic of the tissue of origin. Recent studies have shown new evidence that several tissues may contain cells capable of generating differentiated cells beyond their own tissue boundaries, defining a process termed stem cell plasticity. The pluripotency of adult stem cells have evoked significant excitement over the possibility of novel functional uses of stem cells, with the final purpose to develop new and more effective treatment strategies. However, despite the number of promising studies describing the plasticity of adult stem cells, many questions remain to be answered. In this article, we critically review the current state of the art in the field of adult stem cells, focussing on the present understanding of the concepts of stem cell pluripotency and plasticity.

Adult Stem Cells↗

PubMed2004

Elongation factor 1 (EF1alpha) promoter in a lentiviral backbone improves expression of the CD20 suicide gene in primary T lymphocytes allowing efficient rituximab-mediated lysis.

BACKGROUND AND OBJECTIVES: CD20 has been proposed as a novel suicide gene system for the treatment of graft-versus-host disease (GVHD), a fatal complication of allogeneic bone marrow transplantation: indeed expression of the human non-immunogenic exogenous CD20 protein allows positive immunoselection of transduced cells as well as their killing in vitro with rituximab. Lentiviral vectors are promising tools in the field of gene therapy. We therefore searched for a lentivector giving good efficiency of transduction of human T lymphocytes activated by the sole addition of interleukin (IL)-2 and high expression levels of the CD20 transgene. DESIGN AND METHODS: The T cell line CEM and peripheral T lymphocytes activated by phytohemagglutinin (PHA) and/or IL-2 were transduced with two different vectors carrying the CD20 transgene driven by either the phosphoglycerate kinase (PGK) or elongation factor 1alpha (EF1alpha) promoter, and using different multiplicities of infection (MOIs). RESULTS: Both the PGK- and EF1alpha-CD20 vectors allowed efficient transduction of the CEM cell line and PHA-activated T cells, reaching 99 and 90% in the different targets, respectively. However EF1alpha-CD20 led to much higher expression levels of the transgene (mean fluorescence intensity 588-618 compared to 53 for PGK-CD20). Furthermore lymphocytes activated with IL-2 alone could be efficiently transduced with EF1alpha-CD20, reaching 10-25% positivity for CD20 (mean fluorescence intensity 409-424), allowing adequate immunoselection and strong complement-mediated lysis. INTERPRETATION AND CONCLUSIONS: EF1alpha-CD20 may represent a good candidate vector for gene therapy with the CD20 suicide system in the setting of allogeneic bone marrow transplants.

Antibodies, Monoclonal↗

PubMed2002

A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

B-Lymphocytes↗