Ex Vivo Gene-Edited Cell Therapy for Sickle Cell Disease: Disruption of the BCL11A Erythroid Enhancer with Zinc Finger Nucleases Increases Fetal Hemoglobin in Plerixafor Mobilized Human CD34+ Cells

Abstract Increases in fetal hemoglobin (HbF) are associated with improved clinical outcomes in the inherited hemoglobinopathies. We are developing a novel gene-edited cell therapy to treat patients with sickle cell disease (SCD) and beta-thalassemia (BT) using autologous hematopoietic stem and progenitor cells (HSPCs) genetically modified with zinc finger nucleases (ZFNs) to restore high levels of HbF expression. The ZFNs have been designed to specifically target the GATA motif within an intronic erythroid-specific enhancer (ESE) of BCL11A, the gene encoding a transcriptional regulator of the fetal-to-adult hemoglobin switch. Previously, we reported successful ZFN-mediated, ex vivoBCL11A gene editing in dual mobilized HSPCs, i.e. peripheral stem cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and plerixafor1. The editing procedure was optimized for high on-target/low off-target modification levels and increases in HbF in erythroid progeny. This drug product, ST-400, passed extensive safety testing and is currently in a phase 1/2a clinical trial for transfusion-dependent beta-thalassemia (ClinicalTrials.gov number NCT03432364). An analogous drug product, BIVV003, is being developed as a therapeutic for SCD. In patients with SCD, the use of G-CSF for stem cell mobilization is not recommended due to the risk of clinical complications. Therefore, peripheral HSPCs are obtained from SCD patients via single agent, plerixafor mobilization and apheresis2-5. Understanding the effect of this change in mobilization strategy on ZFN editing efficiency and specificity is a key element in preparing for SCD gene-edited cell therapy. In the present studies, we demonstrate comparability of ZFN editing outcomes in single and dual mobilized HSPCs obtained from healthy donors. In plerixafor mobilized HSPCs from five healthy donors at research scale, ZFN-mediated gene editing induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified, as measured by MiSeq deep sequencing) with high post-editing viability (77%). Similar gene editing efficiencies (>70%) were obtained in HSPCs at clinical manufacturing scale (n=2). Further, in vitro HbF protein levels and HbF+ cell frequencies within erythroid progeny of edited cells were increased by >4 and 3-fold respectively - compared to non-edited cells in the same culture conditions, using reverse phase high-performance liquid chromatography and flow cytometry (n=4 healthy donors at research scale). Single cell clone analysis revealed that ZFN-mediated gene editing targeted both alleles of BCL11A at high frequency (91-94% of edited cells within erythroid progeny) with high levels of replicable GATA-disrupting indel patterns. On average, each edited allele contributed an additional 17.6% increase in HbF production in vitro, with a statistically-significant increase in HbF level for biallelic edited vs. unedited controls (3.4 fold). Critical to BIVV003 use in clinical trials, ZFN-mediated gene editing did not impair single agent mobilized HSPC function in vitro based on measurements of colony forming unit (CFU) production and frequencies of long-term HSC (LT-HSC) and common myeloid progenitor (CMP) cells by flow cytometry. In agreement with this data, injection of BIVV003 into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their differentiated progeny. Overall, these data demonstrate potential efficacy of ZFN-edited HSPCs (BIVV003) as a novel cell therapy for SCD patients.Holmes et al., 2017 (ASH abstract)Fitzhugh et al., 2009Lagresle-Peyrou et al., 2018Hsieh and Tisdale, 2018Yannaki et al., 2012 Disclosures Moran: Bioverativ, a Sanofi Company: Employment. Ling:Bioverativ, a Sanofi Company: Employment. Lessard:Bioverativ, a Sanofi Company: Employment. Vieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ, a Sanofi Company: Employment. Holmes:Sangamo Therapeutics: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Gray:Sangamo Therapeutics: Employment. Levasseur:Bioverativ, a Sanofi Company: Employment. Rimmele:Bioverativ, a Sanofi Company: Employment.

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In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Blood ◽

10.1182/blood.v114.22.4079.4079 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4079-4079

Author(s):

Lei Bao ◽

Mindy M Stamer ◽

Kimberly Dunham ◽

Deepa Kolaseri Krishnadas ◽

Kenneth G Lucas

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Lymphocytes ◽

Cell Therapy ◽

Cytotoxic T Lymphocytes ◽

Cd8 T Cells ◽

Target Cells ◽

Ifn Gamma ◽

Healthy Donors

Abstract Abstract 4079 Poster Board III-1014 MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures: No relevant conflicts of interest to declare.

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Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.

10.1101/2022.01.11.475795 ◽

2022 ◽

Author(s):

Rajeev Rai ◽

Winston Vetharoy ◽

Asma Naseem ◽

Zohar Steinberg ◽

Adrian James Thrasher ◽

...

Keyword(s):

Gene Editing ◽

Culture Media ◽

Ex Vivo ◽

Culture Conditions ◽

Fine Tuning ◽

Great Promise ◽

Hematopoietic Stem ◽

Monogenic Diseases

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

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Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial

Blood ◽

10.1182/blood.v85.1.43.bloodjournal85143 ◽

1995 ◽

Vol 85 (1) ◽

pp. 43-49 ◽

Cited By ~ 179

Author(s):

AF Collins ◽

HA Pearson ◽

P Giardina ◽

KT McDonagh ◽

SW Brusilow ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Cell Number ◽

Response To Treatment ◽

Beta Thalassemia ◽

Thalassemia Patient ◽

Alpha Globin ◽

Pill Counts ◽

Sodium Phenylbutyrate

Butyrate analogues have been shown to increase fetal hemoglobin (HbF) production in vitro and in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals with urea-cycle disorders, has been shown to increase HbF in nonanemic individuals and in individuals with sickle cell disease. We have treated eleven patients with homozygous beta thalassemia (three transfusion dependent) and one sickle-beta- thalassemia patient with 20 g/d (forty 500-mg tablets) of SPB for 41 to 460 days. All patients showed an increase in the percent of F reticulocytes associated with treatment, but only four patients responded by increasing their Hb levels by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2 to 2.8 g/dL). None of the transfusion- dependent thalassemia subjects responded. Increase in Hb was associated with an increase in red blood cell number (mean increase, 0.62 x 10(12)/L), and mean corpuscular volume (mean increase, 6 fL). Changes in percent HbF, absolute HbF levels, or alpha- to non-alpha-globin ratios as measured by levels of mRNA and globin protein in peripheral blood did not correlate with response to treatment. Response to treatment was not associated with the type of beta-globin mutation, but baseline erythropoietin levels of greater than 120 mU/mL was seen in all responders and only two of eight nonresponders to SPB. Compliance with treatment was greater than 90% as measured by pill counts. Side effects of the drug included weight gain and/or edema caused by increase salt load in 2/12, transient epigastric discomfort in 7/12, and abnormal body odor in 3/12 subjects. Two splenectomized patients who were not on prophylactic antibiotics developed sepsis while on treatment. We conclude that SPB increases Hb in some patients with thalassemia, but the precise mechanism of action is unknown.

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Human Bone Marrow-Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-Term In Vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms.

Blood ◽

10.1182/blood.v110.11.1210.1210 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1210-1210

Author(s):

Maria Ester Bernardo ◽

Angela Cometa ◽

Raffaella Villa ◽

Francesca Novara ◽

Antonia Moretta ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

In Vitro Culture ◽

Array Cgh ◽

P53 Gene ◽

Human Bone ◽

Proliferative Capacity ◽

Time Points

Abstract Mesenchymal sem cells (MSCs) are multipotent progenitors with the ability to differentiate along multiple cell lineages and are today considered a useful tool for cell therapy and tissue engineering approaches. Concerns that adult human MSCs may undergo malignant transformation have been recently raised. In fact, human adipose tissue-derived MSCs have been shown to spontaneously transform after long-term in vitro culture. Aim of this study was to investigate the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different time points of in vitro culture. MSCs were isolated from BM of 10 healthy donors and propagated continuously in vitro until reaching a senescence phase or passage (P) 25. MSCs in the senescence phase were closely monitored for 8–12 weeks, to look for the appearance of a crisis phase. MSCs were characterized by morphology, differentiation capacity and immunophenotype at different time-points in culture. The genetic characterization of MSCs was investigated through array comparative genomic hybridization (array-CGH), classical cytogenetics and subtelomeric FISH analysis both before and after prolonged in vitro culture. MSCs were tested for the expression of telomerase activity (TA), hTERT transcripts and alternative mechanisms of telomere lengthening (ALT) at different time-points in culture. MSCs from 5 donors were also analyzed for telomere length at P3 and P15. p53 gene status was also analyzed in MSCs from donors #1 and #2 that rapidly reached senescence in culture. A huge variability between donors was noted in terms of proliferative capacity and in vitro life-span of MSCs. All MSCs maintained the typical spindle-shaped morphology, phenotype and ability to differentiate into osteoblasts and adipocytes throughout the culture period. All MSCs displayed a progressive decrease in the proliferative capacity until reaching senescence, not followed by any further growth. Array-CGH, karyotype and subtelomeric FISH analyses demonstrated that MSCs expanded in vitro did not show chromosomal rearrangements, also after long term culture. TA was not evidenced in the samples tested, including a post-senescence culture. hTERT transcripts (full-length and the additional splice variants a−, b−, a−b−) were found not to be expressed in any of the examined cultures. Telomeres shortened during the culture period. ALT were not evidenced in the MSCs tested, as indicated by the lack of ALT-associated promyelocytic leukemia bodies. Direct DNA sequencing of exons 2–11 in pre-senescence cultures from donors #1 and #2 did not evidence the presence of mutation in the p53 gene. Human BM-derived MSCs do not display an aptitude for spontaneous transformation and can be safely expanded in vitro without any sign of immortalization or development of chromosomal abnormalities. Our results provide support to the concept that the biological properties of human BM-derived MSCs after ex vivo expansion remain suitable for use in cell-therapy approaches; however, it is strongly recommended that phenotype, functional and genetic characteristics of MSCs after in vitro culture and before in vivo infusion are tested, to further guarantee safety for the patient.

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The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽

10.1182/blood.v70.5.1470.bloodjournal7051470 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 1

Author(s):

GF Atweh ◽

XX Zhu ◽

HE Brickner ◽

CH Dowling ◽

HH Jr Kazazian ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Beta Globin ◽

Promoter Mutation ◽

Beta Globin Gene ◽

Gamma Globin ◽

New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

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Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.683631 ◽

2021 ◽

Vol 12 ◽

Author(s):

Snigdha Majumder ◽

Isabelle Jugovic ◽

Domenica Saul ◽

Luisa Bell ◽

Nadine Hundhausen ◽

...

Keyword(s):

T Cells ◽

Gene Editing ◽

Metabolic Reprogramming ◽

Hematopoietic Stem ◽

Retroviral Transduction ◽

Ribonucleoprotein Complexes ◽

Human T Cells ◽

Efficient Gene

Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3+ T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9+CD3+ T cells, CD4+ and CD8+ conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9+CD3+ T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.

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A Mouse Model of PR1-CTL Adoptive Cell Therapy Demonstrates Rapid Reduction of Leukemia Burden in Both Bone Marrow and Extramedullary Organs.

Blood ◽

10.1182/blood.v108.11.3682.3682 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3682-3682

Author(s):

Qing Ma ◽

Eric D. Wieder ◽

Dan J. Jones ◽

Changqing Wang ◽

Dan J. Li ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Adoptive Cell Therapy ◽

Leukemia Cells ◽

Preclinical Model ◽

Mesenteric Lymph Nodes ◽

Rapid Reduction ◽

Healthy Donors ◽

Hypercellular Marrow

Abstract Cytotoxic T lymphocytes (CTL) specific for a nine amino acid HLA-A2-restricted peptide, PR1 (derived from proteinase 3, P3), are capable of killing leukemia cells and contribute to the elimination of CML. PR1-specific CTL (PR1-CTL) elicited in vitro from healthy donors also can lyse P3-expressing AML blasts. Here, we examined whether PR1-CTL can be adoptively transferred to NOD-SCID/HLA-A2 transgenic mice to eliminate blasts as a preclinical model for PR1-CTL cell therapy for AML. Human AML blasts isolated from bone marrow were transferred into irradiated (200 rad) NOD-SCID/HLA-A2 mice and engrafted in bone marrow at various doses (107, 106, 105) between 2–8 weeks. Human CD45 Ab and mouse CD45.1 Ab were used to identify cells of human origin by FACS and IHC, and human CD33 and CD34 Abs were used to identify AML cells. PR1-CTL were elicited from healthy donors that specifically lysed P3-expressing leukemia. These PR1-CTL were predominantly CD45RA−/CCR7−/CD28+ and maintained an activated effector phenotype following adoptive transfer. Transferred AML blasts were significantly decreased in the bone marrow of mice receiving co-transferred PR1-CTL compared to those that received only AML: 0.5×106 AML cells plus 1×104 PR1-CTL or 0.5×106 AML cells (control) were infused into irradiated mice respectively. Mice were sacrificed at 2–4 weeks post-transfer and tissues were analyzed by FACS and IHC. Bone marrow aspirate from mice that received AML alone had 72–88% blasts in hypercellular marrow, whereas mice that received AML plus PR1-CTL had normal hematopoietic elements and only 10–11% blasts in hypocellular marrow. PR1-CTL also migrated to the secondary lymphoid organs including spleen, peripheral and mesenteric lymph nodes, and in the liver. Leukemia cells also infiltrated extramedullary organs. In the liver, intrasinuosidal and periportal collections of leukemic blasts were associated with vascular damage, and PR-CTL infiltrated these same regions by 2 weeks post-transfer. The PR1-CTL in the bone marrow and liver maintained an effector phenotype similar to the pre-infusion CTL, whereas PR1-CTL in secondary lymph organs such as blood and spleen also contained some cells expressing CCR7 that either reverted to a naïve phenotype or had expanded from the precursor population. By 4 weeks post-transfer, leukemia was no longer evident in the liver, although PR1-CTL remained. In conclusion, we found that PR1-CTL generated in vitro can eliminate the AML cells in NOD-SCID/HLA-A2 mice. PR1-CTL can migrate to sites of disease and maintain their capacity to eliminate leukemic blasts. Surface phenotype of PR1-CTL were consistent with their trafficking pattern in both vascular and end-organ tissues. Our results justify a clinical trial with adoptively transferred PR1-CTL in patients with AML.

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The In Vitro and In Vivo Antioxidant Effects of Erythropoietin in Thalassemia.

Blood ◽

10.1182/blood.v110.11.573.573 ◽

2007 ◽

Vol 110 (11) ◽

pp. 573-573 ◽

Cited By ~ 1

Author(s):

Eitan Fibach ◽

Johnny Amer

Keyword(s):

Oxidative Stress ◽

Fetal Hemoglobin ◽

Membrane Lipid ◽

Beta Thalassemia ◽

Dialysis Patients ◽

Erythroid Progenitors ◽

High Exposure ◽

Hemoglobin Production

Abstract Erythropoietin (EPO), a hormone produced on hypoxia mainly in the kidneys, enhances red blood cell (RBC) production (erythropoiesis) by stimulating the proliferation of erythroid progenitors and precursors in the bone marrow. This effect is mediated by the homodimeric EPO receptor, a class 1 cytokine receptor. Recombinant human EPO is widely used for the treatment of anemia, e.g., in patients on dialysis, patients with myelodysplastic syndrome and oncology patients undergoing chemotherapy. Treatment with EPO was also tried experimentally in patients with thalassemia. In these patients, in spite the state of chronic anemia, the levels of EPO is usually low relative to the degree of anemia. Administration of EPO to thalassemic patients have been shown to increase erythropoiesis and in some cases to elevate fetal hemoglobin production. In addition, EPO has been suggested to have cardio- and neuro-protecting effects and to increase RBC survival in dialysis patients. We have previously shown that RBC and platelets derived from patients with beta-thalassemia and sickle cell disease are under oxidative stress; they have elevated potential to generate reactive oxygen species (ROS) and membrane lipid peroxides, and have lower content of reduced glutathione (GSH) than normal RBC. This oxidative stress resulted in high exposure of phosphatidylserine (PS) that is considered a major factor in shortening the life span of thalassemic RBC, and in the tendency of platelets to undergo activation and thus contributes to the high incidence of thromboembolic complications in thalassemia. In the present study, we investigated the in vitro and in vivo effects of EPO as an antioxidant on RBC and platelets from beta-thalassemic patients and mice. Using flow-cytometry methodology, we showed that in vitro treatment of blood cells from beta-thalassemic patients with 1–4 U/ml of EPO for 2 hours increased the GSH content of RBC (2.1-fold) and platelets (1.7-fold) and reduced their ROS (1.5-fold), membrane lipid peroxidation and externalization of PS. Intraperitoneal injection of EPO to heterozygotes (Hbbth3/+) beta-thalassemic mice (3,000U/kg) significantly reduced ROS and increased GSH in their RBC within 3 hours. The in vitro effects of EPO on oxidative stress resulted in reduced sensitivity of thalassemic RBC to undergo hemolysis and phagocytosis by macrophages, and reduced tendency of platelets to undergo activation, as reflected by fewer platelets carrying external PS. Our results suggest that in addition to its effect on erythropoiesis and fetal hemoglobin production, EPO might alleviate symptoms in thalassemia and other hemolytic anemias as a potent antioxidant.

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Engineering T-Cells Beyond Chimeric Antigen Receptor

Blood ◽

10.1182/blood.v128.22.sci-13.sci-13 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-13-SCI-13 ◽

Cited By ~ 1

Author(s):

Chiara Bonini

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Genetic Engineering ◽

Cell Therapy ◽

Cancer Cells ◽

Gene Editing ◽

Adoptive T Cell Therapy ◽

T Cell Therapy

Adoptive T cell therapy exploits the ability of T lymphocytes to recognize and destroy specific targets, on microbes or tumors, through their T cell receptors (TCR), leading to efficient killing and long-term protection against diseases. Unfortunately, tumor antigens are often overexpressed, unmodified self-antigens, subject to tolerance mechanisms; so tumor-specific T lymphocytes are rare cells. Conversely, neoantigens derive from oncogenic mutations can elicit productive T cell responses, but for tumors with a low mutational load, such as the majority of hematological malignancies, such tumor-specific T cells are rarely identified. These limitations can be overcome by genetic engineering of T lymphocyte specificity. Recently, unprecedented clinical results were obtained with chimeric antigen receptor (CAR) engineered T cells in patients affected by B-cell malignancies, raising high expectations among the scientific community, patient associations, biotech companies and general public. While clearly proving the ability of redirected T cells to recognize and efficiently kill cancer cells, CAR therapy has also shown some limitations: the nature of CAR-mediated recognition imposes to restrict the array of targeted antigens to those expressed on the surface of cancer cells. As a consequence, antigens involved in the oncogenic process, that are often expressed as intracellular molecules, cannot be targeted by current CARs. Furthermore, when the natural counterpart of cancer cells cannot be spared, the identification of a proper CAR target on cancer cell surface might become a real challenge. TCR genetic engineering represents a suitable alternative to CAR T cell therapy for several tumors. The core of this approach is the transfer in patients' T cells of genes encoding for rare tumor-specific TCR. TCRs recognize antigen-derived peptides processed and presented on HLA molecules, thus allowing to largely increasing the array of potential targets. However, the simple transfer of tumor specific TCR genes into T cells is affected by other limitations: genetically modified T cells shall express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on lymphocyte surface, thus limiting the efficacy of therapeutic cellular product. To overcome these issues, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to alfa and beta chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors (Provasi et al, Nature Medicine 2012). The TCR gene editing technology, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. Early differentiated T cells, such as memory stem T cells and central memory T cells, cells endowed with long term persistence capacity, can be genetically engineered by TCR gene transfer and TCR gene editing, thus allowing to produce long-lasting living drugs, with the aim of eliminating cancer cells and patrol the organism for tumor recurrence To enter the phase of clinical practice adoptive T cell therapy needs today to face several challenges: compliance to the dynamic and heterogeneous regulatory framework, susceptibility to automated processes, reproducibility, and sustainability shall be relevant variables in determining the fate of these innovative cellular products. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

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Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

Tissue Engineering and Regenerative Medicine ◽

10.1007/s13770-021-00330-7 ◽

2021 ◽

Author(s):

Silvia Barbon ◽

Senthilkumar Rajendran ◽

Thomas Bertalot ◽

Monica Piccione ◽

Marco Gasparella ◽

...

Keyword(s):

Stem Cells ◽

Cell Therapy ◽

Muscle Damage ◽

Adult Stem Cells ◽

Ex Vivo ◽

Myogenic Differentiation ◽

Calcium Flux ◽

Morphological Studies

Abstract Background: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages. Methods: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage. Results: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair. Conclusion: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy.

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