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This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage-associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D-NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D-NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1β. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b + EPCAM + ) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers ( SOX2 , NANOG , miR-302 ) as compared with non-fused (CD11b - EPCAM + ) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10 . Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34 + cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD., Competing Interests: J.D.S., K.L., B.C.B., and S.G. are employees of Rocket Pharmaceuticals. J.A.B. and J.-C.S. are consultants and receive funding from Rocket Pharmaceuticals., (© 2021 The Authors.)

Galectin-1 is a β -galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with β -galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice ( Lgals1 -/- mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1 -/- mice involved an altered Th17/Th1 profile of effector CD4 + T cells. Furthermore, increased frequencies of Foxp3 + CD4 + regulatory T cells in colon lamina propria in Lgals1 -/- mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1 - / - mice was alleviated by adoptive transfer of wild type Foxp3 + CD4 + regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fernandez-Perez, Lopez-Santalla, Sánchez-Domínguez, Alberquilla, Gutiérrez-Cañas, Juarranz, Bueren and Garin.)

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34 + cells/μL required to initiate apheresis. A median of 21.8 CD34 + cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34 + cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34 + cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34 + cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34 + cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols., Competing Interests: J. Sevilla is a consultant and advisor and has received honorarium (Amgen, Novartis, Miltenyi, Sobi, Rocket Pharmaceuticals Inc.) and has licensed medicinal products from Rocket Pharmaceuticals Inc. S.N. and P.R. have licensed medicinal products and receive research funding and equity from Rocket Pharmaceuticals Inc. J.C.S.: Rocket Pharmaceuticals Inc.: consultant/incomes from licensed medicinal products/research funding/equity. J. Schwartz is Medical Director of Rocket Pharmaceuticals Inc. J. Surralles: service agreements (Rocket Pharmaceuticals Inc.). J.A.B.: Rocket Pharmaceuticals Inc.: consultant/incomes from licensed medicinal products/research funding/equity; Roche: honorarium; Pfizer: honorarium., (© 2021 The Author(s).)

Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell ( P KLR ) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients., Competing Interests: J.-C.S. and J.A.B. are consultants and hold shares and receive funding from Rocket Pharma. M.P. serves on the scientific advisory board of CRISPR Therapeutics and Graphite Bio. D.P.D. is employed by Graphite Bio. R.T., M.S.S., and M.A.B. are employed by Integrated DNA Technologies, Inc (IDT), which manufactures reagents similar to some described in the manuscript. R.T. and M.A.B. own equity in DHR, the parent company of IDT. All other authors declare no competing interests., (© 2021 The Author(s).)

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30-60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated., (© 2020 The Authors.)

Background: CD18 is the common beta subunit of β 2 integrins, which are expressed on hematopoietic cells. β 2 integrins are essential for cell adhesion and leukocyte trafficking., Methods: Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34 + cells were classified into CD18 high and CD18 low/neg , and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice., Results: A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18 low/neg CD34 +  and CD18 high CD34 + cells were observed, CD18 low/neg CD34 + cells were characterized by an enhanced long-term repopulating ability in NSG mice., Conclusions: Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date 1,2 . Mutations in FANCA account for more than 60% of FA cases worldwide 3,4 . Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life 5,6 . In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.

Background: Allogeneic haematopoietic stem-cell transplantation is the standard treatment for bone marrow failure (BMF) in patients with Fanconi anaemia, but transplantation-associated complications such as an increased incidence of subsequent cancer are frequent. The aim of this study was to evaluate the safety and efficacy of the infusion of autologous gene-corrected haematopoietic stem cells as an alternative therapy for these patients., Methods: This was an open-label, investigator-initiated phase 1/2 clinical trial (FANCOLEN-1) and long-term follow-up trial (up to 7 years post-treatment) in Spain. Mobilised peripheral blood (PB) CD34 + cells from nine patients with Fanconi anaemia-A in the early stages of BMF were transduced with a therapeutic FANCA-encoding lentiviral vector and re-infused without any cytotoxic conditioning treatment. The primary efficacy endpoint of FANCOLEN-1 was the engraftment of transduced cells, as defined by the detection of at least 0·1 therapeutic vector copies per nucleated cell of patient bone marrow (BM) or PB at the second year post-infusion, without this percentage having declined substantially over the previous year. The safety coprimary endpoint was adverse events during the 3 years after infusion. The completed open-label phase 1/2 and the ongoing long-term clinical trials are registered with ClinicalTrials.gov, NCT03157804; EudraCT, 2011-006100-12; and NCT04437771, respectively., Findings: There were eight evaluable treated patients with Fanconi anaemia-A. Patients were recruited between Jan 7, 2016 and April 3, 2019. The primary endpoint was met in five of the eight evaluable patients (62·50%). The median number of therapeutic vector copies per nucleated cell of patient BM and PB at the second year post-infusion was 0·18 (IQR 0·01-0·20) and 0·06 (0·01-0·19), respectively. No genotoxic events related to the gene therapy were observed. Most treatment-emergent adverse events (TEAEs) were non-serious and assessed as not related to therapeutic FANCA-encoding lentiviral vector. Nine serious adverse events (grade 3-4) were reported in six patients, one was considered related to medicinal product infusion, and all resolved without sequelae. Cytopenias and viral infections (common childhood illnesses) were the most frequently reported TEAEs., Interpretation: These results show for the first time that haematopoietic gene therapy without genotoxic conditioning enables sustained engraftment and reversal of BMF progression in patients with Fanconi anaemia., Funding: European Commission, Instituto de Salud Carlos III, and Rocket Pharmaceuticals., Competing Interests: Declaration of interests PR: has received honoraria as consultant and holds stock options and royalties for licences to Rocket Pharmaceuticals. SN: has received honoraria as consultant and holds stock options and royalties for licences to Rocket Pharmaceuticals. EN: employee of Rocket Pharmaceuticals and owns Rocket Pharmaceuticals equity and equity options. JCS: has received honoraria as consultant and holds stock options and royalties for licences to Rocket Pharmaceuticals. JSu: has received service honoraria from Rocket Pharmaceuticals. GR: employee of Rocket Pharmaceuticals and owns Rocket Pharmaceuticals equity and equity options. JSo: has received honoraria as consultant from Rocket Pharmaceuticals. JDS: employee and officer of Rocket Pharmaceuticals and owns Rocket Pharmaceuticals equity and equity options. JSe: has received support for attending meetings and honoraria as consultant, as member of advisory boards, and holds stock options and royalties for licences to Rocket Pharmaceuticals. JAB: has received honoraria as consultant and holds stock options and royalties for licences to Rocket Pharmaceuticals. AdC: has received honoraria as consultant and holds stock options and royalties for licences to Rocket Pharmaceuticals. The institutions of PR, JZ, SN, AS, PJ-N, ALB, MRa, JCS, JSu, JSe, JAB, and AdC have received funding for research on gene therapy from Rocket Pharmaceuticals. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)