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© 2021 the Author(s)., Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, Background Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. Methods Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. Results Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. Conclusions Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases, This work was supported by the following public grants: Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III Ministerio de Ciencia, Innovación y Universidades and Fondo Europeo de Desarrollo Regional (FEDER) ((RETICS-RD16/0011/0011, PIE15/00048, PI18-01379), and Dirección General de Investigación de la Comunidad de Madrid (AvanCell-CM; Ref S2017/BMD-3692)

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 (PKLR) 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., Graphical abstract, Combination of CRISPR-Cas9 ribonucleoprotein electroporation and donor AGAVE transduction in human hematopoietic stem and progenitor cells from pyruvate kinase deficiency patients allows efficient gene editing with restoration of erythrocyte energetic balance. Maintenance of stem cell properties without detectable off-targets indicates clinical applicability of gene editing for this inherited hemolytic anemia.

© The Author(s)., Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC., This work was supported by grants from the “Ministerio de Economía, Comercio y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER)” (SAF2015-68073-R) and from the “Ministerio de Ciencia, Innovación y Universidades and Fondo Europeo de Desarrollo Regional (FEDER)” (RTI2018-097125-B-I00) and P17-01401 from “Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (FIS-ISCIII)”. The authors also thank the Fundación Botín for promoting translational research at the Hematopoietic Innovative Therapies Division of the CIEMAT. CIBERER is an initiative of the “Instituto de Salud Carlos III” and “FEDER”. CCR was supported by an FPI grant from the Universidad Autónoma de Madrid (UAM).

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., Graphical abstract, Mobilization and collection of HSPCs from FA patients with sufficient HSPC reserve is a safe and efficient procedure, incorporating filgrastim and plerixafor as mobilization agents. Adequate HSPC mobilization correlates with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher BM CD34+ cell numbers.

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance., Author summary Understanding mechanisms that make cells sensitive to xenobiotics (including drugs) is of great importance to both drug development and public health. For the latter, emergence of resistance is particularly important to monitor as well as to predict. Trypanosomes are a major global health burden and for several drugs resistance has emerged and is a considerable concern. Here we have examined the sensitivity to pentamidine, which is mainly mediated by an aquaglyceroporin, a surface channel. We find that the protein is highly sensitive to mutation, rendering the protein unstable, and rendering parasites resistant to pentamidine. As this also includes mutant forms recovered from patients where pentamidine treatment has failed, we suggest that the instability of aquaglyceroporin is an important contributor towards treatment failure.

The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice. The authors would like to thank Miguel A. Martin for the careful maintenance of NSG mice and Omaira Alberquilla for her technical assistance in flow cytometry. The authors also thank the Fundación Botín for promoting translational research at the Hematopoietic Innovative Therapies Division of the CIEMAT. CIBERER is an initiative of the “Instituto de Salud Carlos III” and “Fondo Europeo de Desarrollo Regional (FEDER)”. Finally, although this work was entirely performed with mouse cells, the authors feel deeply grateful to the FA patients and families, clinicians and to Aurora de la Cal as the secretary of the Spanish FA network for always instilling motivation and providing an example for work in this disease. This work was supported by grants from the “7th Framework Program European Commission (HEALTH-F5-2012-305421; EUROFANCOLEN)”, “Ministerio de Sanidad, Servicios Sociales e Igualdad” (EC11/060 and EC11/550), “Ministerio de Economía, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER)” (SAF2015-68073-R), “Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III” (RD12/0019/0023) Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid (AvanCell Project; B2017/BMD3692) and the German Federal Ministry of Education and Research (BMBF-01EO0803) Sí

Background CD18 is the common beta subunit of β2 integrins, which are expressed on hematopoietic cells. β2 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 CD18high and CD18low/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 CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/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.

Development of advanced gene and cell therapies for the treatment of genetic diseases requires confident animal and cellular models to test their efficacy and is crucial in the cases where no primary samples from patients are available. CRISPR/Cas9 technology, has become one of the most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use of two guides tends to produce the precise deletion between the guides via NHEJ. Different distances between guides were tested (from 8 to 500 base pairs). We found that distances between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a (forin vivoprimary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in those diseases where the availability of primary cells is limited.

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.

Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1–6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs., Eileen Nicoletti is an employee of Rocket Pharmaceuticals, Inc. Gayatri Rao is an employee of Rocket Pharmaceuticals, Inc. Juan A. Bueren is the head of the Hematopoietic Innovative Therapies Division at Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas (CIEMAT) which receives funding for Fanconi Anemia (FA) gene therapy and has licensed the PGK-FANCAWpre* lentiviral vector to Rocket Pharmaceuticals, Inc. He is also a consultant for Rocket Pharmaceuticals, Inc. Paula Río is a member of the Hematopoietic Innovative Therapies Division at Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas (CIEMAT) which receives funding for Fanconi Anemia (FA) gene therapy and has licensed the PGK-FANCA-Wpre* lentiviral vector to Rocket Pharmaceuticals, Inc. Susana Navarro is a member of the Hematopoietic Innovative Therapies Division at Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas (CIEMAT) which receives funding for Fanconi Anemia (FA) gene therapy and has licensed the PGK-FANCAWpre* lentiviral vector to Rocket Pharmaceuticals, Inc. Jordi Surrallés does not have any conflicts of interest. Grace Choi is an employee of Rocket Pharmaceuticals, Inc. Jonathan D. Schwartz is an employee of Rocket Pharmaceuticals, Inc.

The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102–106 in the RhoA sequence, displayed an IC(50) of ∼100 μm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential.

Background: Fanconi anemia (FA) is a disorder of defective deoxyribonucleic acid (DNA) repair, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. Approximately 60 to 70% of all cases result from a mutation in the Fanconi Anemia Complementation Group A (FANCA) gene (FA-A). 80% of FA patients experience BMF within the first decade of life. Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative for BMF; however, its efficacy is limited by human leukocyte antigen (HLA)-matched sibling donor availability and transplant-related toxicities. Lentiviral mediated gene therapy utilizing autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) confers a proliferative advantage to gene-corrected HSPCs as demonstrated in preclinical studies and the FANCOLEN-I clinical trial conducted in Madrid, Spain. We report results from ongoing RP-L102 studies using "Process B" manufacturing optimizations including transduction enhancers, commercial grade vector, and modified cell processing. Methods: Patients (pts) with a FANCA gene mutation, age ≥1 year with no HLA-matched sibling donor and at least 30 CD34+ cells/µL in bone marrow (BM) are eligible. Peripheral blood (PB) mononuclear cells are collected via leukapheresis on 2 consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs are enriched, transduced with a lentiviral vector encoding for the FANCA gene (PGK-FANCA-WPRE) and infused without cryopreservation or conditioning. Patients are followed for 3 years post-infusion for safety assessments (replication competent lentivirus [RCL], insertion site analysis [ISA]) and to ascertain evidence of efficacy (increasing PB and BM vector copy number [VCN] and mitomycin-C [MMC] resistance in BM colony forming units [CFUs]), along with stabilization/correction of cytopenias. Results: As of March 2021, 9 pts (aged 2 to 6 years) have received RP-L102. Evidence of engraftment has been identified in 6 pts with ≥6 months of follow up as indicated by PB VCN. 2 of 3 pts with follow up of ≥12 months have shown increased MMC resistance in BM CFUs. 1 pt developed BMF requiring alloHSCT after influenza B infection. 1 pt had a serious Grade 2 transient RP-L102 infusion-related reaction. Updated safety and efficacy data for pts with ≥12 months of follow-up will be presented. Conclusions: RP-L102's safety profile remains favorable. Increasing evidence of engraftment has been confirmed in 6 subjects as demonstrated by PB VCN; without conditioning, 12+ months of follow up is likely required to observe the proliferative advantage of transduced HSPCs. Disclosures Czechowicz: Editas Medicine: Current equity holder in publicly-traded company; Rocket Pharmaceuticals, Inc.: Research Funding; Global Blood Therapeutics: Current equity holder in publicly-traded company; Stemodontics: Consultancy, Current equity holder in publicly-traded company; GV: Consultancy, Current equity holder in publicly-traded company; Spotlight Therapeutics: Consultancy, Current equity holder in publicly-traded company; Beam Therapeutics: Consultancy, Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company, Other: Intellectual property rights; Decibel Therapeutics: Current equity holder in publicly-traded company; Forty Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Other: intellectual property rights; Jasper Therapeutics: Other: Intellectual property rights. Sevilla: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents., Patents & Royalties: J. Sevilla is a consultant and has licensed medical products from Rocket Pharmaceuticals, Inc. ; Amgen: Consultancy; Novartis: Consultancy; Miltenyi: Consultancy; SOBI: Consultancy. Booth: GSK: Honoraria; Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Río: Rocket Pharmaceuticals: Current equity holder in publicly-traded company, Other: Paula Rio has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Research Funding. Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Wagner: Magenta Therapeutics: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company; Vertex Pharmaceuticals: Consultancy; ASC Therapeutics: Consultancy; Bluebird Bio: Consultancy. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding.

Background: Pyruvate kinase deficiency (PKD) is a rare inherited hemolytic anemia caused by mutations in the PKLR gene resulting in decreased red cell pyruvate kinase activity and impaired erythrocyte metabolism. Manifestations include anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current treatments are palliative and limited to blood transfusions, chelation therapy, and splenectomy which are associated with significant side effects. Preclinical studies in a clinically relevant PKD murine model have demonstrated that infusion of gene-modified Lin− bone marrow (BM) cells may ameliorate PKD phenotype. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adult and pediatric subjects with severe PKD. Methods: Six adult and pediatric patients with severe PKD (defined as severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled. Peripheral blood (PB) hematopoietic stem cells (HSCs) are collected on 2 consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. HSCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following release testing of the investigational product (IP), RP-L301, myeloablative therapeutic drug monitoring (TDM) busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis for 2 years post-infusion. Results: As of May 2021, 2 adult patients with severe anemia have received RP-L301. Patient 1 (age 31 years) received 3.9x106 CD34+ cells/kg with mean vector copy number (VCN) of 2.73. Patient 2 (age 47 years) received 2.4x106 CD34+ cells/kg with mean VCN of 2.08. Despite baseline hemoglobin (Hb) levels in the 7.0-7.5 g/dL range, both patients displayed normal-range hemoglobin (Hb), improved hemolysis markers, and have required no red blood cell transfusions post-engraftment at 9- and 6- months follow-up. Both report improved quality of life. PB mononuclear cell VCNs for both patients were >2.0 at last evaluated timepoint (6- and 3-months post-treatment, respectively). No serious adverse events have been attributed to RP-L301. Updated safety and efficacy data will be presented. Conclusions: Hematopoietic stem cell mobilization using G-CSF and plerixafor is feasible and effective in adult PKD patients. RP-L301 was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications. Efficacy was demonstrated by normalized Hb associated with engraftment confirmed by PB and BM VCN. Disclosures Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Glader: Agios: Consultancy. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding.

Background: Leukocyte Adhesion Deficiency-I (LAD-I) is a rare disorder of neutrophil adhesion resulting from ITGB2 mutations encoding for the β2-integrin component, CD18. Severe LAD-I (CD18 Methods: Pediatric patients ≥ 3 months old with severe LAD-I are eligible. HSCs are collected via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor and transduced with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning precedes RP-L201 infusion. Patients are followed for safety and efficacy (i.e., survival to age 2 and at least 1-year post-infusion, increase in PMN leukocyte CD18 expression to at least 10%, peripheral blood [PB] vector copy number [VCN], decrease in infections/hospitalizations, and resolution of skin or periodontal abnormalities). Results: Seven patients (ages 5mos-9yrs) have received RP-L201; all with follow-up ≥3 to 18 months. RP-L201 cell doses ranged from 2.8x106 to 6.5x106 CD34+ cells/kg with VCN from 1.8-3.8 copies/cell. No serious RP-L201 related treatment-emergent adverse events were reported. PB PMN CD18 expression in Patient 1 at 18-months post-treatment was 40% (vs. < 1% at baseline), with PB VCN of 1.44. Baseline skin lesions resolved with no new lesions reported. CD18 expression in the subsequent 6 patients has been 25-80% in neutrophils and stable for each patient from 3 to up to 18 months post-treatment. No new infections have been reported in patients post-infusion. The safety profile of RP-L102 remains highly favorable with no serious adverse events (SAEs) attributed to the investigational product (IP). Conclusion: RP-L201 leads to durable neutrophil CD18 expression and improved clinical course. Additional patient treatment is ongoing in 2021. Disclosures Kohn: UC Regents: Patents & Royalties; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees. Booth: GSK: Honoraria; Takeda: Honoraria; SOBI: Consultancy, Honoraria; Orchard Therapeutics: Consultancy, Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Almarza: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. de Oliveira: Orchard Therapeutics: Research Funding; Bluebird Bio: Research Funding. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

Transduction of hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors (LVs) constitutes a new therapeutic option for the treatment of various monogenic diseases affecting the lymphohematopoietic system. The development of detailed preclinical studies of gene therapy in animal disease models constitutes an essential step in expanding the application of gene therapy in a wide variety of inherited and acquired diseases. Here we describe an efficient protocol to transduce HSPCs from wild-type and Fanconi anemia mice with either gene-marking or therapeutic LVs. In this protocol, purified lineage–, Sca-1+, c-Kit+ bone marrow cells were transduced in vitro for a short period of time under conditions that facilitated efficient transduction of HSPCs capable of engrafting in transplanted recipients.

SummaryCurrent sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34

Purpose: Fanconi anemia rare disease is characterized by bone marrow failure and a high predisposition to solid tumors, especially head and neck squamous cell carcinoma (HNSCC). Patients with Fanconi anemia with HNSCC are not eligible for conventional therapies due to high toxicity in healthy cells, predominantly hematotoxicity, and the only treatment currently available is surgical resection. In this work, we searched and validated two already approved drugs as new potential therapies for HNSCC in patients with Fanconi anemia. Experimental Design: We conducted a high-content screening of 3,802 drugs in a FANCA-deficient tumor cell line to identify nongenotoxic drugs with cytotoxic/cytostatic activity. The best candidates were further studied in vitro and in vivo for efficacy and safety. Results: Several FDA/European Medicines Agency (EMA)-approved anticancer drugs showed cancer-specific lethality or cell growth inhibition in Fanconi anemia HNSCC cell lines. The two best candidates, gefitinib and afatinib, EGFR inhibitors approved for non–small cell lung cancer (NSCLC), displayed nontumor/tumor IC50 ratios of approximately 400 and approximately 100 times, respectively. Neither gefitinib nor afatinib activated the Fanconi anemia signaling pathway or induced chromosomal fragility in Fanconi anemia cell lines. Importantly, both drugs inhibited tumor growth in xenograft experiments in immunodeficient mice using two Fanconi anemia patient–derived HNSCCs. Finally, in vivo toxicity studies in Fanca-deficient mice showed that administration of gefitinib or afatinib was well-tolerated, displayed manageable side effects, no toxicity to bone marrow progenitors, and did not alter any hematologic parameters. Conclusions: Our data present a complete preclinical analysis and promising therapeutic line of the first FDA/EMA-approved anticancer drugs exerting cancer-specific toxicity for HNSCC in patients with Fanconi anemia.

Summary Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34+ cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. 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 life5,6. 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. In an early-phase lentiviral gene therapy trial, gene-corrected autologous hematopoietic stem cells show sustained engraftment and phenotypic correction in non-conditioned patients with Fanconi anemia.

Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only curative treatment for the bone marrow failure in Fanconi anemia (FA) patients. However, recent advances in lentiviral-mediated gene therapy have shown that corrected FA HSCs develop an in vivo proliferation advantage, facilitating the engraftment of corrected HSCs in non-conditioned FA patients. Based on these observations, we proposed that gene editing might constitute a promising alternative to correct patients' hematopoietic stem and progenitor cells (HSPCs) in this disorder. Since non-homologous end joining (NHEJ) is the most frequent repair pathway in HSCs, particularly in FA-HSCs, we aimed at exploiting this DNA repair mechanism to remove/compensate specific mutations in different FANC genes by the use of CRISPR/Cas9 system, thus mimicking spontaneous genetic reversions observed in FA mosaic patients. Our results in lymphoblastic cell lines from five different complementation groups (FANCA, FANCB, FANCC, FANCD2 and FANCD1/BRCA2) demonstrated the efficiency of this approach to generate potentially corrective events in all the different complementation groups studied. Importantly, corrected cells showed a marked proliferative advantage after in vitro culture and the analysis by next generation sequencing confirmed the expansion of cells harboring therapeutic events. Functional studies showing the reversion of mitomycin C sensitivity, FANCD2 foci formation and chromosomal instability supported the phenotypic correction of different mutations by NHEJ-mediated gene editing. Moving towards the clinical application of NHEJ-mediated repair we focused on improving the gene editing efficiency in HSCs. To this aim, chemically modified small guide RNAs (MS-sgRNAs) enabled us to increase the editing efficacy 8-fold compared to efficacies obtained with in vitro transcribed sgRNAs, reaching up to 89% indels in healthy donor hematopoietic stem/progenitor cells. Moreover, the CRISPR/Cas9 system demonstrated high editing capacity in the primitive HSCs capable of engrafting immunodeficient NSG mice, confirming the efficacy of NHEJ-editing to correct the phenotype of long-term repopulating HSCs. Finally, studies conducted in mobilized peripheral blood and bone marrow CD34+ cells from FA patients demonstrated the feasibility to correct FA HSCs by NHEJ-mediated gene editing and confirmed the proliferative advantage of NHEJ-mediated corrected cells both in vitro and in vivo. Our results suggest that NHEJ-mediated gene editing should constitute a versatile and simple therapeutic approach to efficiently correct specific mutations in FA and other monogenic disorders of the hematopoietic system. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Rio:Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene that lead to a reduction of the erythroid pyruvate kinase (RPK) protein activity, which causes an energetic imbalance in PKD erythroid cells. This disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. In selected cases, allogeneic Hematopoietic Stem Cell Transplantation has been shown to correct the disorder. Therefore, autologous HSCT of genetically corrected cells will offer a durable and curative therapeutic option. In fact, a global Phase I clinical trial (clinicaltrials.gov #NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy with severe PKD. Looking for a guided integration of the exogenous therapeutic DNA sequences, herein we conducted a gene editing approach to correct PKD in human Hematopoietic Stem Cells (HSCs). We developed a knock-in approach to insert either a TurboGFP expression cassette or a promotor-less therapeutic codon optimized RPK cDNA (coRPK) at the genomic starting site of the PKLR gene, a gene editing strategy that will correct most of the mutations present in PKD patients. This gene editing approach combines RNP nucleofection and adeno-associated viral vector (AAV6) mediated delivery of homologous donors. In order to assess the safety of the proposed gene editing approach, we performed GUIDE-Seq and rhAmpSeqTM analyses of different single guide RNAs targeting the PKLR starting site. We found PKLR sgRNAs that showed a high targeting efficiency, up to 40% targeted hematopoietic progenitors (in vitro semisolid colony forming units) and a safety profile, with no off-targets detected above threshold values (0.1%) and in the absence of cellular toxicity, when applied to healthy cord blood CD34+ (CB-CD34+) cells. These gene-edited CB-CD34+ cells engrafted efficiently in both primary and secondary immunodeficient NSG recipient mice, demonstrating the gene editing in long-term hematopoietic repopulating HSCs. Furthermore, we evaluated the therapeutic potential of this gene editing strategy to restore the energetic imbalance in erythroid cells derived from PKD patients. CD34+ cells from 4 different PKD patients, were purified, nucleofected with PKLR sgRNA and transduced with AAV-coRPK donor. In vitro differentiated erythroid cells derived from edited PKD CD34+ expressed coRPK mRNA and restored their energetic defect up to normal values obtained in in vitro differentiated erythroid cells from healthy donor cells. Moreover, gene edited mobilized Peripheral Blood CD34+ (mPB-CD34+) cells from a PKD patient engrafted efficiently in immunodeficient NBSGW mice, having human cells that showed the specific integration of coRPK donor 2 months post-transplant. Overall, these results demonstrate the feasibility and safety of PKLR gene editing in human HSPCs and, therefore, its potential clinical application for the treatment of PKD patients. Disclosures Dever: Integral Medicines: Current Employment. Turk:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor. Behlke:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Introduction: LAD-I is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from ITGB2 gene mutations encoding for the β2-integrin component, CD18. Severe LAD-I (i.e., CD18 expression on Methods: Pediatric patients ≥ 3 months old with severe LAD-I (demonstrated by CD18 expression on Results: Two patients (ages 9 and 3) were treated in Phase 1. Both have a history of recurrent severe infections, documented ITGB2 mutations, and baseline CD18, CD11a, and CD11b expression < 1%. Mobilization and apheresis procedures were performed successfully and busulfan was administered at the target AUC. Investigational product comprised of 4.2x106 CD34+ cells/kg with VCN of 3.8 copies/cell (liquid culture) for Patient 1 and 2.8x106 CD34+ cells/kg with VCN of 2.5 for Patient 2 and were infused without complications. No serious treatment-emergent adverse events were reported. Neutrophil engraftment was observed in < 3 weeks in both patients. For Patient 1, PB PMN CD18 expression 6 months post-treatment was 47% (sustained from 45% at 3-months, vs. < 1% at baseline), with PB VCN of 1.3. Skin lesions present at baseline were resolving with no new lesions. Replication competent lentiviral (RCL) testing at 3 months and 6 months post-infusion were negative. Safety and efficacy data 12-months post-treatment for Patient 1 and 6-months post-treatment for Patient 2 will be available at the time of presentation. Conclusion: Initial results from Phase 1 demonstrate preliminary safety and efficacy of RP-L201 for reversal of severe LAD-I. Enrollment of patients in the Phase 2 study is underway. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Almarza:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Sevilla:Novartis: Other: Advisory Board; Sobi: Other: Advisory Board; Rocket Pharma: Consultancy; Amgen: Other: Advisory Board. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18- Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Background: Fanconi anemia (FA) is a rare inherited disorder of defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities and characterized by progressive bone marrow failure (BMF) and a predisposition to hematologic malignancies and solid tumors. Approximately 60-70% of all cases result from mutations in the Fanconi Anemia Complementation Group A (FANCA) gene (FA-A). 80% of FA patients develop BMF within the first decade of life. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially curative treatment for BMF, its utilization and efficacy are limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo lentiviral mediated gene therapy of autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown to confer a survival advantage to gene-modified HSPCs in preclinical studies and, most recently, in the investigator initiated Phase 1/2 FANCOLEN-I clinical trial conducted in Madrid, Spain. Based on the highly favorable safety profile and promising preliminary efficacy data, global studies using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing are underway. Herein, we report updated results from the US Phase 1 clinical trial and preliminary data from the global Phase 2 study in US and EU. Design and Methods: Subjects with a confirmed FANCA gene mutation aged 1 year or older, with no HLA-matched sibling donor and at least 30 CD34+ cells/µL in bone marrow (BM) were eligible for enrollment. Peripheral blood (PB) mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs were enriched, transduced with a lentiviral vector (PGK-FANCA-WPRE) and infused fresh (not cryopreserved) without any antecedent conditioning. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain evidence of efficacy (increasing PB vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: As of August 2020, 2 subjects (aged 5 and 6 years) have received RP-L102 infusion on the Phase 1 study with over 12 months of follow up. Preliminary evidence of gene marking in PB post-RP-L102 infusion at various timepoints has been observed in both subjects. Increased bone marrow (BM) mitomycin-C (MMC) resistance post treatment has also been identified in at least 1 subject. Subject L102-001-1001 has had blood count stabilization over the 12 months following gene therapy administration. Subject L102-001-1002's course has been complicated by influenza B infection with concomitant decreases in blood counts requiring red blood cell transfusions. Transfusion requirements have decreased following resolution of infection. Since November 2019, 5 additional subjects have been enrolled onto the global Phase 2 study and received investigational infusion. Updated preliminary safety and efficacy data including PB VCN, blood counts and BM MMC resistance will be available at the time of presentation for subjects with over 12 months of follow up; drug product (DP) information (VCN and CD34+ cell dose) will be available for all treated subjects. Conclusions: DP has been successfully manufactured in the Phase I (N=2) and Phase 2 (N=5) to meet the required specificationsSafety profile of RP-L102 continues to be highly favorable.Evidence of engraftment has been seen in at least 1 subject with follow up of at least 12 months as indicated by PB genetic markings and increasing BM CFC MMC resistance; 12+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Río:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: PR has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Wagner:BlueRock: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding.

Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Modulation of innate immune responses in rheumatoid arthritis and other immune‐mediated disorders is of critical importance in the clinic since a growing body of information has shown the key contribution of dysregulated innate responses in the progression of the disease. Mesenchymal stromal cells (MSCs) are the focus of intensive efforts worldwide due to their key role in tissue regeneration and modulation of inflammation. In this study, we define innate immune responses occurring during the early course of treatment with a single dose of expanded adipose‐derived MSCs (eASCs) in established collagen‐induced arthritis. eASCs delay the progression of the disease during the early phase of the disease. This is accompanied by a transient induction of Ly6C+ monocytes that differentiate into IL10+F4/80+ cells in arthritic mice. Strikingly, the induced IL10+F4/80+ myeloid cells preferentially accumulated in the draining lymph nodes. This effect was accompanied with a concomitant declining of their frequencies in the spleens. Our results show that eASCs attenuate the arthritic process by inducing an early innate cell signature that involves a transient induction of Ly6C+ monocytes in periphery that differentiate into IL10+F4/80+ macrophages. Our findings demonstrate that early regulatory innate cell responses, involving the monocyte compartment, are targeted by the eASCs during the onset of collagen‐induced inflammation.

Las terapias avanzadas comprenden un grupo de medicamentos biológicos basados en la terapia génica, la terapia celular y la ingeniería de tejidos. En este artículo se presentan algunos conceptos básicos, se describen las estrategias más prometedoras y se detallan los resultados de diferentes ensayos clínicos para un grupo significativo de enfermedades, especialmente las enfermedades hereditarias monogénicas. Se hace un mayor hincapié en la terapia génica ex vivo en enfermedades del sistema hematopoyético, al ser el grupo de enfermedades pioneras que además han orientado la investigación en el campo. Destacamos también algunos ejemplos de éxito de terapia génica in vivo y señalamos la contribución de la terapia celular y de la ingeniería de tejidos al grupo de enfermedades raras de la piel. Pretendemos con ello dar una visión de la situación de las terapias avanzadas en enfermedades raras y señalamos algunas de las vías de futuro orientadas al desarrollo de tratamientos más eficaces y seguros.

Nine Fanconi anemia patients complementation group A (FA-A), age 2-6 years, have been infused with autologous hematopoietic cells after genetic correction with the therapeutic PGK-FANCA.Wpre* lentiviral vector. In all instances patients underwent CD34+ cell mobilization with G-CSF and plerixafor and were subsequently infused in the absence of any pre-conditioning regimen, in order to avoid genotoxic side effects in a population characterized by DNA repair defects and cancer predisposition. The first four patients were treated between January 2016 and March 2017 and were infused with an estimated number of 170,000 and 410,000 transduced CD34+ cells/Kg. The other five patients were treated more recently with cell numbers that ranged between 50,000 to 1.6x106 corrected CD34+ cells/kg. The analyses of the first four patients showed the presence of corrected cells both in BM and PB after six months post-infusion and progressive increases of gene marking were observed thereafter in all these patients until the most recent follow-up (2 to >3 years post-infusion). Gene marking in BM CD34+ cells correlated with the survival of the CFCs to mitomycin-C, with levels up to 70% at 3 years post-infusion. Additionally, progressive decreases in the percentage of PB T cells with diepoxybutane-induced chromosomal breaks were observed in the patients with higher levels of gene marking. Similarly, stabilized PB cell counts have been observed in patients with higher percentages of gene corrected cells. Insertion site analyses revealed the absence of genotoxic events, and demonstrated the engraftment of pluripotent HSCs and a pattern of oligoclonal reconstitution, consistent with the number of infused corrected CD34+ cells and the absence of conditioning. In the five additional patients treated more recently, the presence of gene corrected PB cells has been confirmed; levels of gene marking have been consistent with data observed in the first four treated patients and with the number of infused CD34+ cells. Our results confirm the engraftment of gene corrected HSCs in non-conditioned FA-A patients, in some cases through more than 3 years of follow-up, suggesting the relevance of this therapeutic approach in FA. Disclosures Rio: Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Segovia:Rocket Pharmaceuticals, Inc.: Equity Ownership, Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Wang:GeneWerk: Employment. Casado:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Galy:Genethon: Employment. Cavazzana:SmartImmune: Other: Founder. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH, Heidelberg, Gemrany: Equity Ownership; German Cancer Research Center, Heidelberg, Germany: Employment. Díaz de Heredia:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sevilla:Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Background: Fanconi anemia (FA) is a rare genetic disorder characterized by defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. 80% of FA patients develop BMF. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a curative treatment for BMF, its utilization and efficacy is limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo insertion of a functional FANCA gene into autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown in preclinical studies to provide a survival advantage to the gene-modified stem cells, leading to correction of BMF. Feasibility of this approach was established in the FANCOLEN-1 clinical trial (Spain), although cell doses and transduction levels varied considerably. Modifications to the collection and manufacturing processes were made in the clinical studies to enhance the dose of transduced HSPCs, with the goal of preventing progression of BMF to obviate the need of an allo-HSCT. Design and Methods: RP-L102-0418 (clinicaltrials.gov # NCT03814408) is a U.S. Phase I clinical trial evaluating the feasibility and safety of autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the FANCA gene (PGK-FANCA-WPRE) in two pediatric patients with FA-A. Patients 30/µL were eligible for treatment. Peripheral blood mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and Plerixafor (Mozobil). CD34+ HSPCs were enriched, placed in culture with cytokines, and transduced with PGK-FANCA-WPRE LV. The investigational drug product (DP) (RP-L102) was infused fresh into patients within 4 hours of release, without any prior conditioning regimen. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain early evidence of efficacy (increasing peripheral blood vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: Two FA-A patients (aged 5 and 6 years) were consented and enrolled on the study at Stanford University. Mobilization and apheresis procedures were performed successfully without any serious adverse events. DP was successfully manufactured using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing. Because of higher transduced CD34+ and colony forming cell (CFC) doses, we anticipate early development of BM MMC resistance in the current study patients. Safety and efficacy data 4 to 6 months post-treatment, including peripheral blood VCN, blood counts and bone marrow MMC resistance, will be available at the time of presentation. Conclusions: DP has been successfully manufactured in the Phase I study (N=2) to meet the required specifications.Patients are being monitored for early efficacy assessments; 6+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs.Plans for Phase II portion of the study are in progress. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Beard:Rocket Pharmaceuticals: Employment, Equity Ownership. Law:Rocket Pharmaceuticals: Employment, Equity Ownership. Nicoletti:Rocket Pharmaceuticals, Inc.: Employment, Equity Ownership. Río:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership.

Introduction: Thromboxane A2 [TxA2] is generated from arachidonic acid by cyclooxigenase-1 (COX-1) (prostaglandin H synthase-1) and thromboxane synthase. Aspirin, which irreversibly inhibits COX-1, is a widely used antiplatelet therapy with proven clinical efficacy. Inherited platelet disorders (IPD) are rare diseases caused by alterations of relevant genes in platelet formation and/or function. Despite the relevance of the TxA2 pathway in platelet physiology, few patients with mutations in PTGS1, the gene encoding COX-1, have been identified ( Objective: Characterization of a patient with aspirin-like platelet defect and moderate bleeding, enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with IPD". Methods: The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding (BAT-ISTH=6) and an aspirin-like platelet dysfunction. No coagulation defect or other relevant clinical symptoms were present. Platelet phenotyping included: blood count, PFA-100; platelet aggregation [LTA], glycoproteins (GP), activation and secretion of granules by flow cytometry (FC), TxA2 synthesis by enzyme-immunoassay, synthesis of eicosanoids by tandem gas chromatography with mass spectrometry (LC-MS), western-blot (WB) of platelet lysates, and immunofluorescence (IF) assays. The patient's DNA was analyzed with a HTS-gene panel (Bastida et al, Haematologica 2018). A HEK 293T cell transfection model was established to further assess the pathogenicity of the candidate variant found in the patient. Results: The index case has normal platelet size and count (206x109/L; 11.4 fL). PFA-100 times were normal for COL-ADP and prolonged for COL-EPI (>300s). The FC analysis showed normal expression of GPs (Ib/IX, IIb/IIIa, Ia, GPVI) and reduced fibrinogen*488 binding (20-30%) in response to ADP, TRAP and low dose CRP (2ug/mL). P-selectin and CD63 secretion with agonists was comparable to those of controls. LTA was normal with ristocetin (1.25mg/mL) and TRAP (25uM), reduced by 40-50% with ADP (10uM) and collagen (3ug/mL) and absent with epinephrine (10uM), low dose collagen (1ug/mL) and arachidonic acid (1.6mM). LTA with U46619 (5uM), a direct agonist of the TxA2 receptor, was normal, suggesting a defect in TxA2 synthesis. Indeed, TxA2 levels in LTA supernatants in the patient were very low (5ng/mL; G, [p.Asn143Ser] in PTGS1. This variant, not previously described, affects a conserved residue in the catalytic domain of COX-1, which is one of the three N-glycosylation sites in the enzyme. The variant was not associated with reduced COX-1 expression as evaluated by WB in platelet lysates, and by IF in spread washed platelets and leukocytes. HEK 293T cells transfected with wild-type COX-1 construct (validated by RT-PCR and WB), displayed substantial TxA2 synthesis (500ng/mL; 2.5x105 transfected cells) in response to arachidonic acid. In contrast, similar transfection of p.143Ser COX-1 mutant almost abrogated this TxA2 production (≈50-75ng/mL in 2.5x105 transfected cells). Conclusion: We have identified a novel autosomal dominant COX-1 variant, p.Asn143Ser, associated with functional haploinsufficiency of the enzyme and platelet aggregation defects. To our knowledge, this case represents the third description of variants in PTGS1 (Nance, JTH 2016; Sivapalaratnam, Blood 2018), which cause platelet dysfunction and bleeding. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

The promising ability to genetically modify hematopoietic stem and progenitor cells (HSPCs) by precise gene editing remains challenging due to their sensitivity and poor permissiveness. This represents the first evidence of implementing a gene editing strategy in a murinesafe harborlocus that phenotypically corrects primary cells derived from a mouse model of Fanconi anemia (FA).By co-delivering TALENs and a donor therapeuticFANCAcassette template to theMbs85locus (ortholog of the hAAVS1 safe harborlocus), we achieved efficient gene targeting (23%) in FA mouse embryonic fibroblasts (MEFs). This resulted in the phenotypic correction of these cells, as revealed by the improvement of their hypersensitivity to mitomycinC. Moreover, robust evidence of targeted integration was observed in murine WT and FA-A hematopoietic progenitor cells (HPC) reaching mean targeted integration values of 20.98% and 16.33% respectively, with phenotypic correction of FA HPCs. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy in a murinesafe harborlocus, such as theMbs85gene, of MEFs and murine HPC from a FA mouse model.

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the &ldquo, responder&rdquo, mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-strand breaks, and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here we describe for the first time the possibility to correct mutations in FA genes by generating compensatory indels that restore their coding frame. The efficacy of the strategy was first verified in FA lymphoblastic cell lines and then in primary FA CD34 cells, which acquired proliferative advantage and correction of their characteristic FAcell phenotype. Importantly, in contrast to homologous directed repair (HDR), the efficacy of NHEJ to edit primitive human HSCs was comparable to that observed in more mature progenitor cells, indicating that NHEJ-editing approaches should constitute a good approach for the editing of long-term repopulating HSCs and consequently for the treatment of FA and other monogenic hematopoietic diseases.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol., Graphical Abstract, Ivics and collegues refined the Sleeping Beauty transposon system for gene transfer in human hematopoietic stem and progenitor cells by vectorizing the transposon components as minicircle DNA and synthetic mRNA. The advanced vector system enables efficient and safe non-viral engineering of hematopoietic cells that can be transplanted into immunodeficient mice.

In vivo detection and quantification of inflammation is a major goal in molecular imaging. Furthermore, cell-specific detection of inflammation would be a tremendous advantage in the characterization of many diseases. Here, we show how this goal can be achieved through the synergistic combination of nanotechnology and nuclear imaging. One of the most remarkable features of this hybrid approach is the possibility to tailor the pharmacokinetics of the nanomaterial-incorporated biomolecule and radionuclide. A good example of this approach is the covalent binding of a large amount of a neutrophil-specific, hydrophobic peptide on the surface of 68Ga core-doped nanoparticles. This new nano-radiotracer has been used for non-invasive in vivo detection of acute inflammation with very high in vivo labelling efficiency, i.e. a large percentage of labelled neutrophils. Furthermore, we demonstrate that the tracer is neutrophil-specific and yields images of neutrophil recruitment of unprecedented quality. Finally, the nano-radiotracer was successfully detected in chronic inflammation in atherosclerosis-prone ApoE-/- mice after several weeks on a high-fat diet. This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and from Carlos III Health Research Institute (grant number: DTS16/00059). We thank Simon Bartlett for editorial assistance and manuscript preparation. The CNIC is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505). Sí

Introduction: Fanconi anemia (FA) is a rare inherited disease characterized by congenital abnormalities, bone marrow failure (BMF) and cancer predisposition. Although allogeneic hematopoietic stem cell transplantation (HSCT) is the preferential therapy for restoring the bone marrow (BM) function of FA patients, gene therapy represents a new alternative in FA.Areas covered: This review covers the areas of gene therapy with γ-retroviral and lentiviral vectors. Additionally, the perspectives of gene editing and cell reprogramming for the gene therapy of FA are discussed.Expert opinion: Currently, there is no clinical evidence showing that gene therapy can restore the BM function of FA patients. However, improvements in vector development and advances in manipulation of hematopoietic stem cells (HSCs) have resulted in gene therapy progresses, opening new perspectives for the treatment of FA patients. Although, in FA patients, reduced HSC numbers will be available for gene correction and autologous transplanta...

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.

International audience; Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34(+) cells from FA-A patients with a therapeutic FANCA-lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34(+) cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34(+) cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34(+) graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)‐mediated insertion of a non‐therapeutic EGFP‐reporter donor in the AAVS1 “safe harbor” locus of FA‐A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA‐A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA‐A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. The authors would like to thank Aurora de la Cal for her assistance with thecoordination in the delivery of the samples from the patients. RebecaSánchez-Domínguez for her expertise in flow cytometry and Centro de Trans-fusiones de la Comunidad de Madrid for providing cord blood samples. Theauthors are also indebted to the FA patients, families, and clinicians from theFA spanish network. This work was supported by grants from the“7th Frame-work Program European Commission (HEALTH-F5-2012-305421; EUROFAN-COLEN)”,“Ministerio de Sanidad, Servicios Sociales e Igualdad”(EC11/060andEC11/550),“Ministerio de Economía, Comercio y Competitividad y FondoEuropeo de Desarrollo Regional (FEDER)”(SAF2015-68073-R), and“Fondo deInvestigaciones Sanitarias, Instituto de Salud Carlos III”(RD12/0019/0023). Theauthors also thank the Fundación Marcelino Botín for promoting translationalresearch at the Hematopoietic Innovative Therapies Division of the CIEMAT.CIBERER is an initiative of the Instituto de Salud Carlos III, Spain. Sí

Detectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 x 10(-9)). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 x 10(-16)). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 x 10(-5)), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 x 10(-5)). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients.

Leukocyte adhesion deficiency type-I is a primary immunodeficiency caused by mutations in the ITGB2 gene (CD18 leukocyte integrin) which lead to defects in leukocyte extravasation. To investigate the role of CD18 in hematopoietic stem cell (HSC) biology, we have thoroughly characterized the HSCs of CD18 Itgb2tm1bay hypomorphic mice (CD18HYP) both by flow cytometry and using in vitro and in vivo transplantation assays. Flow cytometry analyses and cultures in methyl cellulose revealed that bone marrow (BM) from CD18HYP mice was enriched in hematopoietic precursors, mainly early quiescent short-term and long-term Hematopoietic progenitors cells. Strikingly, BM competition assays showed a progressive expansion of CD18HYP-derived hematopoiesis in recipient mice. Additionally, we provide evidence that this HSC expansion was not caused by an increased homing capacity of CD18HYP HSCs or by alterations in the hematopoietic environment of CD18HYP mice due to defects in neutrophils clearance. On the contrary, our data demonstrated that the reduced expression of CD18 causes a cell-autonomous expansion in the HSC compartment, thus revealing unexpected regulatory functions for CD18 in mouse HSCs. Stem Cells 2014;32:2794–2798

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2Δ27/Δ27), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2Δ27/Δ27 induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2Δ27/Δ27 iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2Δ27/Δ27 mouse embryonic fibroblasts. Gene-corrected Brca2Δ27/Δ27 iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2Δ27/Δ27 recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2Δ27/Δ27 iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed. Stem Cells 2014;32:436–446

Background and Purpose We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. Experimental Approach Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. Key Results While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. Conclusions and Implications Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of ‘fanconizing’ cancer cells in order to make them more sensitive to other anti-tumour drugs.

Fanconi anemia (FA) is a DNA repair syndrome characterized by bone marrow failure, congenital abnormalities and cancer predisposition. Based on previous experimental results showing the in vivo proliferative advantage of gene corrected FA patients' hematopoietic stem cells (HSCs; Rio, Navarro et al. Blood 2017) a gene therapy trial in non-conditioned FA-A patients was initiated in 2016. Six patients have been treated to-date using fresh and cryopreserved CD34+ cells mobilized to peripheral blood with G-CSF and plerixafor, and transduced with the PGK-FANCA.Wpre* lentiviral vector. Cell doses infused in four patients with a follow-up of at least 12 months varied from 0.6 to 1.4 million CD34+ cells/kg. Transduction efficacies of these samples, determined as vector copies per cell, ranged from 0.17 to 0.53 copies/cell. Despite the absence of patients' conditioning, a marked in vivo expansion of gene-corrected cells was observed in all hematopoietic cell lineages analyzed in BM and PB. Significantly, up to 44% of corrected cells were determined in total PB cells at the most recent follow-up visit (24 month) in the first treated patient. Insertion site analyses in PB cells showed an oligoclonal pattern of hematopoietic reconstitution, and revealed engraftment of multipotent corrected HSCs and no evidence of insertion-site mediated clonal expansion. Functional studies showed significant increases in the resistance of BM progenitors to mitomycin C in all treated patients. Additionally, patients with higher levels of corrected cells also showed significant increases in the chromosomal stability of T cells exposed to diepoxybutane. Finally, analyses discriminating the presence of corrected and uncorrected PB cells in these patients showed marked increases in the total number of corrected leukocytes, contrasting to progressive decreases of uncorrected cells. Our studies demonstrate for the first time that lentiviral-mediated gene therapy results in progressive engraftment and phenotypic correction of HSCs in non-conditioned FA patients, suggesting that this gene therapy approach may constitute a low-toxicity option for the treatment and prevention of BMF in patients with FA. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.