Mara Pavel-Dinu

All Publications

• Failure of metabolic checkpoint control during late-stage granulopoiesis drives neutropenia in reticular dysgenesis. Blood Wang, W., Arreola, M., Mathews, T., DeVilbiss, A. W., Zhao, Z., Martin-Sandoval, M., Mohammed, A., Benegiamo, G., Awani, A., Goeminne, L. J., Dever, D. P., Nakauchi, Y., Porteus, M. H., Pavel-Dinu, M., Al Herz, W., Auwerx, J., Morrison, S. J., Weinacht, K. G. 2024

  Abstract

  Cellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Here, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in reticular dysgenesis patient samples with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated AMP and IMP levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis.

  View details for DOI 10.1182/blood.2024024123

  View details for PubMedID 39378586

• Strategic infection prevention after genetically modified hematopoietic stem cell therapies: recommendations from the International Society for Cell & Gene Therapy Stem Cell Engineering Committee. Cytotherapy John, T. D., Maron, G., Abraham, A., Bertaina, A., Bhoopalan, S. V., Bidgoli, A., Bonfim, C., Coleman, Z., DeZern, A., Li, J., Louis, C., Oved, J., Pavel-Dinu, M., Purtill, D., Ruggeri, A., Russell, A., Wynn, R., Boelens, J. J., Prockop, S., Sharma, A. 2024

  Abstract

  There is lack of guidance for immune monitoring and infection prevention after administration of ex vivo genetically modified hematopoietic stem cell therapies (GMHSCT). We reviewed current infection prevention practices as reported by providers experienced with GMHSCTs across North America and Europe, and assessed potential immunologic compromise associated with the therapeutic process of GMHSCTs described to date. Based on these assessments, and with consensus from members of the International Society for Cell & Gene Therapy (ISCT) Stem Cell Engineering Committee, we propose risk-adapted recommendations for immune monitoring, infection surveillance and prophylaxis, and revaccination after receipt of GMHSCTs. Disease-specific and GMHSCT-specific considerations should guide decision making for each therapy.

  View details for DOI 10.1016/j.jcyt.2024.02.005

  View details for PubMedID 38483362

• Genetically Corrected RAG2-SCID Human Hematopoietic Stem Cells Restore V(D)J-Recombinase and Rescue Lymphoid Deficiency. Blood advances Pavel-Dinu, M., Gardner, C. L., Nakauchi, Y., Kawai, T., Delmonte, O. M., Palterer, B., Bosticardo, M., Pala, F., Viel, S., Malech, H. L., Ghanim, H. Y., Bode, N. M., Kurgan, G. L., Detweiler, A. M., Vakulskas, C. A., Neff, N. F., Sheikali, A., Menezes, S. T., Chrobok, J., Hernandez Gonzalez, E. M., Majeti, R., Notarangelo, L. D., Porteus, M. H. 2023

  Abstract

  Recombination-activating genes (RAG1 and RAG2) are critical in lymphoid cell development and function by initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCID), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phases of the cell cycle at the early stages of T and B cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and one RAG2-SCID patient. V(D)J recombinase activity was restored following gene correction of RAG2-SCID-derived HSPCs, resulting in the development of TCR ab and gd CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of the CDR3 length and preserved usage of distal TRAV genes. We confirmed in vivo rescue of B-cell development, with normal IgM surface expression and a significant decrease in CD56bright NK cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

  View details for DOI 10.1182/bloodadvances.2023011766

  View details for PubMedID 38096800

• High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nature biotechnology Selvaraj, S., Feist, W. N., Viel, S., Vaidyanathan, S., Dudek, A. M., Gastou, M., Rockwood, S. J., Ekman, F. K., Oseghale, A. R., Xu, L., Pavel-Dinu, M., Luna, S. E., Cromer, M. K., Sayana, R., Gomez-Ospina, N., Porteus, M. H. 2023

  Abstract

  Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

  View details for DOI 10.1038/s41587-023-01888-4

  View details for PubMedID 37537500

  View details for PubMedCentralID 3694601

• Discovery of Key Transcriptional Regulators of Alloantigen-Inducible Tregs Used for Cell Therapy Cepika, A., Amaya, L., Waichler, C., Narula, M., Thomas, B. C., Chen, P. P., Mantilla, M. M., Pavel-Dinu, M., Freeborn, R., Porteus, M. H., Bacchetta, R., Mueller, F., Greenleaf, W. J., Chang, H. Y., Roncarolo, M. CELL PRESS. 2023: 370-371

  View details for Web of Science ID 001045144201371

• Base-Editing as a Safe and Highly Effective Alternative Treatment for X-SCID Compared to CRISPR-Cas9 Nuclease Editing with an AAV Donor Bzhilyanskaya, V., Brault, J., Liu, S., Kozhushko, N., Lawson, A., Pavel-Dinu, M., Clark, A. B., Meis, R. J., Ma, M., Lazzarotto, C. R., Tsai, S. Q., Wu, X., Dahl, G., Kleinstiver, B. P., Porteus, M. H., Malech, H. L., De Ravin, S. CELL PRESS. 2023: 572

  View details for Web of Science ID 001045144202381

• International society for cell & gene therapy stem cell engineering committee: Cellular therapies for the treatment of graft-versus-host-disease after hematopoietic stem cell transplant. Cytotherapy Garcia-Rosa, M., Abraham, A., Bertaina, A., Bhoopalan, S. V., Bonfim, C., Cohen, S., DeZern, A., Louis, C., Oved, J., Pavel-Dinu, M., Purtill, D., Ruggeri, A., Russell, A., Sharma, A., Wynn, R., Boelens, J. J., Prockop, S. 2023

  Abstract

  Allogeneic hematopoietic stem cell transplant is a curative approach for many malignant and non-malignant hematologic conditions. Despite advances in its prevention and treatment, the morbidity and mortality related to graft-versus-host disease (GVHD) remains. The mechanisms by which currently used pharmacologic agents impair the activation and proliferation of potentially alloreactive T cells reveal pathways essential for the detrimental activities of these cell populations. Importantly, these same pathways can be important in mediating the graft-versus-leukemia effect in recipients transplanted for malignant disease. This knowledge informs potential roles for cellular therapies such as mesenchymal stromal cells and regulatory T cells in preventing or treating GVHD. This article reviews the current state of adoptive cellular therapies focused on GVHD treatment.We conducted a search for scientific literature in PubMed® and ongoing clinical trials in clinicaltrial.gov with the keywords "Graft-versus-Host Disease (GVHD)," "Cellular Therapies," "Regulatory T cells (Tregs)," "Mesenchymal Stromal (Stem) Cells (MSCs)," "Natural Killer (NK) Cells," "Myeloid-derived suppressor cells (MDSCs)," and "Regulatory B-Cells (B-regs)." All the published and available clinical studies were included.Although most of the existing clinical data focus on cellular therapies for GVHD prevention, there are observational and interventional clinical studies that explore the potential for cellular therapies to be safe modalities for GVHD treatment while maintaining the graft-versus-leukemia effect in the context of malignant diseases. However, there are multiple challenges that limit the broader use of these approaches in the clinical scenario.There are many ongoing clinical trials to date with the promise to expand our actual knowledge on the role of cellular therapies for GVHD treatment in an attempt to improve GVHD-related outcomes in the near future.

  View details for DOI 10.1016/j.jcyt.2023.02.007

  View details for PubMedID 36941149

• Rare immune diseases paving the road for genome editing-based precision medicine. Frontiers in genome editing Pavel-Dinu, M., Borna, S., Bacchetta, R. 2023; 5: 1114996

  Abstract

  Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing platform heralds a new era of gene therapy. Innovative treatments for life-threatening monogenic diseases of the blood and immune system are transitioning from semi-random gene addition to precise modification of defective genes. As these therapies enter first-in-human clinical trials, their long-term safety and efficacy will inform the future generation of genome editing-based medicine. Here we discuss the significance of Inborn Errors of Immunity as disease prototypes for establishing and advancing precision medicine. We will review the feasibility of clustered regularly interspaced short palindromic repeats-based genome editing platforms to modify the DNA sequence of primary cells and describe two emerging genome editing approaches to treat RAG2 deficiency, a primary immunodeficiency, and FOXP3 deficiency, a primary immune regulatory disorder.

  View details for DOI 10.3389/fgeed.2023.1114996

  View details for PubMedID 36846437

• A Curative DNA Code for Hematopoietic Defects: Novel Cell Therapies for Monogenic Diseases of the Blood and Immune System. Hematology/oncology clinics of North America Porteus, M. H., Pavel-Dinu, M., Pai, S. 2022

  Abstract

  Innovations in programmable nucleases have expanded genetic engineering capabilities, raising the possibility of a new approach to curing monogenic hematological diseases. Feasibility studies using exvivo targeted genome-editing, and nonintegrating viral vectors show outstanding potential for correcting genetic conditions at their root cause. This article reviews the latest technological advances in the CRISPR/Cas9 system alone and combined with engineered viruses as editing tools for human hematopoietic stem and progenitor cells (HSPCs). We discuss the early phase in human trials of genome editing-based therapies for hemoglobinopathies.

  View details for DOI 10.1016/j.hoc.2022.05.002

  View details for PubMedID 35773054

• CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells. Frontiers in immunology Brault, J., Liu, T., Liu, S., Lawson, A., Choi, U., Kozhushko, N., Bzhilyanskaya, V., Pavel-Dinu, M., Meis, R. J., Eckhaus, M. A., Burkett, S. S., Bosticardo, M., Kleinstiver, B. P., Notarangelo, L. D., Lazzarotto, C. R., Tsai, S. Q., Wu, X., Dahl, G. A., Porteus, M. H., Malech, H. L., De Ravin, S. S. 2022; 13: 1067417

  Abstract

  Introduction: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs.Methods: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus.Results and discussion: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

  View details for DOI 10.3389/fimmu.2022.1067417

  View details for PubMedID 36685559

• Correction to: Gene Editing Rescues in Vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. Journal of clinical immunology Gardner, C. L., Pavel-Dinu, M., Dobbs, K., Bosticardo, M., Reardon, P. K., Lack, J., DeRavin, S. S., Le, K., Bello, E., Pala, F., Delmonte, O. M., Malech, H., Montel-Hagen, A., Crooks, G., Acuto, O., Porteus, M. H., Notarangelo, L. D. 2021

  View details for DOI 10.1007/s10875-021-01030-6

  View details for PubMedID 33821398

• Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair. Gene therapy Sweeney, C. L., Pavel-Dinu, M., Choi, U., Brault, J., Liu, T., Koontz, S., Li, L., Theobald, N., Lee, J., Bello, E. A., Wu, X., Meis, R. J., Dahl, G. A., Porteus, M. H., Malech, H. L., De Ravin, S. S. 2021

  Abstract

  X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

  View details for DOI 10.1038/s41434-021-00251-z

  View details for PubMedID 33712802

• Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. Journal of clinical immunology Gardner, C. L., Pavel-Dinu, M., Dobbs, K., Bosticardo, M., Reardon, P. K., Lack, J., DeRavin, S. S., Le, K., Bello, E., Pala, F., Delmonte, O. M., Malech, H., Montel-Hagan, A., Crooks, G., Acuto, O., Porteus, M. H., Notarangelo, L. D. 2021

  Abstract

  Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

  View details for DOI 10.1007/s10875-021-00989-6

  View details for PubMedID 33650026

• Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells. Blood De Ravin, S. S., Brault, J., Meis, R. J., Liu, S., Li, L., Pavel-Dinu, M., Lazzarotto, C. R., Liu, T. Q., Koontz, S., Choi, U., Sweeney, C. L., Theobald, N., Lee, G., Clark, A., Burkett, S., Kleinstiver, B. P., Porteus, M., Tsai, S., Kuhns, D. B., Dahl, G. A., Headey, S., Wu, X., Malech, H. L. 2021

  Abstract

  Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.

  View details for DOI 10.1182/blood.2020008503

  View details for PubMedID 33623984

• Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex. Molecular therapy : the journal of the American Society of Gene Therapy de Alencastro, G. n., Puzzo, F. n., Pavel-Dinu, M. n., Zhang, F. n., Pillay, S. n., Majzoub, K. n., Tiffany, M. n., Jang, H. n., Sheikali, A. n., Cromer, M. K., Meetei, R. n., Carette, J. E., Porteus, M. H., Pekrun, K. n., Kay, M. A. 2021; 29 (3): 1016–27

  Abstract

  Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.

  View details for DOI 10.1016/j.ymthe.2020.10.020

  View details for PubMedID 33678249

• Lentivector versus CRISPR/Cas9/AAV6 Gene Editing in X-Linked Severe Combined Immunodeficiency CD34(+) Hematopoietic Cells Liu, T., Brault, J., Pavel-Dinu, M., Liu, S., Meis, R. J., Koontz, S., Corsino, C., Bosticardo, M., Kleinstiver, B., Notarangelo, L. D., Wu, X., Dahl, G. A., Porteus, M., Malech, H. L., De Ravin, S. CELL PRESS. 2020: 355–56

  View details for Web of Science ID 000530089301338

• A Genomic Editing-Based Therapeutic Approach for RAG2 Deficiency Pavel-Dinu, M., Gardner, C., La Guardia, T. A., Vakulskas, C. A., Lee, C., Bao, G., Sheikali, A., Menezes, T., Notarangelo, L. D., Porteus, M. H. CELL PRESS. 2020: 55–56

  View details for Web of Science ID 000530089300107

• Proteins Complex of the Fanconi Anemia Pathway as Determinant of AAV-Mediated Genomic Targeted Integration Puzzo, F., de Alencastro, G., Pavel-Dinu, M., Zhang, F., Pillay, S., Majzoub, K., Tiffany, M., Jang, H., Sheikali, A., Cromer, K. M., Meetei, R., Carette, J. E., Porteus, M. H., Pekrun, K., Kay, M. A. CELL PRESS. 2020: 459

  View details for Web of Science ID 000530089302154

• Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination. Cell stem cell Martin, R. M., Ikeda, K., Cromer, M. K., Uchida, N., Nishimura, T., Romano, R., Tong, A. J., Lemgart, V. T., Camarena, J., Pavel-Dinu, M., Sindhu, C., Wiebking, V., Vaidyanathan, S., Dever, D. P., Bak, R. O., Laustsen, A., Lesch, B. J., Jakobsen, M. R., Sebastiano, V., Nakauchi, H., Porteus, M. H. 2019; 24 (5): 821

  Abstract

  Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

  View details for PubMedID 31051134

• Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination CELL STEM CELL Martin, R. M., Ikeda, K., Cromer, M., Uchida, N., Nishimura, T., Romano, R., Tong, A. J., Lemgart, V. T., Camarena, J., Pavel-Dinu, M., Sindhu, C., Wiebking, V., Vaidyanathan, S., Dever, D. P., Bak, R. O., Laustsen, A., Lesch, B. J., Jakobsen, M. R., Sebastiano, V., Nakauchi, H., Porteus, M. H. 2019; 24 (5): 821-+

  View details for DOI 10.1016/j.stem.2019.04.001

  View details for Web of Science ID 000466726500019

• Gene correction for SCID-X1 in long-term hematopoietic stem cells (vol 10, 1634, 2019) NATURE COMMUNICATIONS Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C. E., Lee, C., Bao, G., Kildebeck, E. J., Punjya, N., Sindhu, C., Inlay, M. A., Saxena, N., DeRavin, S., Malech, H., Roncarolo, M., Weinberg, K. I., Porteus, M. H. 2019; 10

  View details for DOI 10.1038/s41467-019-10080-9

  View details for Web of Science ID 000465840000001

• Gene correction for SCID-X1 in long-term hematopoietic stem cells NATURE COMMUNICATIONS Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C. E., Lee, C., Bao, G., Kildebeck, E. J., Punjya, N., Sindhu, C., Inlay, M. A., Saxena, N., DeRavin, S., Malech, H., Roncarolo, M., Weinberg, K., Porteus, M. H. 2019; 10

  View details for DOI 10.1038/s41467-019-09614-y

  View details for Web of Science ID 000463872400010

• Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature communications Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C. E., Lee, C., Bao, G., Kildebeck, E. J., Punjya, N., Sindhu, C., Inlay, M. A., Saxena, N., DeRavin, S. S., Malech, H., Roncarolo, M. G., Weinberg, K. I., Porteus, M. H. 2019; 10 (1): 1634

  Abstract

  Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

  View details for PubMedID 30967552

• Identification of preexisting adaptive immunity to Cas9 proteins in humans NATURE MEDICINE Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., Behlke, M. A., Dejene, B., Cieniewicz, B., Romano, R., Lesch, B. J., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Weinberg, K. I., Porteus, M. H. 2019; 25 (2): 249-+

  View details for DOI 10.1038/s41591-018-0326-x

  View details for Web of Science ID 000457842100021

• Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature medicine Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., Behlke, M. A., Dejene, B., Cieniewicz, B., Romano, R., Lesch, B. J., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Weinberg, K. I., Porteus, M. H. 2019

  Abstract

  The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

  View details for PubMedID 30692695

• Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature communications Pavel-Dinu, M. n., Wiebking, V. n., Dejene, B. T., Srifa, W. n., Mantri, S. n., Nicolas, C. E., Lee, C. n., Bao, G. n., Kildebeck, E. J., Punjya, N. n., Sindhu, C. n., Inlay, M. A., Saxena, N. n., DeRavin, S. S., Malech, H. n., Roncarolo, M. G., Weinberg, K. I., Porteus, M. H. 2019; 10 (1): 5624

  Abstract

  An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41467-019-13620-5

  View details for PubMedID 31796738

• High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells. Blood advances Jeong, J. n., Jager, A. n., Domizi, P. n., Pavel-Dinu, M. n., Gojenola, L. n., Iwasaki, M. n., Wei, M. C., Pan, F. n., Zehnder, J. L., Porteus, M. H., Davis, K. L., Cleary, M. L. 2019; 3 (19): 2825–35

  Abstract

  Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.

  View details for DOI 10.1182/bloodadvances.2019000450

  View details for PubMedID 31582391

• Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature communications Pavel-Dinu, M. n., Wiebking, V. n., Dejene, B. T., Srifa, W. n., Mantri, S. n., Nicolas, C. E., Lee, C. n., Bao, G. n., Kildebeck, E. J., Punjya, N. n., Sindhu, C. n., Inlay, M. A., Saxena, N. n., DeRavin, S. S., Malech, H. n., Roncarolo, M. G., Weinberg, K. I., Porteus, M. H. 2019; 10 (1): 2021

  Abstract

  The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.

  View details for PubMedID 31028274

• CRISPR-Mediated Targeted Insertion of Cybb cDNAs into the Cybb Locus for Correction of X-CGD Patient CD34(+) Cells Sweeney, C. L., Choi, U., Pavel-Dinu, M., Koontz, S., Li, L., Theobald, N., Lee, J., Wu, X., Porteus, M. H., Malech, H. L., De Ravin, S. CELL PRESS. 2018: 233

  View details for Web of Science ID 000435342203060

• Genome Editing for IL-10 Deficiency in Purified Hematopoietic Stem Cells Romano, R., Pavel-Dinu, M., Bacchetta, R., Porteus, M. H., Roncarolo, M. CELL PRESS. 2018: 237–38

  View details for Web of Science ID 000435342203069

• Genome Editing of Long-Term Human Hematopoietic Stem Cells for X-Linked Severe Combined Immunodeficiency Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C., Lee, C. M., Bao, G., Kildebeck, E., Punjya, N., Sindhu, C., Inlay, M. A., Saxena, N. S., De Ravin, S., Malech, H. L., Roncarolo, M., Weinberg, K., Porteus, M. SPRINGER/PLENUM PUBLISHERS. 2018: 365–66

  View details for Web of Science ID 000431311600087

• A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature medicine Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R. n., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S. n., Camarena, J. n., Lee, C. M., Park, S. H., Wiebking, V. n., Bak, R. O., Gomez-Ospina, N. n., Pavel-Dinu, M. n., Sun, W. n., Bao, G. n., Porteus, M. H., Behlke, M. A. 2018; 24 (8): 1216–24

  Abstract

  Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

  View details for PubMedID 30082871

• Correction of X-Linked Severe Combined Immunodeficiency in Human Hematopoietic Stem and Progenitor Cells Pavel-Dinu, M., Wiebking, V. A., Gomez-Ospina, N., Bak, R. O., Dejene, B. T., Mantri, S., Nicolas, C. E., Punjya, N. R., Lee, C., Weinberg, K. I., Bao, G., DeRavin, S., Porteus, M. H. CELL PRESS. 2017: 345–46

  View details for Web of Science ID 000401083600746

• CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells. Nature Dever, D. P., Bak, R. O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C. E., Pavel-Dinu, M., Saxena, N., Wilkens, A. B., Mantri, S., Uchida, N., Hendel, A., Narla, A., Majeti, R., Weinberg, K. I., Porteus, M. H. 2016

  Abstract

  The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

  View details for DOI 10.1038/nature20134

  View details for PubMedID 27820943

• A crisper look at genome editing: RNA-guided genome modification. Molecular therapy : the journal of the American Society of Gene Therapy Damian, M., Porteus, M. H. 2013; 21 (4): 720-722

  View details for DOI 10.1038/mt.2013.46

  View details for PubMedID 23542565

  View details for PubMedCentralID PMC3616526

• A Crisper Look at Genome Editing: RNA-guided Genome Modification MOLECULAR THERAPY Damian, M., Porteus, M. H. 2013; 21 (4): 719-721

  View details for DOI 10.1038/mt.2013.46

  View details for Web of Science ID 000317110300002

  View details for PubMedCentralID PMC3616526

• Reprogramming towards pluripotency requires AID-dependent DNA demethylation NATURE Bhutani, N., Brady, J. J., Damian, M., Sacco, A., Corbel, S. Y., Blau, H. M. 2010; 463 (7284): 1042-U57

  Abstract

  Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

  View details for DOI 10.1038/nature08752

  View details for Web of Science ID 000275108400028

  View details for PubMedID 20027182

  View details for PubMedCentralID PMC2906123

• SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span CELL Kawahara, T. L., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin, M., McCord, R. A., Ongaigui, K. C., Boxer, L. D., Chang, H. Y., Chua, K. F. 2009; 136 (1): 62-74

  Abstract

  Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

  View details for DOI 10.1016/j.cell.2008.10.052

  View details for PubMedID 19135889

• SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin NATURE Michishita, E., McCord, R. A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T. L., Barrett, J. C., Chang, H. Y., Bohr, V. A., Ried, T., Gozani, O., Chua, K. F. 2008; 452 (7186): 492-U16

  Abstract

  The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

  View details for DOI 10.1038/nature06736

  View details for PubMedID 18337721