Dr. Porteus was raised in California and was a local graduate of Gunn High School before completing A.B. degree in “History and Science” at Harvard University where he graduated Magna Cum Laude and wrote an thesis entitled “Safe or Dangerous Chimeras: The recombinant DNA controversy as a conflict between differing socially constructed interpretations of recombinant DNA technology.” He then returned to the area and completed his combined MD, PhD at Stanford Medical School with his PhD focused on understanding the molecular basis of mammalian forebrain development with his PhD thesis entitled “Isolation and Characterization of TES-1/DLX-2: A Novel Homeobox Gene Expressed During Mammalian Forebrain Development.” After completion of his dual degree program, he was an intern and resident in Pediatrics at Boston Children’s Hospital and then completed his Pediatric Hematology/Oncology fellowship in the combined Boston Chidlren’s Hospital/Dana Farber Cancer Institute program. For his fellowship and post-doctoral research he worked with Dr. David Baltimore at MIT and CalTech where he began his studies in developing homologous recombination as a strategy to correct disease causing mutations in stem cells as definitive and curative therapy for children with genetic diseases of the blood, particularly sickle cell disease. Following his training with Dr. Baltimore, he took an independent faculty position at UT Southwestern in the Departments of Pediatrics and Biochemistry before again returning to Stanford in 2010 as an Associate Professor. During this time his work has been the first to demonstrate that gene correction could be achieved in human cells at frequencies that were high enough to potentially cure patients and is considered one of the pioneers and founders of the field of genome editing—a field that now encompasses thousands of labs and several new companies throughout the world. His research program continues to focus on developing genome editing by homologous recombination as curative therapy for children with genetic diseases but also has interests in the clonal dynamics of heterogeneous populations and the use of genome editing to better understand diseases that affect children including infant leukemias and genetic diseases that affect the muscle. Clinically, Dr. Porteus attends at the Lucille Packard Children’s Hospital where he takes care of pediatric patients undergoing hematopoietic stem cell transplantation.

Clinical Focus

  • Hematopoietic Stem Cell Transplantation
  • Pediatric Hematology-Oncology

Professional Education

  • Board Certification: Pediatric Hematology-Oncology, American Board of Pediatrics (2000)
  • Fellowship:Children's Hospital Boston (1999) MA
  • Residency:Children's Hospital Boston (1996) MA
  • Medical Education:Stanford University School of Medicine (1994) CA

Current Research and Scholarly Interests

Genome Editing and Population Dynamics for Gene Therapy and Cancer Research

Clinical Trials

  • Natural History Study of SCID Disorders Recruiting

    Individuals with Primary Immune Deficiency (PID) may develop severe, life-threatening infections as a result of inherited defects in the genes that normally instruct blood-forming cells to develop and to fight infections. PID diseases include Severe Combined Immune Deficiency (SCID), leaky SCID, Omenn syndrome (OS), and Reticular Dysgenesis (RD). PIDs may be treated by transplantation of bone marrow stem cells from a healthy person or, in some cases, by enzyme replacement or by gene therapy. Patients with SCID were among the first to receive bone marrow stem cell (also called hematopoietic cells) transplantation (HCT) more than 40 years ago, and HCT is the standard treatment today for this group of diseases. Since PID diseases are rare, there are not enough patients at any single center to determine the full range of causes, natural history, or best methods of treatment. For this research study many PID centers across North America have organized into the Primary Immune Deficiency Treatment Consortium (PIDTC) to pool their experience and study PIDs together. The overall goal of this study is the prospective evaluation of children with SCID and related disorders who are treated under a variety of protocols at participating institutions. The study aims to identify variables contributing to the best outcomes for HCT.

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  • Patients Treated for Chronic Granulomatous Disease (CGD) Since 1995 Recruiting

    Chronic granulomatous disease (CGD) is an inherited immune system abnormality in which bone marrow transplantation (BMT) has been shown to be curative. However the risks of transplantation are high and not all patients with CGD may need to undergo this high risk procedure. This study will determine the long term medical condition and daily functioning of participants with CGD after a transplant and if possible, compare these results to participants who do not undergo a transplant.

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  • Patients Treated for SCID (1968-Present) Recruiting

    People with Primary Immune Deficiency (PID) may develop severe, life-threatening infections as a result of inherited defects in the genes that normally instruct blood-forming cells to develop and to fight infections. PID diseases include Severe Combined Immune Deficiency (SCID), leaky SCID, Omenn syndrome (OS), and Reticular Dysgenesis (RD). PIDs may be treated by transplantation of bone marrow stem cells from a healthy person or, in some cases, by enzyme replacement or by gene therapy. Patients with SCID were among the first to receive bone marrow stem cell (also called hematopoietic cells) transplantation (HCT) more than 40 years ago, and HCT is the standard treatment today for this group of diseases. Since PID diseases are rare, there are not enough patients at any single center to determine the full range of causes, natural history, or best methods of treatment. For this research study many PID centers across North America have organized into the Primary Immune Deficiency Treatment Consortium (PIDTC) to pool their experience and study PIDs together. Researchers will collect information on your general health, psychological and developmental health, and the current status of your immune system to help better define future approaches to PID treatments.

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  • A Multicenter, Open-label Study of CMX001 Treatment of Serious Diseases or Conditions Caused by dsDNA Viruses Not Recruiting

    CMX001 is an orally administered lipid conjugate of the synthetic nucleotide analog cidofovir (CDV). The conjugate is believed to be absorbed in the small intestine then delivered to target organs throughout the body where it crosses cell membranes by facilitated and passive diffusion. Inside the cell, CMX001 is cleaved by intracellular phospholipases to release CDV which is converted to the active antiviral agent, CDV-diphosphate (CDV-PP), by intracellular anabolic kinases. Adults and adolescents, regardless of viral infection/disease, will have a maximum weekly dose of 200 mg i.e., 200 mg once weekly OR 100 mg twice weekly; not to exceed 4mg/kg total weekly dose. Pediatric subjects (< 12 years), regardless of viral infection/disease, will have a maximum weekly dose of 4 mg/kg i.e., 4 mg/kg once weekly OR 2 mg/kg twice weekly.

    Stanford is currently not accepting patients for this trial. For more information, please contact Julia Buckingham, (650) 736 - 1556.

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  • Patients Treated for Wiskott-Aldrich Syndrome (WAS) Since 1990 Not Recruiting

    Wiskott - Aldrich syndrome (WAS) is a rare serious medical condition that causes problems both with the immune system and with easy bruising and bleeding. The immune abnormalities cause patients with WAS to be very susceptible to infections. Depending on the specific type of primary immune deficiency diseases, there are effective treatments, including antibiotics, cellular therapy and gene therapy, but studies of large numbers of patients are needed to determine the full range of causes, natural history, or the best methods of treatment for long term success. This multicenter study combines retrospective, prospective and cross-sectional analyses of the transplant experiences for patients with WAS who have already received HCT since 1990, or who will undergo Hematopoietic cell transplant (HCT) during the study period. The retrospective and prospective portions of the study will address the impact of a number of pre and post-transplant factors on post-transplant disease correction and ultimate benefit from HCT and the cross-sectional portion of the study will assess the benefit of HCT 2 years post-HCT in consenting surviving patients.

    Stanford is currently not accepting patients for this trial. For more information, please contact Matthew Porteus, MD, 650-725-6520.

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2017-18 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circulation research Karakikes, I., Termglinchan, V., Cepeda, D. A., Lee, J., Diecke, S., Hendel, A., Itzhaki, I., Ameen, M., Shrestha, R., Wu, H., Ma, N., Shao, N., Seeger, T., Woo, N. A., Wilson, K. D., Matsa, E., Porteus, M. H., Sebastiano, V., Wu, J. C. 2017


    Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

    View details for DOI 10.1161/CIRCRESAHA.116.309948

    View details for PubMedID 28246128

    View details for PubMedCentralID PMC5429194

  • Dual-Method Clone Tracking in Nonhuman Primates Confirms Long-Term Hematopoietic Reconstitution Initiated By Early Engrafting Clones 58th Annual Meeting and Exposition of the American-Society-of-Hematology Adair, J. E., Norgaard, Z. K., Haworth, K. G., Schefter, L. E., Tam, K., Porteus, M., Kiem, H. AMER SOC HEMATOLOGY. 2016
  • 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


    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

  • Invasive Fungal Disease in Pediatric Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY Aftandilian, C., Weinberg, K., Willert, J., Kharbanda, S., Porteus, M., Maldonado, Y., Agarwal, R. 2016; 38 (7): 574-580
  • Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion. EMBO journal Malmersjö, S., Di Palma, S., Diao, J., Lai, Y., Pfuetzner, R. A., Wang, A. L., McMahon, M. A., Hayer, A., Porteus, M., Bodenmiller, B., Brunger, A. T., Meyer, T. 2016; 35 (16): 1810-1821


    Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

    View details for DOI 10.15252/embj.201694071

    View details for PubMedID 27402227

  • Ethical and regulatory aspects of genome editing BLOOD Kohn, D. B., Porteus, M. H., Scharenberg, A. M. 2016; 127 (21): 2553-2560


    Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic.

    View details for DOI 10.1182/blood-2016-01-678136

    View details for Web of Science ID 000378334400009

    View details for PubMedID 27053531

  • Activation of proto-oncogenes by disruption of chromosome neighborhoods SCIENCE Hnisz, D., Weintraub, A. S., Day, D. S., Valton, A., Bak, R. O., Li, C. H., Goldmann, J., Lajoie, B. R., Fan, Z. P., Sigova, A. A., Reddy, J., Borges-Rivera, D., Lee, T. I., Jaenisch, R., Porteus, M. H., Dekker, J., Young, R. A. 2016; 351 (6280): 1454-1458


    Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

    View details for DOI 10.1126/science.aad9024

    View details for Web of Science ID 000372756200050

  • Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis. Nature communications Gomez-Ospina, N., Potter, C. J., Xiao, R., Manickam, K., Kim, M., Kim, K. H., Shneider, B. L., Picarsic, J. L., Jacobson, T. A., Zhang, J., He, W., Liu, P., Knisely, A. S., Finegold, M. J., Muzny, D. M., Boerwinkle, E., Lupski, J. R., Plon, S. E., Gibbs, R. A., Eng, C. M., Yang, Y., Washington, G. C., Porteus, M. H., Berquist, W. E., Kambham, N., Singh, R. J., Xia, F., Enns, G. M., Moore, D. D. 2016; 7: 10713-?


    Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.

    View details for DOI 10.1038/ncomms10713

    View details for PubMedID 26888176

  • Genome Editing: A New Approach to Human Therapeutics ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 56 Porteus, M. 2016; 56: 163-190
  • TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs. Molecular therapy. Nucleic acids Suzuki, S., Sargent, R. G., Illek, B., Fischer, H., Esmaeili-Shandiz, A., Yezzi, M. J., Lee, A., Yang, Y., Kim, S., Renz, P., Qi, Z., Yu, J., Muench, M. O., Beyer, A. I., Guimarães, A. O., Ye, L., Chang, J., Fine, E. J., Cradick, T. J., Bao, G., Rahdar, M., Porteus, M. H., Shuto, T., Kai, H., Kan, Y. W., Gruenert, D. C. 2016; 5


    Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

    View details for DOI 10.1038/mtna.2015.43

    View details for PubMedID 26730810

  • Towards a new era in medicine: therapeutic genome editing GENOME BIOLOGY Porteus, M. H. 2015; 16

    View details for DOI 10.1186/s13059-015-0859-y

    View details for Web of Science ID 000366898900002

    View details for PubMedID 26694713

  • Stem Cell-Specific Mechanisms Ensure Genomic Fidelity within HSCs and upon Aging of HSCs CELL REPORTS Moehrle, B. M., Nattamai, K., Brown, A., Florian, M. C., Ryan, M., Vogel, M., Bliederhaeuser, C., Soller, K., Prows, D. R., Abdollahi, A., Schleimer, D., Walter, D., Milsom, M. D., Stambrook, P., Porteus, M., Geiger, H. 2015; 13 (11): 2412-2424

    View details for DOI 10.1016/j.celrep.2015.11.030

    View details for Web of Science ID 000367101400012

    View details for PubMedID 26686632

  • MLL leukemia induction by genome editing of human CD34+ hematopoietic cells. Blood Buechele, C., Breese, E. H., Schneidawind, D., Lin, C., Jeong, J., Duque-Afonso, J., Wong, S. H., Smith, K. S., Negrin, R. S., Porteus, M., Cleary, M. L. 2015; 126 (14): 1683-1694


    Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.

    View details for DOI 10.1182/blood-2015-05-646398

    View details for PubMedID 26311362

    View details for PubMedCentralID PMC4591792

  • Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature biotechnology Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., Porteus, M. H. 2015; 33 (9): 985-989


    CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

    View details for DOI 10.1038/nbt.3290

    View details for PubMedID 26121415

  • Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature biotechnology Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., Porteus, M. H. 2015; 33 (9): 985-989

    View details for DOI 10.1038/nbt.3290

    View details for PubMedID 26121415

  • A Pediatric Case of T-Cell Prolymphocytic Leukemia PEDIATRIC BLOOD & CANCER Mitton, B., Coutre, S., Willert, J., Schlis, K., Porteus, M., Kharbanda, S., Agarwal-Hashmi, R. 2015; 62 (6): 1061-1062


    T-cell Prolymphocytic Leukemia (T-PLL) is a rare entity, and to date has never been reported in children. Here, we describe the first pediatric case of T-PLL in a 16-year old male and review his clinical course through treatment. He underwent therapy with alemtuzumab and pentostatin, which was successful in inducing initial remission. He then underwent an allogeneic matched sibling stem cell transplant following a myeloablative conditioning regimen and remains disease-free 1.5 years after diagnosis. Pediatr Blood Cancer © 2014 Wiley Periodicals, Inc.

    View details for DOI 10.1002/pbc.25336

    View details for Web of Science ID 000353231500025

    View details for PubMedID 25417638

  • Genome editing of the germline: broadening the discussion. Molecular therapy Porteus, M. H., Dann, C. T. 2015; 23 (6): 980-982


    Genome editing that results in humans with precisely modified germ cells may never become practical. Nonetheless, the implications are great enough that we strongly support the idea of starting the conversation now, providing time for a broad consensus to be developed. We are confident that if diverse voices are heard, a consensus can be reached on a strategy in which societal mores are respected, the desires of parents are integrated, and the health of future generations is maximized.

    View details for DOI 10.1038/mt.2015.83

    View details for PubMedID 26022625

  • Genome editing technologies: defining a path to clinic. Molecular therapy Corrigan-Curay, J., O'Reilly, M., Kohn, D. B., Cannon, P. M., Bao, G., Bushman, F. D., Carroll, D., Cathomen, T., Joung, J. K., Roth, D., Sadelain, M., Scharenberg, A. M., von Kalle, C., Zhang, F., Jambou, R., Rosenthal, E., Hassani, M., Singh, A., Porteus, M. H. 2015; 23 (5): 796-806

    View details for DOI 10.1038/mt.2015.54

    View details for PubMedID 25943494

  • Genome Editing Technologies: Defining a Path to Clinic: Genomic Editing: Establishing Preclinical Toxicology Standards, Bethesda, Maryland 10 June 2014. Molecular therapy : the journal of the American Society of Gene Therapy Corrigan-Curay, J., O'Reilly, M., Kohn, D. B., Cannon, P. M., Bao, G., Bushman, F. D., Carroll, D., Cathomen, T., Joung, J. K., Roth, D., Sadelain, M., Scharenberg, A. M., von Kalle, C., Zhang, F., Jambou, R., Rosenthal, E., Hassani, M., Singh, A., Porteus, M. H. 2015; 23 (5): 796-806

    View details for DOI 10.1038/mt.2015.54

    View details for PubMedID 28142000

  • Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin NATURE COMMUNICATIONS Wienert, B., Funnell, A. P., Norton, L. J., Pearson, R. C., Wilkinson-White, L. E., Lester, K., Vadolas, J., Porteus, M. H., Matthews, J. M., Quinlan, K. G., Crossley, M. 2015; 6

    View details for DOI 10.1038/ncomms8085

    View details for Web of Science ID 000355531400011

    View details for PubMedID 25971621

  • Improved outcomes after autologous bone marrow transplantation for children with relapsed or refractory hodgkin lymphoma: twenty years experience at a single institution. Biology of blood and marrow transplantation Garfin, P. M., Link, M. P., Donaldson, S. S., Advani, R. H., Luna-Fineman, S., Kharbanda, S., Porteus, M., Weinberg, K. I., Agarwal-Hashmi, R. 2015; 21 (2): 326-334


    The purpose of this study is to evaluate the survival of pediatric patients undergoing autologous bone marrow transplantation (auBMT) for relapsed or refractory Hodgkin lymphoma (rrHL) and to identify factors that might contribute to their outcome. We reviewed the records and clinical course of 89 consecutive rrHL patients ≤ 21 years old who underwent auBMT at Stanford Hospitals and Clinics and the Lucile Packard Children's Hospital, Stanford between 1989 and 2012. We investigated, by multiple analyses, patient, disease, and treatment characteristics associated with outcome. Endpoints were 5-year overall and event-free survival. Our findings include that cyclophosphamide, carmustine, and etoposide (CBV) as a conditioning regimen for auBMT is effective for most patients ≤ 21 years old with rrHL (5-year overall survival, 71%). Transplantation after the year 2001 was associated with significantly improved overall survival compared with our earlier experience (80% compared with 65%). Patients with multiply relapsed disease or with disease not responsive to initial therapy fared less well compared with those with response to initial therapy or after first relapse. Administration of post-auBMT consolidative radiotherapy (cRT) also appears to contribute to improved survival. We are able to conclude that high-dose chemotherapy with CBV followed by auBMT is effective for the treatment of rrHL in children and adolescents. Survival for patients who undergo auBMT for rrHL has improved significantly. This improvement may be because of patient selection and improvements in utilization of radiotherapy rather than improvements in chemotherapy. Further investigation is needed to describe the role of auBMT across the entire spectrum of patients with rrHL and to identify the most appropriate preparative regimen with or without cRT therapy in the treatment of rrHL in young patients.

    View details for DOI 10.1016/j.bbmt.2014.10.020

    View details for PubMedID 25445024

  • Quantifying on- and off-target genome editing. Trends in biotechnology Hendel, A., Fine, E. J., Bao, G., Porteus, M. H. 2015; 33 (2): 132-140


    Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. While the ability to make precise and controlled changes at specified sites throughout the genome has grown tremendously in recent years, we still lack a comprehensive and standardized battery of assays for measuring the different genome editing outcomes created at endogenous genomic loci. Here we review the existing assays for quantifying on- and off-target genome editing and describe their utility in advancing the technology. We also highlight unmet assay needs for quantifying on- and off-target genome editing outcomes and discuss their importance for the genome editing field.

    View details for DOI 10.1016/j.tibtech.2014.12.001

    View details for PubMedID 25595557

  • Quantifying on- and off-target genome editing TRENDS IN BIOTECHNOLOGY Hendel, A., Fine, E. J., Bao, G., Porteus, M. H. 2015; 33 (2): 132-140
  • Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature BARZEL, A., Paulk, N. K., Shi, Y., Huang, Y., Chu, K., Zhang, F., Valdmanis, P. N., Spector, L. P., Porteus, M. H., Gaensler, K. M., Kay, M. A. 2015; 517 (7534): 360-364


    Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

    View details for DOI 10.1038/nature13864

    View details for PubMedID 25363772

    View details for PubMedCentralID PMC4297598

  • Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature BARZEL, A., Paulk, N. K., Shi, Y., Huang, Y., Chu, K., Zhang, F., Valdmanis, P. N., Spector, L. P., Porteus, M. H., Gaensler, K. M., Kay, M. A. 2015; 517 (7534): 360-364

    View details for DOI 10.1038/nature13864

    View details for PubMedID 25363772

  • Strategies to increase genome editing frequencies and to facilitate the identification of edited cells. Methods in molecular biology (Clifton, N.J.) Porteus, M. 2015; 1239: 281-289


    The power of genome editing is increasingly recognized as it has become more accessible to a wide range of scientists and a wider range of uses has been reported. Nonetheless, an important practical aspect of the strategy is develop methods to increase the frequency of genome editing or methods that enrich for genome-edited cells such that they can be more easily identified. This chapter discusses several different approaches including the use of cold-shock, exonucleases, surrogate markers, specialized donor vectors, and oligonucleotides to enhance the frequency of genome editing or to facilitate the identification of genome-edited cells.

    View details for DOI 10.1007/978-1-4939-1862-1_16

    View details for PubMedID 25408413

  • Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells. PloS one Breese, E. H., Buechele, C., Dawson, C., Cleary, M. L., Porteus, M. H. 2015; 10 (9)

    View details for DOI 10.1371/journal.pone.0136644

    View details for PubMedID 26351841

  • Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells. PloS one Breese, E. H., Buechele, C., Dawson, C., Cleary, M. L., Porteus, M. H. 2015; 10 (9)


    One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.

    View details for DOI 10.1371/journal.pone.0136644

    View details for PubMedID 26351841

  • Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin. Nature communications Wienert, B., Funnell, A. P., Norton, L. J., Pearson, R. C., Wilkinson-White, L. E., Lester, K., Vadolas, J., Porteus, M. H., Matthews, J. M., Quinlan, K. G., Crossley, M. 2015; 6: 7085-?


    Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter.

    View details for DOI 10.1038/ncomms8085

    View details for PubMedID 25971621

  • Novel Integrated Autologous Hematopoietic Stem Cell Tracking in Nonhuman Primates Reveals Successive Pattern of Multi-Lineage Reconstitution after Total Body Irradiation 56th Annual Meeting and Exposition of the American-Society-of-Hematology Adair, J. E., Porter, S., Haworth, K., Tam, K., Kiem, H., Porteus, M. H. AMER SOC HEMATOLOGY. 2014
  • Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases PLOS ONE Fanslow, D. A., Wirt, S. E., Barker, J. C., Connelly, J. P., Porteus, M. H., Dann, C. T. 2014; 9 (11)
  • Genome Editing of Mouse Fibroblasts by Homologous Recombination for Sustained Secretion of PDGF-B and Augmentation of Wound Healing. Plastic and reconstructive surgery Barker, J. C., Barker, A. D., Bills, J., Huang, J., Wight-Carter, M., Delgado, I., Noble, D. L., Huang, L. J., Porteus, M. H., Davis, K. E. 2014; 134 (3): 389e-401e


    Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. "Genome editing" is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation.This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression.The application of engineered PDGF-B-expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy.These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

    View details for DOI 10.1097/PRS.0000000000000427

    View details for PubMedID 25158716

  • Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States. JAMA-the journal of the American Medical Association Kwan, A., Abraham, R. S., Currier, R., Brower, A., Andruszewski, K., Abbott, J. K., Baker, M., Ballow, M., Bartoshesky, L. E., Bonilla, F. A., Brokopp, C., Brooks, E., Caggana, M., Celestin, J., Church, J. A., Comeau, A. M., Connelly, J. A., Cowan, M. J., Cunningham-Rundles, C., Dasu, T., Dave, N., De La Morena, M. T., Duffner, U., Fong, C., Forbes, L., Freedenberg, D., Gelfand, E. W., Hale, J. E., Hanson, I. C., Hay, B. N., Hu, D., Infante, A., Johnson, D., Kapoor, N., Kay, D. M., Kohn, D. B., Lee, R., Lehman, H., Lin, Z., Lorey, F., Abdel-Mageed, A., Manning, A., McGhee, S., Moore, T. B., Naides, S. J., Notarangelo, L. D., Orange, J. S., Pai, S., Porteus, M., Rodriguez, R., Romberg, N., Routes, J., Ruehle, M., Rubenstein, A., Saavedra-Matiz, C. A., Scott, G., Scott, P. M., Secord, E., Seroogy, C., Shearer, W. T., Siegel, S., Silvers, S. K., Stiehm, E. R., Sugerman, R. W., Sullivan, J. L., Tanksley, S., Tierce, M. L., Verbsky, J., Vogel, B., Walker, R., Walkovich, K., Walter, J. E., Wasserman, R. L., Watson, M. S., Weinberg, G. A., Weiner, L. B., Wood, H., Yates, A. B., Puck, J. M. 2014; 312 (7): 729-738


    Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births.To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments.Epidemiological and retrospective observational study.Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test.Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked.Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia.Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.

    View details for DOI 10.1001/jama.2014.9132

    View details for PubMedID 25138334

  • Transplantation Outcomes for Severe Combined Immunodeficiency, 2000-2009 NEW ENGLAND JOURNAL OF MEDICINE Pai, S., Logan, B. R., Griffith, L. M., Buckley, R. H., Parrott, R. E., Dvorak, C. C., Kapoor, N., Hanson, I. C., Filipovich, A. H., Jyonouchi, S., Sullivan, K. E., Small, T. N., Burroughs, L., Skoda-Smith, S., Haight, A. E., Grizzle, A., Pulsipher, M. A., Chan, K. W., Fuleihan, R. L., Haddad, E., Loechelt, B., Aquino, V. M., Gillio, A., Davis, J., Knutsen, A., Smith, A. R., Moore, T. B., Schroeder, M. L., Goldman, F. D., Connelly, J. A., Porteus, M. H., Xiang, Q., Shearer, W. T., Fleisher, T. A., Kohn, D. B., Puck, J. M., Notarangelo, L. D., Cowan, M. J., O'Reilly, R. J. 2014; 371 (5): 434-446


    The Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth.We collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009).Survival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell-depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pretransplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival.Transplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

    View details for DOI 10.1056/NEJMoa1401177

    View details for Web of Science ID 000339556900008

    View details for PubMedID 25075835

  • Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing. Cell reports Hendel, A., Kildebeck, E. J., Fine, E. J., Clark, J. T., Punjya, N., Sebastiano, V., Bao, G., Porteus, M. H. 2014; 7 (1): 293-305


    Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.

    View details for DOI 10.1016/j.celrep.2014.02.040

    View details for PubMedID 24685129

  • SAPTA: a new design tool for improving TALE nuclease activity. Nucleic acids research Lin, Y., Fine, E. J., Zheng, Z., Antico, C. J., Voit, R. A., Porteus, M. H., Cradick, T. J., Bao, G. 2014; 42 (6)


    Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at

    View details for DOI 10.1093/nar/gkt1363

    View details for PubMedID 24442582

  • Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic acids research Voit, R. A., Hendel, A., Pruett-Miller, S. M., Porteus, M. H. 2014; 42 (2): 1365-1378


    Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and β-thalassemia are common genetic disorders caused by mutations in β-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human β-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the β-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous β-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous β- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.

    View details for DOI 10.1093/nar/gkt947

    View details for PubMedID 24157834

  • Gene/cell therapy approaches for Immune Dysregulation Polyendocrinopathy Enteropathy X-linked syndrome. Current gene therapy Passerini, L., Santoni de Sio, F. R., Porteus, M. H., Bacchetta, R. 2014; 14 (6): 422-428


    Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmune disease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for the function of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastating autoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if not promptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed for IPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative to HSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LV-mediated FOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specific T cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believe that a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of both strategies will be highlighted here.

    View details for PubMedID 25274247

  • Gene/Cell Therapy Approaches for Immune Dysregulation Polyendocrinopathy Enteropathy X-Linked Syndrome CURRENT GENE THERAPY Passerini, L., de Sio, F. R., Porteus, M. H., Bacchetta, R. 2014; 14 (6): 422-428
  • Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nature communications Tomimatsu, N., Mukherjee, B., Catherine Hardebeck, M., Ilcheva, M., Vanessa Camacho, C., Louise Harris, J., Porteus, M., Llorente, B., Khanna, K. K., Burma, S. 2014; 5: 3561-?

    View details for DOI 10.1038/ncomms4561

    View details for PubMedID 24705021

  • Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo GENOME BIOLOGY Porter, S. N., Baker, L. C., Mittelman, D., Porteus, M. H. 2014; 15 (5)
  • Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nature communications Tomimatsu, N., Mukherjee, B., Catherine Hardebeck, M., Ilcheva, M., Vanessa Camacho, C., Louise Harris, J., Porteus, M., Llorente, B., Khanna, K. K., Burma, S. 2014; 5: 3561-?


    Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.

    View details for DOI 10.1038/ncomms4561

    View details for PubMedID 24705021

  • An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level SCIENCE Bauer, D. E., Kamran, S. C., Lessard, S., Xu, J., Fujiwara, Y., Lin, C., Shao, Z., Canver, M. C., Smith, E. C., Pinello, L., Sabo, P. J., Vierstra, J., Voit, R. A., Yuan, G., Porteus, M. H., Stamatoyannopoulos, J. A., Lettre, G., Orkin, S. H. 2013; 342 (6155): 253-257


    Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.

    View details for DOI 10.1126/science.1242088

    View details for Web of Science ID 000325475200047

    View details for PubMedID 24115442

  • Receptor-mediated delivery of engineered nucleases for genome modification NUCLEIC ACIDS RESEARCH Chen, Z., Jaafar, L., Agyekum, D. G., Xiao, H., Wade, M. F., Kumaran, R. I., Spector, D. L., Bao, G., Porteus, M. H., Dynan, W. S., Meiler, S. E. 2013; 41 (19)


    Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.

    View details for DOI 10.1093/nar/gkt710

    View details for Web of Science ID 000326044700005

    View details for PubMedID 23956220

  • Newborn screening for severe combined immunodeficiency and T-cell lymphopenia in California: Results of the first 2 years JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Kwan, A., Church, J. A., Cowan, M. J., Agarwal, R., Kapoor, N., Kohn, D. B., Lewis, D. B., McGhee, S. A., Moore, T. B., Stiehm, E. R., Porteus, M., Aznar, C. P., Currier, R., Lorey, F., Puck, J. M. 2013; 132 (1): 140-U245


    Assay of T-cell receptor excision circles (TRECs) in dried blood spots obtained at birth permits population-based newborn screening (NBS) for severe combined immunodeficiency (SCID).We sought to report the first 2 years of TREC NBS in California.Since August 2010, California has conducted SCID NBS. A high-throughput TREC quantitative PCR assay with DNA isolated from routine dried blood spots was developed. Samples with initial low TREC numbers had repeat DNA isolation with quantitative PCR for TRECs and a genomic control, and immunophenotyping was performed within the screening program for infants with incomplete or abnormal results. Outcomes were tracked.Of 993,724 infants screened, 50 (1/19,900 [0.005%]) had significant T-cell lymphopenia. Fifteen (1/66,250) required hematopoietic cell or thymus transplantation or gene therapy; these infants had typical SCID (n = 11), leaky SCID or Omenn syndrome (n = 3), or complete DiGeorge syndrome (n = 1). Survival to date in this group is 93%. Other T-cell lymphopenic infants had variant SCID or combined immunodeficiency (n = 6), genetic syndromes associated with T-cell impairment (n = 12), secondary T-cell lymphopenia (n = 9), or preterm birth (n = 8). All T-cell lymphopenic infants avoided live vaccines and received appropriate interventions to prevent infections. TREC test specificity was excellent: only 0.08% of infants required a second test, and 0.016% required lymphocyte phenotyping by using flow cytometry.TREC NBS in California has achieved early diagnosis of SCID and other conditions with T-cell lymphopenia, facilitating management and optimizing outcomes. Furthermore, NBS has revealed the incidence, causes, and follow-up of T-cell lymphopenia in a large diverse population.

    View details for DOI 10.1016/j.jaci.2013.04.024

    View details for Web of Science ID 000321052300019

    View details for PubMedID 23810098

  • Generation of an HIV Resistant T-cell Line by Targeted "Stacking" of Restriction Factors MOLECULAR THERAPY Voit, R. A., McMahon, M. A., Sawyer, S. L., Porteus, M. H. 2013; 21 (4): 786-795


    Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

    View details for DOI 10.1038/mt.2012.284

    View details for Web of Science ID 000317110300010

    View details for PubMedID 23358186

    View details for PubMedCentralID PMC3616536

  • Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification MOLECULAR THERAPY-NUCLEIC ACIDS Wilson, K. A., McEwen, A. E., Pruett-Miller, S. M., Zhang, J., Kildebeck, E. J., Porteus, M. H. 2013; 2


    Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013.

    View details for DOI 10.1038/mtna.2013.13

    View details for Web of Science ID 000332461900006

    View details for PubMedID 23632390

  • Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus MOLECULAR THERAPY-NUCLEIC ACIDS Wilson, K. A., Chateau, M. L., Porteus, M. H. 2013; 2


    The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.

    View details for DOI 10.1038/mtna.2013.12

    View details for Web of Science ID 000332461900005

    View details for PubMedID 23612114

  • 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

  • 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

  • A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype VIROLOGY JOURNAL Ellis, B. L., Hirsch, M. L., Barker, J. C., Connelly, J. P., Steininger, R. J., Porteus, M. H. 2013; 10


    The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro.Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + .We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction.In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

    View details for DOI 10.1186/1743-422X-10-74

    View details for Web of Science ID 000316756700001

    View details for PubMedID 23497173

  • Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs GENE THERAPY Ellis, B. L., HIRSCH, M. L., Porter, S. N., Samulski, R. J., Porteus, M. H. 2013; 20 (1): 35-42


    An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

    View details for DOI 10.1038/gt.2011.211

    View details for Web of Science ID 000313053900005

    View details for PubMedID 22257934

  • Gene therapy for primary immunodeficiencies CURRENT OPINION IN PEDIATRICS Kildebeck, E., Checketts, J., Porteus, M. 2012; 24 (6): 731-738


    Primary immunodeficiencies (PIDs) are an often-devastating class of genetic disorders that can be effectively treated by hematopoietic stem cell transplantation, but the lack of a suitable donor precludes this option for many patients. Gene therapy overcomes this obstacle by restoring gene expression in autologous hematopoietic stem cells and has proven effective in clinical trials, but widespread use of this approach has been impeded by the occurrence of serious complications. In this review, we discuss recent advances in gene therapy with an emphasis on strategies to improve safety, including the emergence of gene targeting technologies for the treatment of PIDs.New viral vectors, including lentiviral vectors with self-inactivating long terminal repeats, have been shown to have improved safety profiles in preclinical studies, and clinical trials using these vectors are now underway. Preclinical studies using engineered nucleases to stimulate precise gene targeting have also demonstrated correction of disease phenotypes for X-linked severe combined immunodeficiency, chronic granulomatous disease, and other diseases.Advances in viral vector design and the development of new technologies that allow precise alteration of the genome have the potential to begin a new chapter for gene therapy where effective treatment of PIDs is achieved without serious risk for patients.

    View details for DOI 10.1097/MOP.0b013e328359e480

    View details for Web of Science ID 000311106800012

    View details for PubMedID 23073463

  • Engineering the immune system to cure genetic diseases, HIV, and cancer Editorial overview CURRENT OPINION IN IMMUNOLOGY Porteus, M. H., Fischer, A. 2012; 24 (5): 576-579
  • Development of nuclease-mediated site-specific genome modification. Current opinion in immunology Wirt, S. E., Porteus, M. H. 2012; 24 (5): 609-616


    Genome engineering is an emerging strategy to treat monogenic diseases that relies on the use of engineered nucleases to correct mutations at the nucleotide level. Zinc finger nucleases can be designed to stimulate homologous recombination-mediated gene targeting at a variety of loci, including genes known to cause the primary immunodeficiencies (PIDs). Recently, these nucleases have been used to correct disease-causing mutations in human cells, as well as to create new animal models for human disease. Although a number of hurdles remain before they can be used clinically, engineered nucleases hold increasing promise as a therapeutic tool, particularly for the PIDs.

    View details for DOI 10.1016/j.coi.2012.08.005

    View details for PubMedID 22981684

  • Gene editing: not just for translation anymore. Nature methods McMahon, M. A., Rahdar, M., Porteus, M. 2012; 9 (1): 28-31

    View details for DOI 10.1038/nmeth.1811

    View details for PubMedID 22205513

  • Gene editing: not just for translation anymore NATURE METHODS McMahon, M. A., Rahdar, M., Porteus, M. 2012; 9 (1): 28-31

    View details for DOI 10.1038/nmeth.1811

    View details for Web of Science ID 000298667000014

  • Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells PLOS ONE Hirsch, M. L., Fagan, B. M., Dumitru, R., Bower, J. J., Yadav, S., Porteus, M. H., Pevny, L. H., Samulski, R. J. 2011; 6 (11)


    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

    View details for DOI 10.1371/journal.pone.0027520

    View details for Web of Science ID 000297555800024

    View details for PubMedID 22114676

    View details for PubMedCentralID PMC3219675

  • Zinc fingers hit off target. Nature medicine Cheng, L., Blazar, B., High, K., Porteus, M. 2011; 17 (10): 1192-1193

    View details for DOI 10.1038/nm1011-1192

    View details for PubMedID 21988995

  • Seeing the light: integrating genome engineering with double-strand break repair NATURE METHODS Porteus, M. 2011; 8 (8): 628-630

    View details for DOI 10.1038/nmeth.1656

    View details for Web of Science ID 000293220600013

    View details for PubMedID 21799496

  • Translating the Lessons From Gene Therapy to the Development of Regenerative Medicine MOLECULAR THERAPY Porteus, M. 2011; 19 (3): 439-441

    View details for DOI 10.1038/mt.2011.14

    View details for Web of Science ID 000287911600002

    View details for PubMedID 21358706

  • Homologous recombination-based gene therapy for the primary immunodeficiencies YEAR IN HUMAN AND MEDICAL GENETICS: INBORN ERRORS OF IMMUNITY II Porteus, M. 2011; 1246: 131-140


    The devastating nature of primary immunodeficiencies, the ability to cure primary immunodeficiencies by bone marrow transplantation, the ability of a small number of gene-corrected cells to reconstitute the immune system, and the overall suboptimal results of bone marrow transplantation for most patients with primary immunodeficiencies make the development of gene therapy for this class of diseases important. While there has been clear clinical benefit for a number of patients from viral-based gene therapy strategies, there have also been a significant number of serious adverse events, including the development of leukemia, from the approach. In this review, I discuss the development of nuclease-stimulated, homologous recombination-based approaches as a novel gene therapy strategy for the primary immunodeficiencies.

    View details for DOI 10.1111/j.1749-6632.2011.06314.x

    View details for Web of Science ID 000301519900013

    View details for PubMedID 22236437

  • Creating Higher Titer Lentivirus with Caffeine HUMAN GENE THERAPY Ellis, B. L., Potts, P. R., Porteus, M. H. 2011; 22 (1): 93-100


    The use of lentiviral vectors extends from the laboratory, where they are used for basic studies in virology and as gene transfer vectors gene delivery, to the clinic, where clinical trials using these vectors for gene therapy are currently underway. Lentiviral vectors are useful for gene transfer because they have a large cloning capacity and a broad tropism. Although procedures for lentiviral vector production have been standardized, simple methods to create higher titer virus during production would have extensive and important applications for both research and clinical use. Here we present a simple and inexpensive method to increase the titer by 3- to 8-fold for both integration-competent lentivirus and integration-deficient lentivirus. This is achieved during standard lentiviral production by the addition of caffeine to a final concentration of 2-4 mM. We find that sodium butyrate, a histone deacetylase inhibitor shown previously to increase viral titer, works only ∼50% as well as caffeine. We also show that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) inhibitor NU7026 can also increase viral titer, but that the combination of caffeine and NU7026 is not more effective than caffeine alone. We show that the time course of caffeine treatment is important in achieving a higher titer virus, and is most effective when caffeine is present from 17 to 41 hr posttransfection. Last, although caffeine increases lentiviral vector titer, it has the opposite effect on the titer of adeno-associated virus type 2 vector. Together, these results provide a novel, simple, and inexpensive way to significantly increase the titer of lentiviral vectors.

    View details for DOI 10.1089/hum.2010.068

    View details for Web of Science ID 000286453600012

    View details for PubMedID 20626321

  • Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair GENE THERAPY HIRSCH, M. L., Green, L., Porteus, M. H., Samulski, R. J. 2010; 17 (9): 1175-1180


    Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders.

    View details for DOI 10.1038/gt.2010.65

    View details for Web of Science ID 000281757900012

    View details for PubMedID 20463753

  • Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease MOLECULAR THERAPY Connelly, J. P., Barker, J. C., Pruett-Miller, S., Porteus, M. H. 2010; 18 (6): 1103-1110


    Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

    View details for DOI 10.1038/mt.2010.57

    View details for Web of Science ID 000278545800008

    View details for PubMedID 20389291

  • Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells CELL STEM CELL Zou, J., Maeder, M. L., Mali, P., Pruett-Miller, S. M., Thibodeau-Beganny, S., Chou, B., Chen, G., Ye, Z., Park, I., Daley, G. Q., Porteus, M. H., Joung, J. K., Cheng, L. 2009; 5 (1): 97-110


    We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-mediated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a > 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expression. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs.

    View details for DOI 10.1016/j.stem.2009.05.023

    View details for Web of Science ID 000267879200014

    View details for PubMedID 19540188

  • Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels PLOS GENETICS Pruett-Miller, S. M., Reading, D. W., Porter, S. N., Porteus, M. H. 2009; 5 (2)


    Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.

    View details for DOI 10.1371/journal.pgen.1000376

    View details for Web of Science ID 000266320000016

    View details for PubMedID 19214211

  • Rapid "Open-Source" engineering of customized zinc-finger nucleases for highly efficient gene modification MOLECULAR CELL Maeder, M. L., Thibodeau-Beganny, S., Osiak, A., Wright, D. A., Anthony, R. M., Eichtinger, M., Jiang, T., Foley, J. E., Winfrey, R. J., Townsend, J. A., Unger-Wallace, E., Sander, J. D., Mueller-Lerch, F., Fu, F., Pearlberg, J., Goebel, C., Dassie, J. P., Pruett-Miller, S. M., Porteus, M. H., Sgroi, D. C., Iafrate, A. J., Dobbs, D., McCray, P. B., Cathomen, T., Voytas, D. F., Joung, J. K. 2008; 31 (2): 294-301


    Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

    View details for DOI 10.1016/j.molcel.2008.06.016

    View details for Web of Science ID 000258083400014

    View details for PubMedID 18657511

  • Comparison of zinc finger nucleases for use in gene targeting in mammalian cells MOLECULAR THERAPY Pruett-Miller, S. M., Connelly, J. P., Maeder, M. L., Joung, J. K., Porteus, M. H. 2008; 16 (4): 707-717


    Homologous recombination is a technique used for performing precise genomic manipulations, and this makes it potentially ideal for gene therapy. The rate of spontaneous homologous recombination in human cells has been too low to be used experimentally or therapeutically but, by inducing a DNA double-strand break (DSB) in the target gene this rate can be stimulated. Zinc finger nucleases (ZFNs) are synthetic fusion proteins that can induce DSBs at specific sequences of DNA and stimulate gene targeting. Although the success of ZFNs in this application has been demonstrated, several issues remain. First, an optimal, generalized method of making effective and safe ZFNs needs to be determined. Second, a systematic method of evaluating the efficiency and safety of ZFNs is needed. We compared the gene-targeting efficiencies and cytotoxicity of ZFNs made using modular-assembly and ZFNs made using a bacterial 2-hybrid (B2H) selection-based method, in each case targeting the same single site. We found that a ZFN pair made using the B2H strategy is more efficient at stimulating gene targeting and less toxic than a pair made using modular-assembly. We demonstrate that a pair of three-finger B2H ZFNs is as efficient at stimulating gene targeting as ZFNs with more fingers, and induce similar or lower rates of toxicity.

    View details for DOI 10.1038/mt.2008.20

    View details for Web of Science ID 000254929600014

    View details for PubMedID 18334988

  • Spermatogonial Stem Cell Self-Renewal Requires OCT4, a Factor Downregulated During Retinoic Acid-Induced Differentiation STEM CELLS Dann, C. T., Alvarado, A. L., Molyneux, L. A., Denard, B. S., Garbers, D. L., Porteus, M. H. 2008; 26 (11): 2928-2937


    The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture and for colonization activity following cell transplantation, using lentiviral-mediated short hairpin RNA expression to knock down OCT4 in an in vitro model for SSCs ("germline stem" [GS] cells). Expression of promyelocytic leukemia zinc-finger (PLZF), a factor known to be required for SSC self-renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, premeiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state and RA triggers spermatogonial differentiation through the direct or indirect downregulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells. Disclosure of potential conflicts of interest is found at the end of this article.

    View details for DOI 10.1634/stemcells.2008-0134

    View details for Web of Science ID 000261156500023

    View details for PubMedID 18719224

  • A look to future directions in gene therapy research for monogenic diseases PLOS GENETICS Porteus, M. H., Connelly, J. P., Pruett, S. M. 2006; 2 (9): 1285-1292


    The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise.

    View details for DOI 10.1371/journal.pgen.0020133

    View details for Web of Science ID 000240867700002

    View details for PubMedID 17009872

  • Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks EMBO JOURNAL Potts, P. R., Porteus, M. H., Yu, H. 2006; 25 (14): 3377-3388


    The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex is thought to promote HR by maintaining the close proximity of sister chromatids at DSBs. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this is unclear. Here, we show that RNAi-mediated knockdown of the SMC5/6 complex components in human cells increases the efficiency of gene targeting due to a specific requirement for hSMC5/6 in sister chromatid HR. Knockdown of the hSMC5/6 complex decreases sister chromatid HR, but does not reduce nonhomologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. The hSMC5/6 complex is itself recruited to nuclease-induced DSBs and is required for the recruitment of cohesin to DSBs. Our results establish a mechanism by which the hSMC5/6 complex promotes DNA repair and suggest a novel strategy to improve the efficiency of gene targeting in mammalian somatic cells.

    View details for DOI 10.1038/sj.emboj.7601218

    View details for Web of Science ID 000239625900013

    View details for PubMedID 16810316

  • Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly NATURE PROTOCOLS Wright, D. A., Thibodeau-Beganny, S., Sander, J. D., Winfrey, R. J., Hirsh, A. S., Eichtinger, M., Fu, F., Porteus, M. H., Dobbs, D., Voytas, D. F., Joung, J. K. 2006; 1 (3): 1637-1652


    Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

    View details for DOI 10.1038/nprot.2006.259

    View details for Web of Science ID 000251155400069

    View details for PubMedID 17406455

  • Gene targeting using zinc finger nucleases NATURE BIOTECHNOLOGY Porteus, M. H., Carroll, D. 2005; 23 (8): 967-973


    The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.

    View details for DOI 10.1038/nbt1125

    View details for Web of Science ID 000231019900029

    View details for PubMedID 16082368

  • Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells NUCLEIC ACIDS RESEARCH Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M. H., Chandrasegaran, S. 2005; 33 (18): 5978-5990


    Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

    View details for DOI 10.1093/nar/gki912

    View details for Web of Science ID 000233046100040

    View details for PubMedID 16251401

  • Chimeric nucleases stimulate gene targeting in human cells SCIENCE Porteus, M. H., Baltimore, D. 2003; 300 (5620): 763-763

    View details for Web of Science ID 000182579800039

    View details for PubMedID 12730593

  • Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks MOLECULAR AND CELLULAR BIOLOGY Porteus, M. H., Cathomen, T., Weitzman, M. D., Baltimore, D. 2003; 23 (10): 3558-3565


    Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

    View details for DOI 10.1128/MCB.23.10.3558-3565.2003

    View details for Web of Science ID 000182696100017

    View details for PubMedID 12724414

  • Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus GENOMICS McGuinness, T., Porteus, M. H., Smiga, S., Bulfone, A., Kingsley, C., Qiu, M. S., Liu, J. K., Long, J. E., Xu, D. W., Rubenstein, J. L. 1996; 35 (3): 473-485


    There are at least five murine Dlx genes that are related to the Drosophila Distal-less homeobox gene. The Dlx genes are primarily expressed in the developing forebrain, derivatives of the cranial neural crest and restricted epidermal craniofacial and limb domains. Dlx-2 is required for differentiation of subsets of cranial neural crest and forebrain cells. Previous genomic studies have shown that Dlx-1 and Dlx-2 are linked on mouse chromosome 2, near the HoxD cluster. Here we report a detailed analysis of the nucleotide sequence (approximately 14 kb), organization, and transcription of the murine Dlx-1 and Dlx-2 locus. In addition, we show that Dlx-1 makes multiple sense transcripts and at least one antisense transcript, whereas Dlx-2 makes one major transcript. The sequence of the human Dlx-2 gene is reported and is compared to that of the murine gene. Finally, sequence analysis of the deduced protein sequences reveals several candidate functional domains.

    View details for Web of Science ID A1996VB75800009

    View details for PubMedID 8812481



    CSF lymphocytes from patients with Coccidioides immitis meningitis exhibited a significant antigen-specific response to in vitro stimulation with C. immitis antigens. In some patients, lesser responses to control antigens (Candida and PPD) were also detected. Antigen-specific responses by CSF lymphocytes were seen early in the course of this disease as well as several years after patients had entered remission. When compared to CSF cells, the response of autologous peripheral blood mononuclear cells was similar but of a much smaller magnitude and at times undetectable. Fluorescence activated cell sorting revealed an increased percentage of CD3+ (T-cells), CD4+ (helper/inducer) and CD3+/HLA-DR+ (activated T-cell) cells in the CSF of C. immitis meningitis patients compared to their blood. Most of the antigen-specific proliferative response resided in the CD4+ lymphocyte subset. CSF T-cell proliferation assays may have a role in the diagnosis of C. immitis meningitis.

    View details for Web of Science ID A1995QZ83200008

    View details for PubMedID 7544405

  • The spatial localization of Dlx-2 during tooth development CONNECTIVE TISSUE RESEARCH Thomas, B. L., Porteus, M. H., Rubenstein, J. L., Sharpe, P. T. 1995; 32 (1-4): 27-34


    The spatial distribution of Dlx-2 protein during murine tooth development has been investigated using immunohistochemistry with Dlx-2 antibodies. In common with several other homeobox genes expressed in toothgerms, Dlx-2 shows a multiphasic distribution in both epithelially and mesenchymally derived structures. This localization shows a number of similarities with the expression of Msx-2 and suggests a role for Dlx-2 in tooth initiation and tissue patterning.

    View details for Web of Science ID A1995VT03000005

    View details for PubMedID 7554927



    Recently, the Dlx family of homeobox genes have been identified as candidates for regulating patterning and differentiation of the forebrain. We have made a polyclonal antiserum to the protein product of the Dlx-2 gene. Using this antiserum, we have characterized the spatial and temporal pattern of DLX-2 protein expression during murine development and in the adult mouse brain. These studies demonstrate that, like the mRNA from the Dlx-2 gene, DLX-2 protein is expressed in mouse embryonic forebrain, limbs, tail, genital tubercle, and branchial arches. Within the embryonic forebrain, DLX-2 protein is expressed within specific transverse and longitudinal domains. Analysis of expression within the wall of the forebrain shows that DLX-2 is expressed in proliferative regions including the ventricular and subventricular zones. DLX-2 is expressed in the same cells as MASH-1, a marker of relatively undifferentiated cells, but in a reciprocal fashion to MAP-2, a marker of terminal neuronal differentiation. A number of DLX-2-expressing cells, but not all, can be labeled with bromodeoxyuridine (BrdU). Using the patterns of DLX-2, MASH-1, MAP-2 expression, and bromodeoxyuridine incorporation, we identify four molecularly distinct populations of cells that may correspond to different stages of neuronal differentiation in the mouse basal forebrain, in which DLX-2 is expressed at the transition from proliferation to terminal differentiation.

    View details for Web of Science ID A1994PQ38100006

    View details for PubMedID 7965042



    The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins, and one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telecephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development, and based upon them we suggest a model for forebrain segmentation.

    View details for Web of Science ID A1993LM27300037

    View details for PubMedID 7687285



    The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.

    View details for Web of Science ID A1993KW17900001

    View details for PubMedID 8098616



    Dlx-2 (also called Tes-1), a mammalian member of the Distal-less family of homeobox genes, is expressed during murine fetal development in spatially restricted domains of the forebrain. Searching for a candidate neurological mutation that might involve this gene, we have assigned the human and mouse loci to regions of conserved synteny on human chromosome 2, region cen--q33, and mouse chromosome 2 by Southern analysis of somatic cell hybrid lines. An EcoRI dimorphism, discovered in common inbred laboratory strains, was used for recombinant inbred strain mapping. The results place Dlx-2/Tes-1 near the Hox-4 cluster on mouse chromosome 2.

    View details for Web of Science ID A1992JH14800031

    View details for PubMedID 1354641



    In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

    View details for Web of Science ID A1992GZ10000002

    View details for PubMedID 1372074



    A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.

    View details for Web of Science ID A1991GB93300005

    View details for PubMedID 1678612



    We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

    View details for Web of Science ID A1990DX66200027

    View details for PubMedID 2168539



    To study the movement of subretinal fluid, we have injected fluid into the subretinal space through a glass micropipette and monitored its resorption. This technique has been criticized as a model of non-rhegmatogenous detachment because the small retinal hole made by the micropipette might allow efflux of subretinal fluid into the vitreous. The present experiments answer this criticism: we found that sealing the micropipette hole with cyanoacrylate, mucilage or an air bubble had no effect on the rate of subretinal fluid resorption, and detachments with two to five micropipette holes did not resorb faster than those with only one.

    View details for Web of Science ID A1984SC67800016

    View details for PubMedID 6697753