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

  • 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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  • The Adv Halt Trial Not Recruiting

    The primary objective of this study is to evaluate the safety and efficacy of preemptive treatment with CMX001 versus placebo for the prevention of AdV disease in recipients of HSCT with asymptomatic AdV viremia.

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

    View full details

2015-16 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • 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 PubMedID 26940867

  • 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

  • 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 Web of Science ID 000365451100009

    View details for PubMedID 26311362

  • 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)
  • 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

  • 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

  • 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 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

  • 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

  • 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 Web of Science ID 000348632700018

  • 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

  • Promoter less 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-U476
  • 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)


    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

  • 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 Web of Science ID 000349460300005

  • Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States JAMA-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 Web of Science ID 000340438700021

  • 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 Web of Science ID 000334298200028

    View details for PubMedID 24685129

  • Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice NATURE COMMUNICATIONS Tomimatsu, N., Mukherjee, B., Hardebeck, M. C., Ilcheva, M., Camacho, C. V., Harris, J. L., Porteus, M., Llorente, B., Khanna, K. K., Burma, S. 2014; 5

    View details for DOI 10.1038/ncomms4561

    View details for Web of Science ID 000335220000003

  • 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 Web of Science ID 000334758600010

    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 Web of Science ID 000331138100059

  • 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
  • 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

  • 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

    View details for DOI 10.1016/j.coi.2012.09.004

    View details for Web of Science ID 000319248200011

    View details for PubMedID 23084086

  • 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

  • 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

  • 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



    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



    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



    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