Ham-Wasserman Lecture

Abstract Known since the beginning of the first millennium, the hemophilias are among the most frequent inherited disorders of blood coagulation and definitely the most severe. In the 1970s, with the availability of concentrated preparations of the deficient coagulation factors VIII and IX and with the large-scale adoption of home treatment, hemophilia care became one of the most gratifying examples of successful secondary prevention of a chronic disease. Unfortunately, in the early 1980s it was recognized that factor concentrates prepared from plasma pooled from thousands of donors transmitted the hepatitis and the human immunodeficiency viruses. The scientific community reacted promptly to the devastation brought about by hepatitis and AIDS. The last 15 years of the second millennium have witnessed the development of methods that, when applied during concentrate manufacturing, inactivate viruses escaping the screening procedures. The adoption of these measures has reduced dramatically the risk of transmission of bloodborne infections. The production of recombinant factors and their availability for patients’ treatment epitomize progress in hemophilia care through DNA technology. Methods based on the polymerase chain reaction (PCR) have unraveled an array of gene lesions associated with hemophilia, permitting improved secondary control of the disease through carrier detection in women from affected families and prenatal termination of their affected male infants. This article will review the aforementioned areas of progress and discuss unresolved problems (such as treatment of patients with antibodies, the risk of new infectious complications, and the issue of secondary tumors). Hopes and expectations for further improvement in the third millennium and particularly the prospects of hemophilia cure though gene replacement therapy will also be mentioned.

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Mass spider silk production through targeted gene replacement in Bombyx mori

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806805115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8757-8762 ◽

Cited By ~ 31

Author(s):

Jun Xu ◽

Qinglin Dong ◽

Ye Yu ◽

Baolong Niu ◽

Dongfeng Ji ◽

...

Keyword(s):

Bombyx Mori ◽

Large Scale ◽

Spider Silk ◽

Natural Fibers ◽

Gene Replacement ◽

Chain Gene ◽

Scale Production ◽

Transcription Activator ◽

Efficient System ◽

Silk Production

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

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Human iPSC modeling reveals mutation-specific responses to gene therapy in Best disease

10.1101/796581 ◽

2019 ◽

Cited By ~ 1

Author(s):

Divya Sinha ◽

Benjamin Steyer ◽

Pawan K. Shahi ◽

Katherine Mueller ◽

Rasa Valiauga ◽

...

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Gene Replacement ◽

Patient Specific ◽

Genomic Alteration ◽

Inherited Disorders ◽

Induced Pluripotent ◽

Best Disease

AbstractDominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients.SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

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Recombinant factor VIIa

Thrombosis and Haemostasis ◽

10.1160/th05-01-0032 ◽

2005 ◽

Vol 93 (06) ◽

pp. 1027-1035 ◽

Cited By ~ 33

Author(s):

Marco Zaffanello ◽

Dino Veneri ◽

Massimo Franchini

Keyword(s):

Recombinant Factor Viia ◽

Factor Viia ◽

Current Knowledge ◽

Coagulation Factors ◽

Factor Vii ◽

Recombinant Activated Factor Vii ◽

Activated Factor Vii ◽

Viii And Ix ◽

Bleeding Episodes ◽

Uncontrolled Bleeding

SummaryRecombinant activated factor VII (rFVIIa, Novo Seven®) has been successfully used to treat bleeding episodes in patients with antibodies against coagulation factors VIII and IX. In recent years, rFVIIa has also been employed for the management of uncontrolled bleeding in a number of congenital and acquired haemos- tatic abnormalities. Based on a literature search, this review examines the current knowledge on therapy with rFVIIa, from the now well-standardized uses to the newer and less well-characterised clinical applications.

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Erratum: BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method

Genes & Development ◽

10.1101/gad.300863.117 ◽

2017 ◽

Vol 31 (8) ◽

pp. 846-846

Author(s):

Chae-Seok Lim ◽

Xi Kang ◽

Vincent Mirabella ◽

Huaye Zhang ◽

Qian Bu ◽

...

Keyword(s):

Mutation Analysis ◽

Large Scale ◽

Gene Replacement ◽

Replacement Method ◽

Human Mutation

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From Lineage to Territory

Indian Historical Review ◽

10.1177/0376983617726460 ◽

2017 ◽

Vol 44 (2) ◽

pp. 173-197

Author(s):

Manu V. Devadevan

Keyword(s):

State Formation ◽

Large Scale ◽

Indian Subcontinent ◽

Indian Region ◽

Political Control ◽

The Political ◽

Complex Process ◽

Systematic Exploration ◽

First Millennium ◽

Scale Expansion

The evolution of territorial self-consciousness was among the most significant and historically far-reaching developments of the later half of the first millennium CE in the Indian subcontinent. However, discussions concerning this complex process have not had the benefit of systematic exploration. Although a handful of perspectives exist, they have been presented impressionistically in the context of debates on feudalism and state formation. In this article, this question is examined in relation to the rise of territoriality in the eastern Indian region of Kaliṅga. On the basis of the evidence occurring in inscriptions from the region, it is argued that large-scale expansion of agriculture and the spread of landed property, the prospects thus generated by the political control that could be exercised over its resources and the consolidation of such prospects at different local, supralocal and regional levels were the causes that resulted in the rise of Kaliṅga as a geopolitically self-conscious territory.

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Infectious and non-infectious complications in primary immunodeficiency disorders: an autopsy study from North India

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204708 ◽

2017 ◽

Vol 71 (5) ◽

pp. 425-435 ◽

Cited By ~ 5

Author(s):

Kirti Gupta ◽

Amit Rawat ◽

Parimal Agrawal ◽

Ankur Jindal ◽

Ritambhra Nada ◽

...

Keyword(s):

Primary Immunodeficiency ◽

Bacterial Infections ◽

Clinical Symptoms ◽

Fungal Infections ◽

Wide Spectrum ◽

Case Reports ◽

Infectious Complications ◽

North India ◽

Inherited Disorders ◽

Primary Immunodeficiency Disorders

BackgroundPrimary immunodeficiency disorders (PID) include a wide spectrum of inherited disorders characterised by functional abnormalities of one or more components of the immune system. Recent updates from the genomic data have contributed significantly to its better understanding with identification of new entities. Diagnosis is always challenging due to their variable clinical presentation. With the evolution of molecular diagnosis, many of these children are being diagnosed early and offered appropriate therapy. However, in developing countries, early diagnosis is still not being made: as a result these patients succumb to their disease. Autopsy data on PID is notably lacking in the literature with histopathological evaluation of PID being limited to rare case reports.ObjectiveTo analyse the clinical, immunologic (including mutational) and morphologic features at autopsy in 10 proven and suspected cases of primary immunodeficiency disorders diagnosed at our Institute over the past decade.MethodsStudy includes a detailed clinico-pathological analysis of 10 proven and suspected cases of primary immunodeficiency disorders.ResultsA varied spectrum of infectious and non-infectious complications were identified in these cases of which fungal infections were found to be more frequent compared with viral or bacterial infections. Rare and novel morphological findings, like granulomatous involvement of the heart in a patient with chronic granulomatous disease, systemic amyloidosis in a teenage girl with X-linked agammaglobulinemia, are highlighted which is distinctly lacking in the literature.ConclusionsThe present study is perhaps the first autopsy series on PID. Even in the molecular era, such analysis is still important, as correlation of pathological features with clinical symptoms provides clues for a timely diagnosis and appropriate therapeutic intervention.

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A Cell-based Model of Hemostasis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615947 ◽

2001 ◽

Vol 85 (06) ◽

pp. 958-965 ◽

Cited By ~ 790

Author(s):

Dougald Monroe ◽

Maureane Hoffman

Keyword(s):

Large Scale ◽

Coagulation Factors ◽

Cellular Receptors ◽

Platelet Surface ◽

Surface Receptors ◽

Coagulation Proteins ◽

A Cell ◽

And Control ◽

Prevailing View

SummaryBased on our work and that of many other workers, we have developed a model of coagulation in vivo. Many workers have demonstrated mechanisms by which cells can influence the coagulation process. Nonetheless, the prevailing view of hemostasis remains that the protein coagulation factors direct and control the process with cells serving primarily to provide a phosphatidylserine containing surface on which the procoagulant complexes are assembled. By contrast, we propose a model in which coagulation is regulated by properties of cell surfaces. This model emphasizes the importance of specific cellular receptors for the coagulation proteins. Thus, cells with similar phosphatidylserine content can play very different roles in hemostasis depending on their complement of surface receptors. We propose that coagulation occurs not as a “cascade”, but in three overlapping stages: 1) initiation, which occurs on a tissue factor bearing cell; 2) amplification, in which platelets and cofactors are activated to set the stage for large scale thrombin generation; and 3) propagation, in which large amounts of thrombin are generated on the platelet surface. This cell based model explains some aspects of hemostasis that a protein-centric model does not.

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A curated transcriptome dataset collection to investigate inborn errors of immunity

10.1101/526004 ◽

2019 ◽

Author(s):

Salim Bougarn ◽

Sabri Boughorbel ◽

Damien Chaussabel ◽

Nico Marr

Keyword(s):

Web Application ◽

Large Scale ◽

Mononuclear Cells ◽

Ex Vivo ◽

Clinical Manifestations ◽

Inherited Disorders ◽

Transcriptional Responses ◽

Control Subjects ◽

A Genome ◽

Wide Range

ABSTRACTPrimary immunodeficiencies (PIDs) are a heterogeneous group of inherited disorders, frequently caused by loss-of-function and less commonly by gain-of-function mutations, which can result in susceptibility to a broad or a very narrow range of infections but also in inflammatory, allergic or malignant diseases. Owing to the wide range in clinical manifestations and variability in penetrance and expressivity, there is an urgent need to better understand the underlying molecular, cellular and immunological phenotypes in PID patients in order to improve clinical diagnosis and management. Here we have compiled a manually curated collection of public transcriptome datasets mainly obtained from human whole blood, peripheral blood mononuclear cells (PBMCs) or fibroblasts of patients with PIDs and of control subjects for subsequent meta-analysis, query and interpretation. A total of nineteen (19) datasets derived from studies of PID patients were identified and retrieved from the NCBI Gene Expression Omnibus (GEO) database and loaded in GXB, a custom web application designed for interactive query and visualization of integrated large-scale data. The dataset collection includes samples from well characterized PID patients that were stimulated ex vivo under a variety of conditions to assess the molecular consequences of the underlying, naturally occurring gene defects on a genome-wide scale. Multiple sample groupings and rank lists were generated to facilitate comparisons of the transcriptional responses between different PID patients and control subjects. The GXB tool enables browsing of a single transcript across studies, thereby providing new perspectives on the role of a given molecule across biological systems and PID patients. This dataset collection is available at: http://pid.gxbsidra.org/dm3/geneBrowser/list.

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THE EFFECT OF THE VENOM OF THE ORIENTAL HORNET ON COAGULATION FACTORS

10.1055/s-0038-1644338 ◽

1987 ◽

Author(s):

A Kornberg ◽

S Kaufman ◽

L Silber ◽

J Ishay

Keyword(s):

Human Plasma ◽

Degradation Products ◽

Anticoagulant Activity ◽

Coagulation Factors ◽

Plasma Fibrinogen ◽

Thrombin Time ◽

Clotting Factors ◽

Viii And Ix

The extract from the venom sac of Vespa orientalis (VSE) inactivates exogenous and endogenous thromboplastin (Joshua and Ishay, Toxicon, 13:11-20,1975). The prolongation of both prothrombin time (PT) and recalcification time suggests inactivation of other factors. The aim of the present study is to investigate the effect of VSE on clotting factors. A lyophilized VSE with protein concentration of 5 mg/ml was used. Studies were performed in vitro with human plasma and in vivo in cats. Routine methods were employed for the assay of PT, activated tissue thromboplastin (APTT), thrombin time (TT), fibrinogen degradation products (FDP), fibrinogen and factors V,VII,VIII,IX,X. Human plasma was incubated with various concentrations of VSE (0,1,5,10,50,100 μg/ml) for 60 min and for various incubation times (0,5,15,30,+ 60,90,120 min) with 50 μg/ml VSE (n=8). 1 μg/ml VSE prolonged PT from 13.5 to 16 sec (p<0.05) and APTT from 62 to 180 sec. PT was maximal (17.7 sec) with 10 μg/ml and APTT (442 sec) with 50 μg/ml VSE. Factors V,VII,X decreased gradually from 94-105% to 11%,11% and 29% with 100 μg/ml VSE and VIII and IX to 1% even with 1 μg/ml VSE. After 5 min with constant concentration of VSE (50 μg/ml) PT was 14.9 sec (normal 13 sec) and APTT 165 sec (normal 54 sec). Both were maximal (17.5 and 298 sec) after 60 min. Factors VII and X decreased to 13% and 32% and VIII and IX to >1% after 60 min of incubation. Injection of 5 mg/kg VSE to cats (n=6-8) resulted in prolongation of PT from 9.4 to 11.2 sec and of APTT from 19.5 to 63 sec after 5 min. Both were maximal after 90 min (12.3 and 127 sec). Factors V,VII and X decreased from 100% to 7.6%, 13% and 37% and VIII and IX to 1% after 10 min. In all experiments TT and plasma fibrinogen were not affected and FDP were normal. Heating of VSE for 5 min at 80°C abolished completely the anticoagulant activity but dialysis for 24 hr at 4°C had no effect on it. The activity was eluted on Sephadex-25 both in void and post void volumes. The results show that VSE has a potent anticoagulant activity against various factors. Factors VIII and IX are markedly decreased. The effect on V, VII and X is moderate. Plasma fibrinogen is not affected. The nature and clinical significance of the anticoagulant activity merit further investigation.

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