ULTRAHIGH THROUGHPUT CELLULAR MANIPULATION VIA MASSIVELY PARALLEL MICROINJECTION
Bradley Christopher
University Of California Riversidecity: Riverside country: United States (us)
Grant 5R21RR026253-03 from National Center For Research Resources
Keywords: Address; Area; Automation; base; Biological; biological systems; Biomedical Research; Cell Therapy; cell type; Cells; Clinical; clinical application; Clinical Research; Communities; Computers; Computers and Advanced Instrumentation; cost; design; Development; Device or Instrument Development; Devices; Discipline; Disease; disorder control; Drug Delivery Systems; drug discovery; Elements; Future; gene therapy; Genetic; Genetic Engineering; Goals; Gold; Health; Hematology; Hematopoietic stem cells; Hereditary Disease; Human; improved; Injection of therapeutic agent; innovation; instrument; instrumentation; Investigation; Lead; Libraries; Life; Methodology; Microfabrication; Microinjections; Mission; Modification; novel; oncology; operation; Outcome Study; Performance; Preclinical Drug Evaluation; prevent; Process; Production; prototype; public health relevance; Reliance; Research; Research Personnel; Risk; RNA Interference; Robotics; Safety; Screening procedure; Site; Solid; Speed (motion); Staging; success; System; Techniques; Technology; Testing; Therapeutic; Time; Transfection; Transgenic Animals; Transgenic Organisms; Translations; Validation; Viral Vector
Relevance: The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH´s mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent
Project start date: 2010-01-20
Project end date: 2013-01-19
Budget start date: 20-JAN-2012
Budget end date: 19-JAN-2013
5R21RR026253-03 (2012): $164392
Sponsored Links Excellgen http://Excellgen.com
ULTRAHIGH THROUGHPUT CELLULAR MANIPULATION VIA MASSIVELY PARALLEL MICROINJECTION
Bradley Christopher, Assistant Professor
University Of California Riversidecity: Riverside country: United States (us)
Grant 5R21RR026253-02 from National Center For Research Resources
Abstract: The manipulation of cells via introduction of exogenous materials serves as a critical enabler for a broad spectrum of applications, including drug discovery, transgenics, and cell-based therapeutics. However, in many applications progress is nevertheless constrained by the limitations of current manipulation techniques. The long-term goal of this proposal is to address this issue through development of advanced instrumentation for microinjection-based manipulation. Although microinjection represents the "gold standard" for cellular manipulation, it has been largely relegated to niche applications by its labor-intensiveness and low throughput (~3 cells/min), which result from reliance upon skilled operators and serialized injection methodologies. The objective of this proposal is to develop ultrahigh throughput (UHT) microinjection instrumentation that addresses these limitations through automation, massive parallelization, and monolithic integration. This instrumentation will be based upon a microelectromechanical systems (MEMS) device core, composed of a massively parallel array of cell Capture Sites with monolithically integrated Injectors, which will enable simultaneous capture and injection of many thousands of cells/min with minimal need for human or robotic involvement. Guided by preliminary studies demonstrating feasibility of the first functional element of this instrumentation, namely UHT cell capture, the proposed effort seeks to take the next steps forward through pursuit of a staged research plan that gradually introduces additional functionalities. The Specific Aims are 1) Develop prototype for UHT cell capture and permeabilization; and 2) Develop prototype for UHT microinjection. Novel microfabrication processes will be developed for the MEMS device cores and computer-controlled external subsystems will be developed that add high-speed cell handling functionality. Instrument functionality will then be validated using live cell testing (i.e. capture, injection, release, and transfection efficiency, as well as viability). The proposed research is innovative because it represents the first attempt to address microinjection´s limitations through not only automation, but also massive parallelization and monolithic integration of all functionality into a single MEMS device. This research is significant because it may a) simplify microinjection sufficiently to make it accessible to a broader range of researchers; b) considerably enhance current applications where throughput is often a limiting factor, e.g. transgenics; and c) serve as a fundamental enabler for applications where progress is constrained by safety or efficacy concerns associated with current manipulation techniques, e.g. ex vivo cell therapies based on genetic modification. PUBLIC HEALTH RELEVANCE The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH´s mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent
Keywords: Address; Advanced Instrumentation; Area; Au element; Automation; base; Biological; biological systems; Biomedical Research; Biotechnology, Genetic Engineering; Blood Precursor Cell; Cancer, Oncology; Cell Therapy; cell type; cell-based therapy; Cells; Clinical; clinical applicability; clinical application; Clinical Research; Clinical Study; Communities; Computer Instrumentation; Computers; Computers and Advanced Instrumentation; cost; design; designing; Development; device development; Device or Instrument Development; Devices; Discipline; Disease; disease control; disease/disorder; Disorder; disorder control; Drug Delivery; Drug Delivery Systems; drug discovery; Drug Evaluation, Preclinical; Drug Screening; Drug Targeting; Drug Targetings; Elements; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Future; gene therapy; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genetic; Genetic Condition; Genetic Diseases; genetic disorder; Genetic Engineering; Genetic Intervention; genetic therapy; Goals; Gold; Health; heavy metal lead; heavy metal Pb; Hematology; Hematopoietic stem cells; Hereditary Disease; hereditary disorder; Human; Human, General; improved; Injection of therapeutic agent; Injections; innovate; innovation; innovative; instrument; instrument development; instrumentation; Instrumentation, Other; Intervention, Genetic; Investigation; Investigators; Lead; Libraries; Life; Man (Taxonomy); Man, Modern; Method LOINC Axis 6; Methodology; Methods and Techniques; Methods, Other; Microfabrication; Microinjections; Mission; Modification; Molecular Biology, Gene Therapy; Molecular Biology, Genetic Engineering; Molecular Disease; novel; oncology; operation; Outcome Study; Pb element; Performance; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Preclinical Drug Evaluation; prevent; preventing; Process; Production; Progenitor Cells, Hematopoietic; prototype; public health relevance; Quelling; Recombinant DNA Technology; Reliance; Research; Research Personnel; Researchers; Risk; RNA Interference; RNA Silencing; RNA Silencings; RNAi; Robotics; Safety; screening; Screening procedure; screenings; Sequence-Specific Posttranscriptional Gene Silencing; Site; Solid; Speed; Speed (motion); Staging; success; System; System, LOINC Axis 4; Techniques; Technology; Testing; Therapeutic; Therapy, Cell; Therapy, DNA; Time; Transfection; transgenic; Transgenic Animals; Transgenic Organisms; Translations; Validation; Viral Vector
Relevance: The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH´s mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent
Project start date: 2010-01-20
Project end date: 2013-01-19
Budget start date: 20-JAN-2011
Budget end date: 19-JAN-2012
PFA/PA: RFA-RR-09-001
5R21RR026253-02 (2011): $166053
Grants awarded to Bradley Christopher
HIGH EXPRESSION RECOMBINANT FACTOR VIII
Bradley Christopher, Assistant Professor
Expression Therapeuticscity: Tucker country: United States (us)
Grant 5R42HL090112-03 from National Heart, Lung, And Blood Institute
Abstract: The hemophilias (A and B) are rare bleeding disorders toward which much scientific and medical effort has been devoted. In 1840, blood transfusion was used for the first time to stop post-operative bleeding in a hemophilia patient and in 1968 the first commercial coagulation factor concentrate became available. The cloning of the fVIII gene in 1984 facilitated the development of recombinant fVIII protein products that became commercially available in 1992. This was viewed as a dramatic therapeutic improvement due to the perceived safety advantage recombinant products have over plasma-derived products, which proved responsible for the infection of thousands of patients with hemophilia with human immunodeficiency virus and/or hepatitis C virus during the 1980´s. Since the development of recombinant fVIII, progress in the treatment of hemophilia A has slowed. The current limitations to treatment are 1) access to fVIII-replacement products, 2) the cost of fVIII- replacement products, 3) the development of anti-fVIII immune responses that block treatment efficacy and 4) morbidity due to joint disease. Unless the worldwide fVIII supply increases and prices drop significantly, hemophilia A will remain an important heath burden to human society. Therefore, the search for improved therapeutics is warranted. One strategy for improving hemophilia A care is to develop improved recombinant- protein products, e.g. manufactured more efficiently or have increased hemostatic efficacy. The mission of Expression Therapeutics is to develop products that will improve the treatment of individuals with hemophilia A. Our technology is based on the identification of sequence elements within fVIII that can be modified to increase its´ biosynthesis. The goal of the current study is to provide preclinical data necessary to get FDA approval to conduct a phase 1 human clinical trial using a high-expression fVIII-replacement product that can be manufactured more efficiently than traditional human recombinant fVIII products. These data will support the commercialization of a novel fVIII-replacement product that will improve the treatment of hemophilia A. Hemophilia A is a bleeding disorder caused by the insufficiency of a blood clotting factor, termed factor VIII (fVIII). Current treatment for hemophilia relies on infusion of plasma-derived or recombinant fVIII products to restore circulating fVIII activity. Currently, treatment is offered to less than one-third of all patients due to excessive product cost. Unless the worldwide fVIII supply increases and prices drop significantly, hemophilia A will remain an important heath burden to human society, and therefore, the search for improved therapeutics is warranted
Keywords: A Mouse; Adult; Affect; Anabolism; arthropathies; base; Biological Products; Blood Clot; Blood coagulation; Blood Coagulation Factor; Blood Transfusion; Canis familiaris; Caring; Cell Culture System; Cells; Cessation of life; Chemicals; Child; Clinical; Clinical Research; Clinical Trials; Clip; Cloning; commercialization; cost; cost effective; Data; Development; Disease; domain mapping; Dose; Drops; Drug Kinetics; Elements; Factor VIII; Family suidae; Genes; glycosylation; Goals; Hemophilia A; Hemorrhage; Hemostatic Agents; Hepatitis C virus; HIV; Human; human F8 protein; Hybrids; Immune response; immunogenicity; improved; Individual; Infection; Infusion procedures; Insecta; Intravenous infusion procedures; Lentivirus Vector; Link; Longevity; Mammalian Cell; Medical; Mission; Modeling; Monkeys; Morbidity - disease rate; Mus; nonhuman primate; novel; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Plasma; Plasma Proteins; Population; Post-Translational Protein Processing; Postoperative Period; pre-clinical; Price; Process; Production; Proteins; public health relevance; recombinant antihemophilic factor VIII; Recombinant Proteins; Recombinants; Relative (related person); Replacement Therapy; Safety; Secretory Cell; Societies; standard care; sulfation; System; Tail; Technology; Therapeutic; Time; Toxic effect; Toxicology; Treatment Efficacy; Tyrosine; United States; Yeasts
Relevance: Hemophilia A is a bleeding disorder caused by the insufficiency of a blood clotting factor, termed factor VIII (fVIII). Current treatment for hemophilia relies on infusion of plasma-derived or recombinant fVIII products to restore circulating fVIII activity. Currently, treatment is offered to less than one-third of all patients due to excessive product cost. Unless the worldwide fVIII supply increases and prices drop significantly, hemophilia A will remain an important heath burden to human society, and therefore, the search for improved therapeutics is warranted
Project start date: 2007-07-01
Project end date: 2012-06-30
Budget start date: 1-JUL-2011
Budget end date: 30-JUN-2012
PFA/PA: PA-09-081
5R42HL090112-03 (2011): $885011
HEMATOPOIETIC GENE THERAPY FOR HEMOPHILIA A
Bradley Christopher, Assistant Professor
Emory Universitycity: Atlanta country: United States (us)
Grant 5R01HL092179-03 from National Heart, Lung, And Blood Institute
Abstract: Hemophilia A is a congenital bleeding disorder caused by genetic mutations affecting a plasma protein, termed factor VIII (fVIII), whose function is to facilitate blood clotting. State of the art treatment for hemophilia A consists of frequent intravenous infusions of fVIII containing products. The current limitations to treating hemophilia are 1) the cost of fVIII products, 3) the development of immune responses against fVIII that block treatment efficacy, 3) morbidity due to joint disease resulting from repeated bleeding into individual joints and 4) the limitation of treatment to 30% of the world population. Due to the limited amount of fVIII needed to provide clinical benefit to the patient, hemophilia A is an attractive disease target for gene therapy, and three phase 1 clinical trials have been conducted. The outcome of these trials has been disappointing due to the extremely low, non-therapeutic levels of fVIII produced in each of the gene therapy strategies. We recently showed that a modified porcine fVIII transgene, designated BDDpfVIII, facilitates very high-level protein expression, and we demonstrated proof-of-concept that this transgene functions extremely efficiently in a mouse model of hemophilia A following transplantation of genetically-modified hematopoietic stem cells (HSCs). Specifically, we have shown that the expression of BDDpfVIII is superior to other bioengineered human fVIII expression constructs and that genetic modification and transplantation of HSCs results in curative fVIII levels. Additionally, curative fVIII activity levels are achieved after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pre-transplantation conditioning with targeted immunosuppression, even in the context of pre-existing anti-human fVIII inhibitors. Therefore, we have overcome the major hurdle of low-level expression using a transgene that encodes a protein that has been used successfully in patients with hemophilia A. In the current application, we propose to more fully characterize the use of the high-expression construct and further our understanding of the critical parameters involved with this novel gene therapy strategy and study the biology of non-physiological BDDpfVIII expression in hematopoietic (blood) cells. To advance our studies toward clinical significance, we propose to 1) test clinically relevant HSC transplant conditioning regimens that more closely resemble those used routinely in human bone marrow transplant protocols and 2) test recombinant lentiviral vectors that have been demonstrated to display a reduction of insertional mutagenesis compared to oncoretroviruses. Finally, the optimized lentiviral vector(s) encoding BDDpfVIII will be tested for the ability to genetically modify human HSCs. Hemophilia A is a bleeding disorder caused by insufficiency of a blood clotting factor, designated factor VIII, for which gene therapy offers a potential cure. However, pre-clinical studies and clinical trials showed that a major limitation to a successful gene therapy treatment is the extremely poor expression of fVIII from the human fVIII transgene, which we recently overcame by introducing a porcine fVIII transgene. We demonstrated that use of the porcine transgene results in up to 100-fold greater fVIII production than the human version, and more recently, we demonstrated that a similar differential is observed following gene transfer into blood stem cells using recombinant retroviruses. Our current studies focus on better understanding the critical pharmacologic parameters involved in generating curative fVIII levels with this procedure using a mouse model of hemophilia A with the goal of understanding how this application can best be translated to clinical successes
Keywords: A Mouse; Affect; Anabolism; Antibodies; arthropathies; base; Biology; Biomedical Engineering; Blood; Blood Cells; Blood Clot; Blood coagulation; Blood Coagulation Factor; Bone Marrow Transplantation; CD34 gene; Cell Lineage; Cells; Clinical; Clinical Trials; clinically relevant; clinically significant; conditioning; cost; Data; design; Development; Disease; Drug Kinetics; Engineering; Engraftment; expression vector; Factor VIII; Family suidae; Gene Mutation; gene therapy; gene therapy clinical trial; Gene Transfer; gene transfer vector; Genetic; genetically modified cells; Goals; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemophilia A; Hemorrhage; HIV; Human; human F8 protein; Immune response; Immune Tolerance; Immunization; Immunologic Deficiency Syndromes; In Vitro; in vivo; Individual; inhibitor/antagonist; Insertional Mutagenesis; Intravenous infusion procedures; Joints; Lentivirus Vector; Mesenchymal Stem Cells; Methods; Modeling; Modification; Morbidity - disease rate; mouse model; Mus; Natural immunosuppression; novel; Outcome; Pathway interactions; Patients; Performance; Phase I Clinical Trials; Plasma Proteins; Population; preclinical study; Procedures; Production; Promotor (Genetics); Property; protein expression; Proteins; Protocols documentation; public health relevance; Reagent; Recombinants; Regimen; Reporting; Retroviral Vector; Retroviridae; Safety; simian human immunodeficiency virus; SIV; stem; stem cell biology; Stem cell transplant; Stem cells; success; System; Testing; Therapeutic; Toxic effect; Transgenes; Transgenic Organisms; Translating; Transplantation; Transplantation Conditioning; Treatment Efficacy; Viral Genes; Xenograft procedure
Relevance: Hemophilia A is a bleeding disorder caused by insufficiency of a blood clotting factor, designated factor VIII, for which gene therapy offers a potential cure. However, pre-clinical studies and clinical trials showed that a major limitation to a successful gene therapy treatment is the extremely poor expression of fVIII from the human fVIII transgene, which we recently overcame by introducing a porcine fVIII transgene. We demonstrated that use of the porcine transgene results in up to 100-fold greater fVIII production than the human version, and more recently, we demonstrated that a similar differential is observed following gene transfer into blood stem cells using recombinant retroviruses. Our current studies focus on better understanding the critical pharmacologic parameters involved in generating curative fVIII levels with this procedure using a mouse model of hemophilia A with the goal of understanding how this application can best be translated to clinical successes
Project start date: 2009-04-01
Project end date: 2014-02-28
Budget start date: 1-MAR-2011
Budget end date: 29-FEB-2012
PFA/PA: PA-07-070
5R01HL092179-03 (2011): $387500
5R01HL092179-02 (2010): $387500
ULTRAHIGH THROUGHPUT CELLULAR MANIPULATION VIA MASSIVELY PARALLEL MICROINJECTION
Bradley Christopher
University Of California Riversidecity: Riverside country: United States (us)
Grant 8R21GM103973-03 from National Institute Of General Medical Sciences
Keywords: Address; Area; Automation; base; Biological; biological systems; Biomedical Research; Cell Therapy; cell type; Cells; Clinical; clinical application; Clinical Research; Communities; Computers; Computers and Advanced Instrumentation; cost; design; Development; Device or Instrument Development; Devices; Discipline; Disease; disorder control; Drug Delivery Systems; drug discovery; Elements; Future; gene therapy; Genetic; Genetic Engineering; Goals; Gold; Health; Hematology; Hematopoietic stem cells; Hereditary Disease; Human; improved; Injection of therapeutic agent; innovation; instrument; instrumentation; Investigation; Lead; Libraries; Life; Methodology; Microfabrication; Microinjections; Mission; Modification; novel; oncology; operation; Outcome Study; Performance; Preclinical Drug Evaluation; prevent; Process; Production; prototype; public health relevance; Reliance; Research; Research Personnel; Risk; RNA Interference; Robotics; Safety; Screening procedure; Site; Solid; Speed (motion); Staging; success; System; Techniques; Technology; Testing; Therapeutic; Time; Transfection; Transgenic Animals; Transgenic Organisms; Translations; Validation; Viral Vector
Relevance: The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH´s mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent
Project start date: 2010-01-20
Project end date: 2013-01-19
Budget start date: 20-JAN-2012
Budget end date: 19-JAN-2013
8R21GM103973-03 (2012): $182658