John
F. Engelhardt, Ph.D.
Director of the Center for Gene Therapy
Professor, Department of Anatomy and Cell Biology
Link
to the Engelhardt Lab web site
Training:
B.S., Iowa State University, Ames, IA (Biochemistry), 1985
Ph.D., Johns Hopkins University, Baltimore, MD (Human Genetics), 1990
Research Associate, University of Michigan, Ann Arbor, MI (HHMI), 1992
Research Associate, University of Michigan, Ann Arbor, MI (Internal Medicine), 1993
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Dr. Engelhardt's research focuses on the development of gene therapies for inherited and environmentally induced diseases. Included are two major research areas: 1) The study of lung molecular and cellular biology as it relates to the pathogenesis and treatment of cystic fibrosis (CF) lung disease and 2) Molecular mechanism of redox-mediated environmental injuries such as ischemia/reperfusion injury and sepsis and the development of molecular therapies for these disorders. Interests in lung biology include understanding signals that establish epithelial stem cell niches in the airway. Stem cell niches play a role in regulating proliferation and differentiation of stem cells during development and in the adult following organ injury. Airway glandular stem cell niches may play important roles in several human diseases associated with aberrant expansion of submucosal glands leading to excessive mucous production in the airway. These diseases include cystic fibrosis, chronic bronchitis, and asthma. Our laboratory has focused on elucidating transcriptional signals important for early establishment of the airway glandular stem cell niche. We have found that a related family of High Mobility Group (HMG) transcription factors called, TCF/Lef-1, may coordinate events leading to gland formation in the airway. Our studies have begun to dissect how certain mesenchymally secreted signals (Wnts, BMPs, and Noggin) coordinate the regulation of HMG transcription factors required for development of airway glands and establishment of glandular stem cell niches. A second area of interest includes the development of redox mediated gene therapies to the liver. Such studies are aimed at the treatment of environmentally induced liver injuries caused by sepsis and ischemia/reperfusion. Rodent models are used to better understand the mechanisms of free radical damage and inflammatory mediated injury. Of particular interest in this area are mechanisms of redox-regulated NF k B activation involving the NADPH oxidase complex. The NADPH oxidase complex is responsible for generating intracellular superoxides in response to extracellular signals. Transgenic mouse models together with recombinant adenoviral vectors expressing free radical scavenging enzymes and dominant negative or active signal transduction intermediates are used to approach these questions. Most recently, Dr. Engelhardt's group has identified several key redox-regulated signaling intermediates (Rac1, NIK, c-Src) responsible for NF k B activation in response to LPS, pro-inflammatory cytokines, and/or ischemia/reperfusion. This program heavily utilizes recombinant viral vectors not only to address basic aspects of pathophysiology, but also to generate more applied gene therapies capable of promoting organ regeneration and repair while minimizing the deleterious inflammatory responses to injury. |
Link to a complete list of Dr. Engelhardt's peer-reviewed journal articles
Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway
Zhang LN, Karp P, Gerard CJ, Pastor E, Munson K, Yan Z, Godwin S, Thomas C, Zabner , J, Peluso R, Carter B, and Engelhardt JF . (2004). Dual Therapeutic Utility of Proteasome Modulating Agents for Pharmico-Gene Therapy of the Cystic Fibrosis Airway. Molecular Therapy 10 :6, 990-1002. (full text)
Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.
Figure 1. Doxorubicin treatment increases CpG methylation of the g-ENaC gene promoter. (A) Schematic diagram of the g-ENaC gene promoter. The transcription start site is labeled as +1; exon 1 is shown as a gray box; the positions of the CpG islands studied in this report are shown as black rectangles; the positions of restriction enzymes used to study CpG methylation are shown as vertical lines; the positions of primers used in the methylation-sensitive PCR analysis are shown by arrows at 3471 and 3161 bp; the probe used for Southern blot analysis is shown by the bar at 631 to +401 bp. (B) Results from methylation-sensitive PCR analysis with primary CF polarized airway epithelia for the 3471 to 3161 bp region of the g-ENaC gene promoter evaluating methylation at the CpG island 1. MboI digestion of genomic DNA prior to PCR analysis (lanes 1–3) served as a positive control and gave rise to two PCR products (more than one product is likely, due to the GC-rich content of the PCR fragment). When no DNA is added as template (lane 10), no PCR product is seen. Codigestion of Dox-treated genomic DNA samples with MboI/HpaII (lanes 8 and 9) gave rise to PCR products similar to those seen in the positive control (lanes 1–3), indicating that the HpaII sites are protected from digestion by methylation. The extent of protection from HpaII was significantly less in cells not treated with Dox (lane 7). In contrast, all samples, regardless of Dox treatment, gave very little PCR production following MboI digestion since this enzyme is not methylation sensitive (lanes 4–6). (C) Southern blot analysis of CpG island 2 ( 229 to +270 bp) in the g-ENaC promoter was analyzed in CuFi-1 cells following a 16-h treatment with 5 AM doxorubicin. Cells were harvested at 0 days (untreated) or 1 and 3 days posttreatment. Open arrow, the fragments resulting from MspI digestion of MspI/HpaII sites or HpaII digestion of unmethylated MspI/HpaII sites. Solid arrow, the fragments that were protected from HpaII digestion by methylation of MspI/HpaII sites.
Airway Stem Cell and Cystic Fibrosis Research.
(Funded by NIDDK 2RO1 DK 47967 "Models
of Submucosal Gland Development and Function"; NHLBI P50 HL 61234
"Antibacterial Functions of Submucosal Glands in the Airway";
and the Cystic Fibrosis Foundation)
Our program in lung research centers around elucidating pathophysiologic mechanisms of cystic fibrosis and the development of gene therapies for the disease. Specific interests pertain to submucosal gland involvement in the pathogenesis of this disorder and the development of in utero gene therapies targeting submucosal gland stem cells. To this end, research has focused on determining mechanisms of gland development in the airway and the identification of airway progenitor/stem cells capable of submucosal gland development and differentiation. This area of interest has led to projects that attempt to identify the molecular characteristic of gland stem cells and the molecular mechanisms of epithelial/mesenchyme interactions involved in organogenesis of submucosal glands. Additionally, a focus has been to develop animal models capable of addressing mechanisms of pathophysiology in cystic fibrosis and modeling gene therapy for this disorder. Such models include the human bronchial xenograft, transgenic mice and the ferret. Newly developed programs are attempting to generate a CF ferret model using nuclear transfer cloning technologies together with targeted somatic cell mutations in ferret cell lines.
Related Publications
Filali M, Cheng N, Abbott D, Leontiev V, and Engelhardt JF, Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter, J Biol Chem, 277, 36, 33398-33410, 2002.
Ritchie T, Zhou W, McKinstry E, Hosch M, Zhang Y, Nathke I, and Engelhardt JF, Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway, Exp Lung Res, 27, 121-141, 2001.
Engelhardt JR, Stem Cell Niches in the Mouse Airway, Am J Respir Cell Mol Biol, 24, 649-652, 2001.
Duan D, Yue Y, Zhou W, Labed B, Ritchie T, Grosschedl R, and Engelhardt JF: Submucosal Gland Development in the Airway is Controlled by Lymphoid Enhancer Binding Factor-1 (Lef-1). Development, 126:4441-4453, 1999.
Duan D, Sehgal A, Yao J, and Engelhardt JF. The Lef1 transcription factor
expression defines submucosal gland progenitor cell phenotypes for gene
therapy in the airway. Am. J. Resp. Cell. Mol. Biol,. 18:750-758,
1998.
Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollingworth MA,
and Engelhardt JF. MUC5B and MUC7 are Differentially Expressed in Mucous
and Serous Cells of Submucosal Glands in Human Bronchial Airways. Am.
J. Resp. Cell. Mol. Biol., 19:30-37, 1998.
Presente A, Sehgal A, Dudus L, and Engelhardt J. Differentially Regulated
Epithelial Expression of an Eph Family Tyrosine Kinase (Fek2) During Tracheal
Surface Airway and Submucosal Gland Development, Am. J. Resp. Cell.
and Mol. Biol., 16:53-61, 1997.
Sehgal A, Presente A, Dudus L, and Engelhardt JF. Isolation of differentially
expressed cDNAs during ferret tracheal development: Application of Differential
Display PCR. Experimental Lung Research, 22:419-434, 1996.
Sehgal A, Presente A, and Engelhardt JF. Developmental Expression Patterns
of CFTR in Ferret Tracheal Surface Airway and Submucosal Glands. Am.
J. Resp. Cell. and Mol. Biol., 15:122-131, 1996.
Engelhardt JF, Schlossberg H, Yankaskas JR, and Dudus L. Progenitor cells
of the adult human airway involved in submucosal gland development. Development,
121:2031-2046, 1995.
Pathogenesis and treatment of cystic fibrosis lung
disease using gene therapy.
(Funded by CFF grant ENGELH97Z0 "Mechanisms
of Fluid and Electrolyte Regulation in the Airway" and NIH SCOR in
Cystic Fibrosis P50 HL 61234, Project 3: "Antibacterial Functions
of Submucosal Glands in the Airway")
Since a major goal of this laboratory is the development of gene therapies for cystic fibrosis (CF), the elucidation of primary defects in electrolyte transport, fluid transport, and mucus biochemistry has aided in dissecting mechanisms that influence the pathogenesis of bacterial infections in CF lung disease. CF patients suffer from a genetic defect in a chloride channel called the cystic fibrosis transmembrane regulator (CFTR). The laboratory uses several model systems to approach questions pertaining to CFTR function including, transgenic mice, human bronchial xenografts, ferret tracheal xenografts, and xenopus oocytes. A current focus includes studies of the mechanisms of fluid and electrolyte transport in the airway using CF and Non-CF bronchial xenograft models. This genetically defined model system has allowed for the characterization of primary defects that influence bacterial infection in the CF airway and the evaluation of gene therapy approaches with functionally relevant endpoints.
Related Publications
Liu X, Jiang Q, Mansfield SG, Puttaraju M, Zhang Y, Zhou W, Cohn JA, Garcia-Blanco MA, Mitchell LG and Engelhardt JF, Partial correction of endogenous delta-F508 CFTR in human cystic fibrosis airway epithelial by spliceosome-mediated RNA trans-splicing, Nature Biotechnology, 20, 47-52, 2002.
Engelhardt JF, The lung as a metabolic factory for gene therapy, J Clin Invest, 110, 429-432, 2002.
Wang X, Zhang Y, Amberson A, and Engelhardt JF, New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition, J Resp Cell Mol Biol, 24, 195-202, 2001.
Duan D, Yan Z, Yue Y, Ding W, and Engelhardt JF, Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation, Journal of Virology, 75, 16, 7662-7671, 2001.
Duan D, Yue Y, and Engelhardt JF, Expanding AAV Packaging Capacity with Trans-splicing or Overlapping Vectors: A Quantitative Comparison, Molecular Therapy, 4, 4, 383-391, 2001.
Engelhardt JF, Sen CK, and Oberley L, Redox-modulating gene therapies for human diseases, Antioxidant and Redox Signaling, 3, 3, 341-346, 2001.
Duan D, Yue Y, Yan Z, and Engelhardt JF, A new dual vector approach to enhance recombinant AAV mediated gene expression through intermolecular cis-activation, Nat. Med., 6, 595-598, 2000.
Sanlioglu S, Benson PK, Yang J, Atkinson EM, Reynolds T, and Engelhardt JF, Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation, J Virol, 74, 9184-9196, 2000.
Naren AP, Anke D, Cormet-Boyaka E, Boyaka PN, McGhee JR, Zhou W, Akagawa K, Jujiwara T, Thome U, Engelhardt JF, Nelson DJ, and Kirk KL, Syntaxiin 1A is expressed in airway epithelial cells where it modulates CFTR Cl-currents, J Clin Invest, 105, 377-386, 2000.
Sanlioglu S, Benson P, and Engelhardt JF, Loss of ATM function enhances recombinanat adeno-associated virus transduction and integration through pathways similar to UV irradiation, J Virol, 268, 68-78, 2000.
Yan Z, Zhang Y, Duan D, and Engelhardt JF, Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy, PNAS, 97, 6716-6721, 2000.
Jiang Q, Li J, Dubroff R, Ahn YJ, Foskett JK, Engelhardt JF, and Kleyman TR, Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes, J Biol Chem, 275, 13266-13274, 2000.
Duan D., Yue, Y., Yan Z, Yang, J, Engelhardt JF. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest, 105: 1573-1587, 2000.
Zhang Y and Engelhardt JF. Airway Surface Fluid Volume and Chloride Content in Cystic Fibrosis and Normal Bronchial Xenografts. Am. J. Physiol: Cell Physiol., 276: C469-C476, 1999.
Zhang Y, Jiang Q, Dudus, L, Yankaskas JR, and Engelhardt JF. Vector Specific
Complementation Profiles of Two Independent Primary Defects in Cystic
Fibrosis airways. Human Gene Therapy, 9:635-648, 1998.
Jiang Q, Mak D, Devidas S, Schwiebert EM, Bragin A, Zhang Y, Skach WR,
Guggino WB, Foskett JK, and Engelhardt JF. Cystic fibrosis transmembrane
conductance regulator-associated ATP release is controlled by a chloride
sensor. J. Cell Biol., 143(3):645-57, 1998.
Zhang Y, Yankaskas J, Wilson JM and Engelhardt JF. In vivo Analysis of Fluid Transport in Cystic Fibrosis Airway Epithelia of Bronchial Xenografts. Am J Physiology: Cell Physiology, 270:C1326-1335, 1996.
Zhang Y, Doranz B, Yankaskas JR, and Engelhardt JF. Genotypic
analysis of respiratory mucous sulfation defects in cystic fibrosis. J.
Clin Invest., 96:2997-3004, 1995.
Generation of a Transgenic Ferret Model of Cystic Fibrosis.
(Funded by NIH/NIDDK RO1 DK47967 "Models of Submucosal Gland Development
and Function" and NIH SCOR in Cystic Fibrosis P50 HL 61234, Project
3: "Antibacterial Functions of Submucosal Glands in the Airway".)
The need for an improved animal model for investigation of CF lung disease stems from the critical shortage of human CF airway tissue for research and the lack of lung pathology in current mouse models. A ferret CF animal model will supplement current in vitro (polarized airway epithelial cultures) and ex vivo (human and ferret airway xenografts) model systems for study of the pathogenic mechanisms of CF, as well as for the development of gene therapy strategies for the lung. Previous studies have demonstrated that ferrets, unlike mice, have identical cell types and a similar distribution of submucosal glands as in the human airway, making them a more advantageous model for CF airway disease than other current animal models of CF, such as transgenic and knockout mice. A combination of the most recently developed somatic-cell embryo cloning techniques with allele-specific gene targeting based on homologous recombination will be utilized. The initial research strategy is to develop a transgenic ferret model for cystic fibrosis by introducing the G551D mutation into ferret fetal fibroblasts using a novel homologous recombination technique. Subsequently, nuclei from the manipulated cells are transplanted into enucleated oocytes, and embryos are cultured in vitro and transplanted into surrogate mothers for development. Thus far, preliminary studies have demonstrated our ability to superovulate ferrets, to culture the ferret embryos in vitro, and to produce live offspring with high efficiency after transferring manipulated embryos to "pseudopregnant" females. These studies have laid the foundation for the successful generation of a transgenic CF ferret. The availability of this animal model will undoubtedly further our understanding of the pathophysiology of cystic fibrosis and will greatly enhance the efforts to conquer this disease.
Figure 4. Development of enucleation
procedures for ferret oocytes.
(A) A ferret oocyte immobilized with a holding
pipette prior to enucleation.
(B) Ferret oocyte during removal of cytoplasmic
material with a transfer pipet. The procedure did not
produce obvious trauma to the oocyte.
Figure 5. Ferret oocytes were enucleated and
ferret fibroblast nuclei were used to
reconstitute the embryo. Following
activation and in vitro culture, cloned
embryos matured to both morula an
blastocyst stages. The arrow marks a
hatched blastocyst.
Related Publications
Ziyi L, Qinshi J, Rezaei Sabet, M, Zhang Y, Ritchie TC, and Engelhardt JF, Conditions for in Vitro Maturation and Artificial Activation of Ferret Oocytes, Biology of Reproduction, 66, 1380-1386, 2002.
Ritchie T, Zhou W, McKinstry E, Hosch M, Zhang Y, Nathke I and Engelhardt JF. Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway. Experimental Lung Research, 27: 121-141, 2001.
Engelhardt JF. Stem cell niches in the mouse airway. Am J Respir Cell Mol Biol, 24: 649-652, 2001.
Wang X, Zhang Y, Amberson A and Engelhardt JF. New models of the tracheal airway define the glandular contribution to airway surfface fluid and electrolyte composition. Am J Resp Cell Mol Biol, 24: 195-202, 2001.
Li Z, Jiang Q, Zhang Y, Liu X and Engelhardt JF. Successful production of offspring following superovulation and in vitro culture of embryos from domestic ferrets. Reproduction, 122: 611-618, 2001.
Li Z, Jiang Q, Sabet M, Zhang Y, Ritchie T and Engelhardt JF. Conditions for in vitro maturation and artificial activation of ferret oocytes. Biology of Reproduction, (in press).
Jiang Q, Li J, Dubroff R, Ahn YJ, Foskett JK, Engelhardt JF and Kleyman TR. Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes. J Biol Chem, 275: 13266-13275, 2000.
Naren AP, Anke D, Cormet-Boyaka E, Boyaka PN, McGhee JR, Zhou W, Akagawa K, Jujiwara T, Thome U, Engelhardt JF, Nelson DJ and Kirk KL. Syntaxin 1A is expressed in airway epithelial cells where it modulates CFTR Cl- currents. J Clin Invest, 105: 377-386, 2000.
Zhang Y and Engelhardt JF. Airway surface fluid voluume and chloride content in cystic fibrosis and normal bronchial xenografts. Am J Physio: Cell Physiol, 276: C469-C476, 1999.
Duan D, Yue Y, Zhou W, Labed B, Ritchie T, Grosschedl R and Engelhardt JF. Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor-1 (lef-1). Development, 126: 4441-4453, 1999.
Adeno-associated Virus Biology and Vector Development
(Funded by 2R01 HL58340 "Airway Epithelial Biology of AAV Integration")
Determination of vector/target cell interactions that limit the utility of AAV gene therapy to the airway. These studies have focused on the mechanisms of viral entry, trafficking to the nucleus, gene conversion and integration for adeno-associated virus. Several new discoveries regarding the mechanisms of AAV genome conversion have led to the development of innovative strategies to expand AAV packaging capacity using dual vector heterodimerization approaches. Studies in the laboratory are also focused on understanding machanisms of AAV genome concatamerization and circularization as a platform for improving dual vector trans-splicing and cis activation techniques.
Figure 6. Circular concatamerization of rAAV genomes in muscle
Using two independent rAAV vectors encoding either alkaline phosphatase
or GFP transgenes, studies in Engelhardt Lab have demonstrated that concatamerization
of AAV genomes occurs through intramolecular recombination between independent
viral genomes. This figure demonstrates co-localization of GFP (Green)
and Alkaphos (Red) transgene expression in muscle fibers of mice. The
superimposed schematic bifunctional circular concatamer is shown with
a Nomarski image of myofibers overlapping inside the structure. Rescued
circular concatamers from these experiments demonstrate the characteristic
head-to-tail orientation of genomes consistent with AAV latent infection
(Yang, et al., 1999). Importantly, bifunctional genomes were found. This
is the cornerstone of newly developed technologies to expand AAV packaging
capacity using dual vector delivery approaches.
Movie 1: Endosomal trafficking in live cells
This movie localizes a Rab11-GFP fusion protein in live Hela cells
incubated in Texas Red labeled transferrin. Rab11 marks the late recycling
endosome. Similar studies using Cy3-labeled AAV are in progress to understand
the pathway by which this virus moves through cells to the nucleus.
Related Publications
Yan, Z., Ritchie, T. C., Duan, D. and Engelhardt, J. F, Recombinant AAV-mediated gene delivery using dual vector heterodimerization, Methods Enzymol, 346, 334-335, 2002.
Yang GS, Schmidt M, Yan Z, Lindbloom JD, Harding TC, Donahue BA, Engelhardt JF, Kotin R and Davidson BL, Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size, Journal of Virology, 76, 15, 7651-7660, 2002.
Doerschug K, Sanlioglu S, Flaherty DM, Wilson RL, Yarovinsky T, Monick MM, Engelhardt JF, and Hunninghake GW., Adenovirus Vectors Shorten Survival Time in a Murine Model of Sepsis, J. Immunology, 169, 6539-6545, 2002.
Yan Z, Zak R, Luxton GWG, Ritchie T, Bantel-Schaal U and Engelhardt JF, Ubiquitination of Both Adeno-associated Virus Types 2 and 5 Capsid Proteins Affect the Transduction Efficiency of Recombinant Vectors, Journal of Virology, 76, 5, 2043-2053, 2002.
Sanlioglu S, Monick MM, Luleci G, Hunninghake GW and Engelhardt, JF, Rate Limiting Steps of AAV Transduction and Implications for Human Gene Therapy, Current Gene Therapy, 1, 137-147, 2001.
Duan D, Yue Y, and Engelhardt JF, Expanding AAV Packaging Capacity with Trans-splicing or Overlapping Vectors: A Quantitative Comparison, Molecular Therapy, 4, 4, 383-391, 2001.
Lam EWN, Hammad HM, Zwacka R, Darby CJ, Baumgardner KR, Davidson BL, Oberley TD, Engelhardt JF, and Oberley LW, Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors, J Dent Res, 79, 1410-1417, 2000.
Duan D, Yue Y, Yan Z and Engelhardt JF. A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Nature Medicine, 6: 595-598, 2000.
Yan Z, Zhang Y, Duan D and Engelhardt JF. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc natl acad sci USA, 97: 6716-6721, 2000.
Duan D, Sharma P, Dudus L, Zhang Y, Sanlioglu S, Yan Z, Yue Y, Ye Y, Lester R, Yang J, Fisher KJ and Engelhardt JF. Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4-ORF6 and E2a gene expression. J Virology, 73: 161-169, 1999.
Sanlioglu S, Duan D and Engelhardt JF. Two independent molecular pathways for rAAV genome conversion occur following UV-C and E4ORF6 augmentation of rAAV transduction. Human Gene Therapy, 10: 591-602, 1999.
Sanlioglu S and Engelhardt JF. Cellular redox state alters recombinant adeno-associated virus transduction through tyrosine phosphatase pathways. Gene Therapy, 6: 1427-1437, 1999.
Duan D, Yan Z, Yue Y and Engelhardt JF. Structural analysis of AAV circular intermediates. Virology, 261: 8-14, 1999.
Yang J, Zhou W, Zhang Y, Zidon T, Ritchie T, and Engelhardt JF. Concatatmerization of adeno-associated virus circular genomes occurs through internolecular recombination. J Virology, 11: 9468-77, 1999.
Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y, Fisher KJ and Engelhardt JF. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virology, 72: 8568-8577, 1998.
Duan D, Yue Y, McCray PB and Engelhardt JF. Polarity influences the efficiency of recombinant adeno-associated virus infection in differentiated airway epithelia. Human Gene Therapy, 9: 2761-2776, 1998.
Duan D, Fisher KJ, Burda JF and Engelhardt JF. Structural and functional heterogeneity of integrated recombinant AAV genomes. Virus Research, 48: 41-56, 1997.
Gene Therapy for Ischemia/Reperfusion Injury
(Funded by NIH/NIDDK RO1 DK51315 "Gene Therapy of Reperfusion Injury
In Liver" and NIH SCOR in Lung Injury,
P50 HL60316, project 2 "Reactive Oxygen Species
Control of NFkB/TNF Signal Transduction Cascades".)
A last major area of research in Dr. Engelhardt's laboratory involves the development of redox mediated gene therapies to the liver and heart. These investigations are aimed at the treatment of environmentally induced injuries, such as sepsis and ischemia/reperfusion damage associated with organ transplantation and cardiac infarct. Rodent models are used to better understand the mechanisms of free radical damage and inflammatory mediated injury. Of particular interest in these mechanisms are redox regulated pathways of signal transduction (such as MAP kinase and NFkB) that determine the course of cellular apoptosis and regeneration. Inherent in the redox activation of these pathways are the subcellular compartmentalization of reactive oxygen species generated following injury. Alterations in the phosphorylation status of signal transduction molecules such as IkB and c-Jun appear to be central mechanisms invoked by redox activation of serine/threonine and tyrosine kinases. The identification of these redox regulated effector molecules and the subsequent consequences on cytokine gene activation are areas under investigation. Transgenic mouse models, together with recombinant adenoviral vectors expressing free radical scavenging enzymes, are used to approach these questions. These mechanisms will lay the foundation for the rational development of gene therapies aimed at decreasing organ damage and enhancing tissue regeneration following ischemic or endotoxin injury.
Related Publications
Yang JQ, Buettner GR, Domann FE, Li Q, Engelhardt JF, Darby Weydert C and Oberley LW, v-Ha-ras Mitogenic Signaling Through Superoxide and Derived Reactive Oxygen Species, Molecular Cardinogenesis, 33, 206-218, 2002.
Zanetti M, Zwacka RM, Engelhardt JF, Katusic Z, and O'Brien T, Superoxide anions and endothelial cell proliferation in normoglycemia and hyperglycemia, Anterioscler Thromb Vasc Biol, 21, 195-200, 2001.
Zhou W, Zhang Y, Hosch MS, Lang A, Zwacka RM, and Engelhardt JF, Subcellular site of superoxide dismutase expression differentially controls AP-1 activity in the liver and injury following ischemia/reperfusion, Hepatology, 33, 4, 902-914, 2001.
Li Q, Sanlioglu S, Li S, Ritchie T, Oberley L, and Engelhardt JF, GPx-1 gene delivery modulates NFkB activation following diverse environmental injuries through a specific subunit of the IKK complex, Antioxidant and Redox Signaling, 3, 3, 415-432, 2001.
Sanlioglu S, Williams C, Samavati L, Butler N, Wang G, McCray P Jr., Ritchie T. Hunninghake G, Zandi E, and Engelhardt JF, Lipopolysaccharide Induces Rac1-dependent Reactive Oxygen Species Formation and Coordinates Tumor Necrosis Factor-alpha Secretion through IKK Regulation of NK-kappa B, J Biol Chem, 276, 32, 30188-30198, 2001.
Engelhardt, JF. Redox mediated gene therapies for environmental injury: approaches and concepts. Antioxidant and Rdox Signaling, 1: 5-27, 1999.
Fan C, Zwacka RM and Engelhardt JF. Theraputic approaches for ischemia/reperfusion injury in the liver. Journal of Molecular Medicine, 77: 577-92, 1999.
Zwacka R, Dudus L, Epperly MW, Greenberger JS and Engelhardt JF. Redox gene therapy protects human IB-3 lung epithelial cells against ionizing radiation-induced apoptosis. Human Gene Therapy, 9: 1381-1386, 1998a.
Zwacka RM, Zhou W, Zhang Z, Darby CJ, Dudus L, Halldorson J, Oberley L and Engelhardt JF. Redox gene therapy of liver ischemia/reperfusion injury reduces AP1 and NFkB activation. Nature Medicine, 4: 698-704, 1998b.
Zwacka RM, Zhang Y, Halldorson J and Engelhardt JF. Ishcemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and NF-kB independently of IkB degradation. Hepatology, 28: 1022-1030, 1998.
Zwacka R, Zhang Y, Halldorson JB, Schlossberg H, Dudus D and Engelhardt JF. T-lymphocyte-mediated ischemia/reperfusion induced inflammatory responses in mouse liver. J Clin Inv, 100: 279-289, 1997.