Current state of U.S. Food and Drug Administration regulation for cellular and gene therapy products: potential cures on the horizon.

Cellular & Gene Therapies (CGTs) are complex products, which have been key foci of the International Society for Cell & Gene Therapy (ISCT). For this ISCT North American Legal & Regulatory Affairs Committee review publication, CGTs include but are not limited to somatic cell-based therapies, pluripotent cell-derived cell-based therapies, gene- or non-gene-modified or gene edited versions of these cell-based therapies, in vivo gene therapies, organ/tissue engineered products, and relevant combination products. These products are regulated by the Food and Drug Administration (FDA) in the United States. This publication reviews selected laws, regulations, guidance, definitions, processes, types of meetings and submissions, and other key factors that the FDA follows and implements to regulate and support development of these types of products. These factors may be considered in order to help current and potential product developers/sponsors/applicants navigate through FDA regulatory pathways. We also review expedited programs including types of Designations available at the FDA, and their specific eligibility criteria. We include FDA and other stakeholder resources to consider regarding CGT regulation, to help prepare for CGT development and subsequent FDA approval.

International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection.

Surgical considerations for the neglected or chronic Achilles tendon rupture: a combined technique for reconstruction.

The Achilles tendon is among the most commonly injured tendons in the human body. The most common reason for delayed treatment is a missed diagnosis or a deficiency in presentation. The neglected or chronically ruptured Achilles tendon presents a unique treatment challenge. The surgical approach varies greatly depending on the extent of degeneration and the resultant gap between the opposing tendon ends. Most surgeons have recommended the use of a tendon transfer or augmentation to strengthen the Achilles tendon repair. The following technique uses a flexor hallucis longus tendon transfer with gastrocnemius aponeurosis turndown flap augmentation. This technique has been commonly performed by us with success.

Technical considerations for surgical intervention of Jones fractures.

Jones fractures are a common injury treated by foot and ankle surgeons. Surgical intervention is recommended because of the high rate of delayed union, nonunion, and repeat fracture, when treated conservatively. Percutaneous intramedullary screw fixation is commonly used in the treatment of these fractures. We present techniques that can increase the surgical efficiency and decrease the complications associated with percutaneous delivery of internal fixation.

Surgical approach for combined ankle and subtalar joint chronic mechanical instability.

Combined ankle and subtalar joint instability can lead to severe disability of the lower extremity. Multiple procedures have been described for hindfoot and ankle instability, including anatomic and non-anatomic reconstructions. The authors present their technique consisting of a free autogenous split peroneus longus tendon graft combined with a modified Brostrom-Gould repair.

Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs.

BACKGROUND: The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post-transcriptional gene expression, offers a promising alternative to achieving this goal. METHODS: Here, the combination of nuclear transfer cloning and RNAi technology was used to produce pigs that may not propagate PERV. Small interfering RNAs (siRNA) were expressed as short hairpin RNAs (shRNA) against the gag and pol PERV genes, respectively, under the control of a RNA polymerase III (pol III), or a pol II promoter. PERV gag and pol model-genes, in combination with a Green Fluorescent Protein (GFP) reporter system, were developed to assess in vitro PERV target knockdown. Two shRNAs were selected, and transgenic pigs were produced that expressed the anti-gag and -pol shRNAs, in tandem, under the control of a ubiquitous pol II promoter. RESULTS: The anti-gag and -pol shRNAs, effectively knocked down expression of the PERV model-genes, and also endogenous PERV within cells in vitro. PERV knockdown was achieved whether the shRNA was expressed under the control of a RNA pol III, or a pol II promoter. Three litters of cloned pigs were produced. The shRNA construct was expressed by all the transgenic cloned animals, and within all the tissues of transgenic animals tested. PERV expression at the mRNA and PERV particulate levels in the pigs was virtually undetectable, compared with the infectious levels expressed by the positive control PK15 cell line in vitro. Immunofluorescence and Western blotting, with an anti-PERV-envelope antibody, did not detect PERV in pig tissues or cells whether activated or not, as compared to the positive control on PK15 cells. CONCLUSIONS: The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.

Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.

BACKGROUND: Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection. METHODS: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. RESULTS: Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. CONCLUSIONS: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.

Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.

DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38 degrees C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 microg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.

Fgl2 deficiency causes neonatal death and cardiac dysfunction during embryonic and postnatal development in mice.

We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we previously reported in fibrinogen-like protein 2 (Fgl2) knockout (KO) mice. We therefore used ultrasound biomicroscopy to assess left ventricular (LV) cardiac structure and function during development in Fgl2 KO and wild-type (WT) mice. The only deaths observed between embryonic day (E)8.5 (onset of heart beating) and postnatal day (P)28 (weaning) were within 3 days after birth, when 33% of Fgl2 KO pups died. Histopathology and Doppler assessments suggested that death was due to acute congestive cardiac failure without evidence of valvular or other obvious cardiac structural abnormalities. Heart rates in Fgl2 KO embryos were significantly reduced at E8.5 and E17.5, and irregular heart rhythms were significantly more common in Fgl2 KO (21/26) than WT (2/21) embryos at E13.5. Indexes of systolic and/or diastolic cardiac function were also abnormal in KO mice at E13.5 and E17.5, in postnatal mice studied at P1, and in KO mice surviving to P28. M-mode analysis showed no difference in LV diastolic chamber dimension, although posterior wall thickness was thinner at P7 and P28 in Fgl2 KO mice. We conclude that Fgl2 deficiency is not associated with obvious structural cardiac defects but is associated with a high incidence of neonatal death as well as contractile dysfunction and rhythm abnormalities during embryonic and postnatal development in mice.

The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis.

Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin-deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin-/- mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.

Targeted deletion of Fgl-2/fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts.

BACKGROUND: Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. METHODS AND RESULTS: We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2-/-) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2-/- mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2-/- grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2-/- grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2-/- hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2-/- long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2-/- xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. CONCLUSIONS: Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.

Induction of xenogeneic neonatal tolerance to transgenic human leukocyte antigen class I grafts.

BACKGROUND: The immune response against xenografts is vigorous and poorly controlled with conventional immunosuppressants. Therefore, success in xenotransplantation will depend on developing additional approaches such as induction of immunologic unresponsiveness or tolerance. Although classic protocols of neonatal tolerance induction in mice are very tolerogenic in many allogeneic models, they have generally failed in xenogeneic models. The purpose of these studies was to determine whether failure results from an intrinsic property of xenogenic major histocompatibility complex (MHC) molecules themselves or, instead, is caused by some limitation in species-specific molecular interactions distinct from the polymorphic domains of xenogenic MHC molecules. METHODS: Our approach was to test the ability of lymphoid cells from a transgenic (Tg) mouse donor expressing a xeno-MHC class I molecule encoding the polymorphic alpha1/alpha2 for human leukocyte antigen (HLA)-B7 to induce neonatal tolerance in non-Tg syngeneic C57BL/6 recipients. Because the donor and recipient strains are genetically identical (C57BL/6, H-2b) except for Tg human MHC HLA-B7, any species-specific molecular incompatibility in this mouse anti-human class I xeno-combination that could potentially interfere with induction of tolerance has been eliminated. RESULTS: Our results show that HLA-B7 Tg-, but not C57BL/6 syngeneic-, injected neonates were unresponsive as adults to HLA-B7-expressing target cells in vitro and specifically accepted HLA-B7-expressing Tg skin grafts. In addition, neonatal injection of donor cells resulted in peripheral chimerism. CONCLUSIONS: These experiments demonstrate that, as long as species-specific molecular interactions are maintained, recognition of the polymorphic domains of xenogeneic MHC does not represent a barrier to neonatal tolerance induction.

MSC-based product characterization for clinical trials: an FDA perspective.

Proposals submitted to the FDA for MSC-based products are undergoing a rapid expansion that is characterized by increased variability in donor and tissue sources, manufacturing processes, proposed functional mechanisms, and characterization methods. Here we discuss the diversity in MSC-based clinical trial product proposals and highlight potential challenges for clinical translation.

Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

Cytokine-induced hepatic apoptosis is dependent on FGL2/fibroleukin: the role of Sp1/Sp3 and STAT1/PU.1 composite cis elements.

Previous studies from our laboratory have shown that fulminant hepatitis caused by the mouse hepatitis virus, MHV-3, is dependent on production of the novel immune coagulant fgl2/fibroleukin. In this study, we investigate the role of IFN-gamma and TNF-alpha in the induction of fgl2 expression and fgl2-dependent hepatic apoptosis. Infusion of IFN-gamma in combination with TNF-alpha through the portal vein of fgl2+/+ mice led to widespread hepatic apoptosis and fibrin deposition. Livers from fgl2-/- mice were normal, although strong expression of the fgl2 knockout reporter gene Lac Z was seen in both resident hepatic macrophages and endothelial cells. In vitro, IFN-gamma and TNF-alpha induced fgl2 expression in a macrophage and endothelial cell-specific manner. In macrophages (peritoneal and RAW 264.7 cells), IFN-gamma, but not IFN-alpha, LPS, TNF-alpha, or IL-1 induced fgl2 mRNA transcription and protein expression, while in endothelial cells TNF-alpha, but not IFN-gamma, induced fgl2 transcription. In addition, while TNF-alpha enhanced IFN-gamma-induced macrophage fgl2 transcription, IFN-gamma also enhanced TNF-alpha-induced endothelial cell fgl2 transcription. The induction of fgl2 by IFN-gamma in macrophages involved a STAT1-dependent pathway, involving the composite cis elements Sp1/Sp3 and GAS/PU.1. The latter interacted with IFN-gamma-dependent Sp1/Sp3, STAT1, and the ETS family of transcription factors member PU.1. The interaction of PU.1 with the IFN-gamma-activated sequence/ETS family of transcription factors site determined the macrophage-specific induction of fgl2 by IFN-gamma. Overall, this study demonstrates that IFN-gamma and TNF-alpha induce hepatocyte apoptosis in vivo, which is dependent on induction of fgl2, and defines the molecular basis of transcription of fgl2 in vitro.

Endothelial induction of fgl2 contributes to thrombosis during acute vascular xenograft rejection.

Thrombosis is a prominent feature of acute vascular rejection (AVR), the current barrier to survival of pig-to-primate xenografts. Fibrinogen-like protein 2 (fgl2/fibroleukin) is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis and cytokine-induced fetal loss. We hypothesized that induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with AVR. We first examined fgl2 as a source of procoagulant activity in the pig-to-primate combination. The porcine fgl2 (pfgl2) was cloned and its chromosomal locus was identified. Recombinant pfgl2 protein expressed in vitro was detected on the cell surface and generated thrombin from human prothrombin. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells (ECs). Cultured porcine ECs activated by human TNF-alpha in vitro demonstrated induction of pfgl2 expression and enhanced activation of human prothrombin. The availability of gene-targeted fgl2-deficient mice allowed the contribution of fgl2 to the pathogenesis of AVR to be directly examined in vivo. Hearts heterotopically transplanted from fgl2(+/+) and fgl2(+/-) mice into Lewis rats developed AVR with intravascular thrombosis associated with induction of fgl2 in graft vascular ECs. In contrast, xenografts from fgl2(-/-) mice were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium plays a role in the pathogenesis of AVR-associated thrombosis. Manipulation of fgl2, in combination with other interventions, may yield novel strategies by which to overcome AVR and extend xenograft survival.

The fgl2 prothrombinase/fibroleukin gene is required for lipopolysaccharide-triggered abortions and for normal mouse reproduction.

Increased fgl2 prothrombinase activity in maternal decidua and fetal trophoblasts may trigger abortions by proinflammatory cytokines induced by bacterial lipopolysaccharide (LPS) in mice and is implicated in human recurrent miscarriages and pre-eclampsia. Defining the physiological and pathological role of the fgl2/fibroleukin gene required an fgl2-knockout mouse and data on normal pattern of fgl2 expression during pregnancy. Expression of fgl2 protein was determined by immunostaining with specific antibody. Fgl2 knockout mice were generated and typed by PCR for presence of the altered gene. Immunostaining of timed CBAxDBA/2 mouse matings in a low-abortion-rate colony showed a distinct pattern of development of fgl2 protein expression in maternal decidua, and in embryonic tissues in early pregnancy. Outbred (mixed background) heterozygous fgl2 +/-x+/- matings with a similar low abortion rate showed selective occult loss of both +/- and, to a greater extent, -/- embryos prior to gestation day 11.5, in association with haemorrhage at the anti-mesometrial pole of fgl2-deficient embryo. LPS injected on day 6.5 caused classical abortions at mid-pregnancy in fgl2 +/+x+/+ matings, but not -/-x-/- matings. Physiological expression of fgl2 in fetal trophoblast may prevent occult loss in early pregnancy, along with other coagulation factors, but fgl2 expression is required for LPS to induce abortion pathology.

Gene transcription of fgl2 in endothelial cells is controlled by Ets-1 and Oct-1 and requires the presence of both Sp1 and Sp3.

The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome. Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages. To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5'-RACE and primer extension. A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription. A complex positive regulatory domain (PRD) spanning a 39-bp sequence from -87 to -49 (relative to the transcription start site) was identified. Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct-1, and Ets-1. Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets-1 and requires the presence both of the Sp1 and Sp3 transcription factors. The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated. Moreover, viral induced fgl2 expression also requires the presence of this PRD. These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral-induced fgl2 expression in endothelial cells.