Glenn F Pierce

Publications (27)217.91 Total impact

• Article: Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs.

  Jennifer A Dumont, Tongyao Liu, Susan C Low, Xin Zhang, George Kamphaus, Paul Sakorafas, Cara Fraley, Douglas Drager, Thomas Reidy, Justin McCue, Helen W G Franck, Elizabeth P Merricks, Timothy C Nichols, Alan J Bitonti, Glenn F Pierce, Haiyan Jiang
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  ABSTRACT: Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.
  Blood 01/2012; 119(13):3024-30. · 9.90 Impact Factor
• Article: Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients.

  Jerry S Powell, Neil C Josephson, Doris Quon, Margaret V Ragni, Gregory Cheng, Ella Li, Haiyan Jiang, Lian Li, Jennifer A Dumont, Jaya Goyal, Xin Zhang, Jurg Sommer, Justin McCue, Margaret Barbetti, Alvin Luk, Glenn F Pierce
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  ABSTRACT: Current factor VIII (FVIII) products display a half-life (t(1/2)) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.
  Blood 01/2012; 119(13):3031-7. · 9.90 Impact Factor
• Article: Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients.

  Amy D Shapiro, Margaret V Ragni, Leonard A Valentino, Nigel S Key, Neil C Josephson, Jerry S Powell, Gregory Cheng, Arthur R Thompson, Jaya Goyal, Karen L Tubridy, Robert T Peters, Jennifer A Dumont, Donald Euwart, Lian Li, Bengt Hallén, Peter Gozzi, Alan J Bitonti, Haiyan Jiang, Alvin Luk, Glenn F Pierce
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  ABSTRACT: Current factor IX (FIX) products display a half-life (t(1/2)) of ∼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG(1), to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t(1/2) and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is ∼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.
  Blood 11/2011; 119(3):666-72. · 9.90 Impact Factor
• Article: Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment.

  Baisong Mei, Clark Pan, Haiyan Jiang, Hendri Tjandra, Jonathan Strauss, Yaoqi Chen, Tongyao Liu, Xin Zhang, Joanne Severs, Jim Newgren, Jianmin Chen, Jian-Ming Gu, Babu Subramanyam, Michael A Fournel, Glenn F Pierce, John E Murphy
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  ABSTRACT: A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.
  Blood 03/2010; 116(2):270-9. · 9.90 Impact Factor
• Article: Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

  Valder R Arruda, Hansell H Stedman, Virginia Haurigot, George Buchlis, Stefano Baila, Patricia Favaro, Yifeng Chen, Helen G Franck, Shangzhen Zhou, J Fraser Wright, Linda B Couto, Haiyan Jiang, Glenn F Pierce, Dwight A Bellinger, Federico Mingozzi, Timothy C Nichols, Katherine A High
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  ABSTRACT: Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.
  Blood 03/2010; 115(23):4678-88. · 9.90 Impact Factor
• Article: Treatment of nonhealing diabetic foot ulcers with a platelet-derived growth factor gene-activated matrix (GAM501): results of a phase 1/2 trial.

  Gerit Mulder, Arthur J Tallis, V Tracy Marshall, David Mozingo, Laurie Phillips, Glenn F Pierce, Lois A Chandler, Barbara K Sosnowski
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  ABSTRACT: The results from a Phase 1/2 study of a replication-defective adenovirus encoding human platelet-derived growth factor (PDGF)-B formulated in a bovine collagen (Ad-5PDGF-B; 2.6% collagen; GAM501) gel for nonhealing neuropathic diabetic foot ulcers is reported. The primary objectives of the study were to evaluate the safety, maximum-tolerated dose, and preliminary biological activity of GAM501. Fifteen patients enrolled into the study with chronic, nonhealing ulcers received either a single administration of GAM501 at one of three dose levels, or up to four administrations of GAM501 at 1-week intervals. All patients received standard of care treatment including debridement and were required to wear an off-loading shoe. GAM501 was found to be safe and well tolerated with no evidence of systemic or local toxicity at all doses so no maximum-tolerated dose was reached. Serum antibody titers to platelet-derived growth factor-B homodimer and collagen were negative and adenoviral DNA was not detected in the blood. In the 12 patients that completed the study, ulcer closure was observed by Month 3 in 10 patients, seven of whom received a single application of GAM501. In conclusion, GAM501 did not appear to have any toxicity at doses that showed biological activity. GAM501 holds promise as a potentially effective treatment for nonhealing diabetic foot ulcers.
  Wound Repair and Regeneration 10/2009; 17(6):772-9. · 2.91 Impact Factor
• Article: Enhanced efficacy of recombinant FVIII in noncovalent complex with PEGylated liposome in hemophilia A mice.

  Junliang Pan, Tongyao Liu, Ji-Yun Kim, Daguang Zhu, Chandra Patel, Zhi-Hua Cui, Xin Zhang, James O Newgren, Aaron Reames, Dodie Canivel, Gary Jesmok, Glenn F Pierce, Jurg M Sommer, Haiyan Jiang
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  ABSTRACT: Recombinant FVIII formulated in PEG-ylated liposomes (rFVIII-PEG-Lip) was reported to increase the bleed-free days from 7 to 13 days (at 35 IU/kg rFVIII) in severe hemophilia A patients. To understand the underlying mechanism, we sought to recapitulate its efficacy in hemophilia A mice. Animals treated with rFVIII-PEG-Lip achieved approximately 30% higher survival relative to rFVIII after tail vein transection inflicted 24 hours after dosing. The efficacy of rFVIII-PEG-Lip represents an approximately 2.5-fold higher "apparent" FVIII activity, which is not accounted for by its modestly increased (13%) half-life. The enhanced efficacy requires complex formation between rFVIII and PEG-Lip before the administration. Furthermore, PEG-Lip associates with the majority of platelets and monocytes in vivo, and results in increased P-selectin surface expression on platelets in response to collagen. Rotational thromboelastometry (ROTEM) analysis of whole blood from rFVIII-PEG-Lip-treated animals at 5 minutes up to 72 hours after dosing recapitulated the 2- to 3-fold higher apparent FVIII activity. The enhanced procoagulant activity is fully retained in plasma unless microparticles are removed by ultracentrifugation. Taken together, the efficacy of rFVIII-PEG-Lip is mediated mainly by its sensitization of platelets and the generation of procoagulant microparticles that may express sustained high-affinity receptors for FVIII.
  Blood 09/2009; 114(13):2802-11. · 9.90 Impact Factor
• Article: The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.

  Lingxia Chen, Fuxiang Zhu, Juan Li, Hui Lu, Haiyan Jiang, Rita Sarkar, Valder R Arruda, Jinhui Wang, Jennifer Zhao, Glenn F Pierce, Qiulan Ding, Xuefeng Wang, Hongli Wang, Steven W Pipe, Xiang-Qin Liu, Xiao Xiao, Rodney M Camire, Weidong Xiao
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  ABSTRACT: Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10-100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.
  Molecular Therapy 11/2007; 15(10):1856-62. · 6.87 Impact Factor
• Article: Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver.

  Federico Mingozzi, Nicole C Hasbrouck, Etiena Basner-Tschakarjan, Shyrie A Edmonson, Daniel J Hui, Denise E Sabatino, Shangzhen Zhou, J Fraser Wright, Haiyan Jiang, Glenn F Pierce, Valder R Arruda, Katherine A High
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  ABSTRACT: Adeno-associated virus (AAV)-mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti-T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti-IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs.
  Blood 11/2007; 110(7):2334-41. · 9.90 Impact Factor
• Article: CD8(+) T-cell responses to adeno-associated virus capsid in humans.

  Federico Mingozzi, Marcela V Maus, Daniel J Hui, Denise E Sabatino, Samuel L Murphy, John E J Rasko, Margaret V Ragni, Catherine S Manno, Jurg Sommer, Haiyan Jiang, Glenn F Pierce, Hildegund C J Ertl, Katherine A High
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  ABSTRACT: Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.
  Nature Medicine 05/2007; 13(4):419-22. · 22.46 Impact Factor
• Article: Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.

  Guang Qu, Jennifer Bahr-Davidson, Joseph Prado, Alex Tai, Floro Cataniag, Jennifer McDonnell, Jingmin Zhou, Bernd Hauck, Jac Luna, Jurg M Sommer, Peter Smith, Shangzhen Zhou, Peter Colosi, Katherine A High, Glenn F Pierce, J Fraser Wright
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  ABSTRACT: Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose(xl) are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 x 10(14)vg and 2.52 x 10(15) empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 x 10(14)vg containing 0.25 x 10(14) empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.
  Journal of Virological Methods 04/2007; 140(1-2):183-92. · 2.01 Impact Factor
• Article: Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.

  Haiyan Jiang, Linda B Couto, Susannah Patarroyo-White, Tongyao Liu, Dea Nagy, Joseph A Vargas, Shangzhen Zhou, Ciaran D Scallan, Jurg Sommer, Sharmila Vijay, Federico Mingozzi, Katherine A High, Glenn F Pierce
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  ABSTRACT: In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.
  Blood 12/2006; 108(10):3321-8. · 9.90 Impact Factor
• Article: Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5.

  Michael A Lochrie, Gwen P Tatsuno, Alejandra E Arbetman, Kris Jones, Cheryl Pater, Peter H Smith, Jennifer W McDonnell, Shang-Zhen Zhou, Shu Kachi, Michiko Kachi, Peter A Campochiaro, Glenn F Pierce, Peter Colosi
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  ABSTRACT: Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.
  Virology 10/2006; 353(1):68-82. · 3.35 Impact Factor
• Article: Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B.

  Haiyan Jiang, Glenn F Pierce, Margareth C Ozelo, Erich V de Paula, Joseph A Vargas, Peter Smith, Jürg Sommer, Alvin Luk, Catherine S Manno, Katherine A High, Valder R Arruda
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  ABSTRACT: In a phase I study, administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and FIX expression for at least 10 months after vector injection, the last time point assessed by muscle biopsy. In hemophilia B dogs we have demonstrated FIX in both muscle biopsies and circulation >4 years following AAV2-FIX injection. Because circulating FIX levels remained less than 1% of normal in human subjects from the study, the duration of AAV2-mediated transgene expression in humans is unknown. We sought to determine if FIX gene transfer and expression persisted locally at injection sites. Muscle biopsies were obtained from one subject 3.7 years following treatment and revealed transgene FIX DNA and protein by quantitative PCR, DNA fluorescence in situ hybridization, and immunohistochemistry for FIX. These results demonstrate, for the first time, multiyear FIX expression by AAV2 vector in humans and suggest that improved muscle delivery provides effective treatment for protein deficiencies or muscle-specific diseases.
  Molecular Therapy 10/2006; 14(3):452-5. · 6.87 Impact Factor
• Article: Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs.

  Haiyan Jiang, David Lillicrap, Susannah Patarroyo-White, Tongyao Liu, Xiaobing Qian, Ciaran D Scallan, Sandra Powell, Tracey Keller, Morag McMurray, Andrea Labelle, Dea Nagy, Joseph A Vargas, Shangzhen Zhou, Linda B Couto, Glenn F Pierce
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  ABSTRACT: Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.
  Blood 08/2006; 108(1):107-15. · 9.90 Impact Factor
• Source

  Available from: Ciaran D Scallan

  Article: Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice.

  Ciaran D Scallan, Haiyan Jiang, Tongyao Liu, Susannah Patarroyo-White, Jurg M Sommer, Shangzhen Zhou, Linda B Couto, Glenn F Pierce
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  ABSTRACT: Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.
  Blood 04/2006; 107(5):1810-7. · 9.90 Impact Factor
• Article: Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.

  Catherine S Manno, Glenn F Pierce, Valder R Arruda, Bertil Glader, Margaret Ragni, John J Rasko, John Rasko, Margareth C Ozelo, Keith Hoots, Philip Blatt, [......], Jürg M Sommer, Michael Tigges, Denise Sabatino, Alvin Luk, Haiyan Jiang, Federico Mingozzi, Linda Couto, Hildegund C Ertl, Katherine A High, Mark A Kay
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  ABSTRACT: We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.
  Nature Medicine 04/2006; 12(3):342-7. · 22.46 Impact Factor
• Source

  Available from: Ciaran D Scallan

  Article: Improved coagulation in bleeding disorders by Non-Anticoagulant Sulfated Polysaccharides (NASP).

  Tongyao Liu, Ciaran D Scallan, George J Broze, Susanna Patarroyo-White, Glenn F Pierce, Kirk W Johnson
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  ABSTRACT: Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, severe von Willebrand disease, and other rare factor deficiencies. A novel approach to improve coagulation in such clotting disorders has been identified that, parodoxically, involves heparinlike sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). A mechanism involving blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI) by NASPs, was conceived as an approach for improving procoagulant behavior in hemophilic settings. A subset of NASPs, including pentosan polysulfate (PPS) and fucoidan inhibited both full-length and Kunitz 1 and 2 (K1K2) TFPI and, at concentrations from 4-500 nM, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced factor VII levels when tested in dilute prothrombin time (dPT) assays. Fucoidan did not reduce normal plasma APTT times implying specificity for extrinsic pathway control. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Increased survival of factor deficient mice following a bleeding challenge was observed. Accordingly, administration of select NASP(s), via mechanism(s) not fully understood, represents a unique means of improving coagulation in bleeding disorders.
  Thrombosis and Haemostasis 02/2006; 95(1):68-76. · 5.04 Impact Factor
• Source

  Available from: ncbi.nlm.nih.gov

  Article: Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization.

  Michael A Lochrie, Gwen P Tatsuno, Brian Christie, Jennifer Wellman McDonnell, Shangzhen Zhou, Richard Surosky, Glenn F Pierce, Peter Colosi
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  ABSTRACT: Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.
  Journal of Virology 02/2006; 80(2):821-34. · 5.40 Impact Factor
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  Available from: ncbi.nlm.nih.gov

  Article: Novel caprine adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties.

  Alejandra E Arbetman, Michael Lochrie, Shangzhen Zhou, Jennifer Wellman, Ciaran Scallan, Mohammad M Doroudchi, Britta Randlev, Susannah Patarroyo-White, Tongyao Liu, Peter Smith, Howard Lehmkuhl, Lea Ann Hobbs, Glenn F Pierce, Peter Colosi
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  ABSTRACT: Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8 administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.
  Journal of Virology 01/2006; 79(24):15238-45. · 5.40 Impact Factor