Kinetics of Recombinant Adeno-Associated Virus-Mediated Gene Transfer

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to β-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 μg of hFIX/106 cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.

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Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes.

Blood ◽

10.1182/blood.v104.11.3985.3985 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3985-3985

Author(s):

Hengjun Chao ◽

Wei Chen ◽

James R. Tunstead ◽

Christopher E. Walsh

Keyword(s):

Gene Transfer ◽

Factor Ix ◽

Hemophilia B ◽

Cmv Promoter ◽

Adeno Associated Virus ◽

Immunodeficient Mice ◽

Human Factor Ix ◽

B Mice ◽

Knockout Animals ◽

Virion Particles

Abstract Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.

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Hemophilia B Gene Therapy Modelled in Null and Missense Mutant Mice: Kinetics of Transgenic Factor IX Expression Influences Tolerance to a Greater Extent Than Dose or Serotype of Adeno-Associated Virus (AAV) Vector.

Blood ◽

10.1182/blood.v106.11.5526.5526 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5526-5526

Author(s):

Paul E. Monahan ◽

Tai-Ping Zhang ◽

Da-Yun Jin ◽

Tong Gui ◽

Darrel W. Stafford

Keyword(s):

Gene Therapy ◽

Factor Ix ◽

Vector Particle ◽

Adeno Associated Virus ◽

Reactive Material ◽

Inhibitor Development ◽

Transgenic Protein ◽

Human Factor Ix ◽

Igg1 Subclass ◽

Kinetics Of

Abstract A major concern regarding the safety of gene therapy for protein deficiencies, including hemophilia, is the possibility of immune responses against the therapeutic transgenic protein. We have previously reported the use of hemophilic mice expressing defective missense human factor IX (hFIX) protein (Cross-reactive material positive, CRM +) or having a complete deletion of FIX product (CRM-) to examine the influence of the underlying mutation upon the risk of inhibitor development following intramuscular FIX gene therapy. When inhibitor antibodies have developed following adeno-associated virus (AAV) virus FIX gene therapy, the vector dose per injection site has been implicated as an important influence. To examine this, we treated CRM + missense R333Q-hFIX strain hemophilic mice with a single IM injection of AAV serotype2.hFIX. Doses of 1 x 1011 and 8 x 1011 vector genomes/animal resulted in increases of 50 ng/ml and 100 ng/ml hFIX, respectively, without inhibitor development. Identical vector doses given to the FIXKO CRM- strain resulted in inhibitors in all animals, predominantly IgG1 subclass, and zero circulating FIX. Using identical gene sequences and injection parameters, 1 x 1011 vg/animal of an AAV1 serotype hFIX vector was given to R333Q-hFIX mice and FIXKO mice. This resulted in physiologic hFIX levels in all animals at two weeks (1st timepoint examined), peaking at 3.9 μg/ml in FIXKO mice and 21.8 μg/ml in R333Q-hFIX mice. No mice developed inhibitors, despite the development of non-inhibitory IgG1 and IgG2 anti-factor IX. Despite subsequent challenge with repeated IV hFIX protein injections and with 1 x 1011 AAV2.hFIX, inhibitor antibodies could not be elicited in these animals. To examine the role of kinetics of trangene expression upon inhibitor development, stepwise decreases in the AAV1 vector dose were used, to try to reproduce the level of expression achieved in AAV2-treated animals that developed inhibitors [see Table]. At doses of 1 x 109 and 1 x 1010 AAV1hFIX, onset of expression was slow in R333Q-hFIX, averaging only10 ng/ml and 60 ng/ml above background at 2 weeks, and remaining below 300 ng/ml over months. Although lowering the vector dose of AAV1 reproduced the pattern of slow, low level transgene expression seen with the higher doses of AAV2, no inhibitors ever developed in the mice with CRM+ background. Nevertheless, FIXKO CRM- mice failed to achieve tolerance after the lower AAV1 doses. Neither the AAV1 serotype nor the vector particle number independently determined the inhibitor trigger. The results suggest the influence of the kinetics of onset and level of transgenic protein achieved are of primary importance in establishing tolerance in this application. Factor IX Expression and Inhibitor Formation Relative to Underlying FIX Mutation Mouse strain/Dose vector (vg/animal) FIX incr: 2 weeks (μg/ml) FIX incr: Peak (μg/ml) Bethesda Inhibitor (BIU range) R333Q, AAV2 1x10e11 0.01 0.05 0/5 mice (0 BIU) FIXKO, AAV2 1x10e11 0 0 5/5 mice (3–34 BIU) R333Q, AAV1 1x10e11 15.8 21.8 0/5 mice (0 BIU) R333Q, AAV1 1x10e10 0.06 0.25 0/4 mice (0 BIU) R333Q, AAV1 1x10e9 0.02 0.12 0/4 mice (0 BIU) FIXKO, AAV1 1x10e11 2.5 3.9 0/5 mice (0 BIU) FIXKO, AAV1 1x10e10 0.04 0.4 4/5 mice (2–16 BIU) FIXKO, AAV1 1x10e9 0 0 3/4 mice (2–6 BIU)

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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

Journal of Virology ◽

10.1128/jvi.79.24.15238-15245.2005 ◽

2005 ◽

Vol 79 (24) ◽

pp. 15238-15245 ◽

Cited By ~ 41

Author(s):

Alejandra E. Arbetman ◽

Michael Lochrie ◽

Shangzhen Zhou ◽

Jennifer Wellman ◽

Ciaran Scallan ◽

...

Keyword(s):

Protein Expression ◽

Neutralization Assay ◽

Biological Properties ◽

Scid Mice ◽

Factor Ix ◽

In Vivo Model ◽

Adeno Associated Virus ◽

Human Factor Ix

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-8administration 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.

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Hemophilia gene therapy comes of age

Blood Advances ◽

10.1182/bloodadvances.2017009878 ◽

2017 ◽

Vol 1 (26) ◽

pp. 2591-2599 ◽

Cited By ~ 28

Author(s):

Lindsey A. George

Keyword(s):

Clinical Trial ◽

Gene Therapy ◽

Gene Transfer ◽

Factor Ix ◽

Large Animal Model ◽

Large Animal ◽

Adeno Associated Virus ◽

Target Disease ◽

American Society ◽

Clinical Trial Results

Abstract Concurrent with the development of recombinant factor replacement products, the characterization of the F9 and F8 genes over 3 decades ago allowed for the development of recombinant factor products and made the hemophilias a target disease for gene transfer. The progress of hemophilia gene therapy has been announced in 3 American Society of Hematology scientific plenary sessions, including the first “cure” in a large animal model of hemophilia B in 1998, first in human sustained vector-derived factor IX activity in 2011, and our clinical trial results reporting sustained vector-derived factor IX activity well into the mild or normal range in 2016. This progression to clinically meaningful success combined with numerous ongoing recombinant adeno-associated virus (rAAV)–mediated hemophilia gene transfer clinical trials suggest that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized. Although several novel therapeutics have recently emerged for hemophilia, gene therapy is unique in its potential for a one-time disease-altering, or even curative, treatment. This review will focus on the prior progress and current clinical trial investigation of rAAV-mediated gene transfer for hemophilia A and B.

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Neonatal gene transfer with a retroviral vector results in tolerance to human factor IX in mice and dogs

Blood ◽

10.1182/blood-2003-06-2181 ◽

2004 ◽

Vol 103 (1) ◽

pp. 143-151 ◽

Cited By ~ 36

Author(s):

Jun Zhang ◽

Lingfei Xu ◽

Mark E. Haskins ◽

Katherine Parker Ponder

Keyword(s):

Gene Transfer ◽

Antibody Formation ◽

Human Factor ◽

Retroviral Vector ◽

Factor Ix ◽

High Dose ◽

Human Factor Ix ◽

B Mice ◽

Different Strains ◽

Induce Antibody

Abstract The effect of neonatal gene transfer on antibody formation was determined using a retroviral vector (RV) expressing human factor IX (hFIX). Normal mice from different strains injected intravenously with RV as newborns achieved therapeutic levels of hFIX without antibody production and were tolerant as adults to challenge with hFIX. Neonatal hemophilia B mice that received different amounts of RV achieved stable and dose-related expression of hFIX without anti-hFIX antibody formation. After protein challenge, antibody formation was markedly reduced for animals that expressed hFIX at levels higher than 14 ng/mL (0.3% of normal). However, antibodies developed for animals that received the lowest dose of RV and expressed hFIX at approximately 2 ng/mL before protein challenge. In dogs, neonatal injection of a high dose of RV resulted in 500 ng/mL hFIX in plasma without antibody formation. We conclude that neonatal gene transfer with RV does not induce antibody responses to hFIX in mice or dogs and that mice achieving levels greater than 3 × 10–10 M hFIX are usually tolerant to protein injection as adults. Low-dose gene therapy or frequent protein injections in the neonatal period might induce tolerance to subsequent injections of protein with a low risk for adverse effects.

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Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

Blood ◽

10.1182/blood.v116.21.3765.3765 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3765-3765 ◽

Cited By ~ 2

Author(s):

Jonathan D. Finn ◽

Patricia Favaro ◽

J. Fraser Wright ◽

Federico Mingozzi ◽

Katherine A. High ◽

...

Keyword(s):

T Cells ◽

Gene Transfer ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Rescue Therapy ◽

Factor Ix ◽

Large Animal ◽

Solid Organ ◽

Large Animal Models ◽

Human Factor Ix

Abstract Abstract 3765 Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue' agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures: High: Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

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Dose response results of self complementary adeno-associated virus (AAV) vector-mediated factor IX gene transfer in non-human primates

Haemophilia ◽

10.1111/j.1365-2516.2009.01996_6.x ◽

2009 ◽

Vol 15 (2) ◽

pp. 635-635

Author(s):

ULRIKE REISS ◽

ANDREW DAVIDOFF ◽

JOHN GRAY ◽

ARTHUR NIENHUIS ◽

AMIT NATHWANI

Keyword(s):

Gene Transfer ◽

Dose Response ◽

Factor Ix ◽

Aav Vector ◽

Adeno Associated Virus

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Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

Journal of Experimental Medicine ◽

10.1084/jem.179.6.1867 ◽

1994 ◽

Vol 179 (6) ◽

pp. 1867-1875 ◽

Cited By ~ 82

Author(s):

S Z Zhou ◽

S Cooper ◽

L Y Kang ◽

L Ruggieri ◽

S Heimfeld ◽

...

Keyword(s):

Gene Transfer ◽

Progenitor Cells ◽

Umbilical Cord Blood ◽

Recombinant Virus ◽

High Efficiency ◽

Hematopoietic Progenitor Cells ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Hematopoietic Progenitor ◽

Adeno Associated Virus

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

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Functional Pan-Universality of the Age-Related Regulatory Mechanisms of Gene Expression.

Blood ◽

10.1182/blood.v104.11.1943.1943 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1943-1943

Author(s):

Kotoku Kurachi ◽

Jean-Marc Fontaine ◽

Takahiro Abe ◽

Sumiko Kurachi

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Critical Issue ◽

Factor Ix ◽

Expression Vectors ◽

Gene Expressions ◽

Cmv Promoter ◽

Age Related ◽

Time Period ◽

Human Factor Ix

Abstract Through analyses of human factor IX (hFIX) and protein C (hPC) gene expressions in transgenic mice, we recently discovered the first molecular mechanism of age-dimension gene regulation, involving two critical genetic elements ASE and AIE required for age-related stable and increase patterns of gene expression, respectively. The next most critical issue to be tested was whether or not these elements, particularly ASE due to its possible utility, similarly functions with grossly different genes. We constructed hFIX expression vectors carrying a CMV promoter with or without ASE, and tested them in transgenic mice. Vectors with no ASE showed an age-dependent gradual decrease in hFIX expression, reaching the background levels in 3–9 months. Vectors with ASE, however, showed age-stable hFIX expression over the same time period. These findings supported the pan-universality of ASE function. As we previously reported, ASE regulates gene expression not only temporally but also spacially. With ASE, CMV-driven expression showed the highest in the heart muscle, whereas without ASE, the highest in the skeletal muscle. These results led to development of the Age Dimension Technology, a new field for exploring the unique applications of the age-related knowledge.

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AAV8-Factor IX Hepatic Gene Transfer with Transient Immunosuppression Is Safe but Is Abrogated by Low Titers of Neutralizing Antibody in Rhesus Macaques.

Blood ◽

10.1182/blood.v106.11.5532.5532 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5532-5532

Author(s):

Haiyan Jiang ◽

Susannah Patarroyo-White ◽

Tongyao Liu ◽

Bin Yang ◽

Dea Nagy ◽

...

Keyword(s):

Gene Transfer ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Small Animal ◽

Neutralizing Antibody ◽

Cross Reactivity ◽

Factor Ix ◽

Post Surgery ◽

Short Course ◽

Human Factor Ix

Abstract AAV8 vectors have potential in human gene transfer due to improved transduction efficiencies as demonstrated in small animal models, and are also reported to show less cross-reactivity to AAV2 neutralizing antibodies prevalent in humans. To assess this hypothesis, the efficacy and safety of AAV8-human Factor IX (hFIX) was evaluated in rhesus macaques. At doses of 5–20x1012 vg/kg, AAV8-hFIX achieved therapeutic to supraphysiological levels of circulating human FIX via intra-hepatic artery delivery in AAV8-naïve macaques. However, transduction was abrogated in macaques with pre-existing AAV8 neutralizing antibodies at titers as low as 1:5. Intrahepatic delivery of AAV8-hFIX resulted in gene transfer primarily in liver, and was well distributed among individual hepatic lobes. Two macaques that experienced traumatic catheterization developed immediate and delayed (week 4–6) transient transaminitis post surgery. Nevertheless, a significant percentage of AAV8-hFIX transduced hepatocytes persisted despite the liver damage and partial loss of hFIX expression. Transduction was well tolerated in all other macaques. Transient immunosuppression with tacrolimus and mycophenolate mofetil did not impair AAV8-hFIX transduction. The results [1] support AAV8-mediated gene transfer in humans but indicate that both the efficacy achieved and the resistance to pre-existing neutralizing antibodies of AAV8-hFIX are unlikely to be significantly enhanced over AAV2 vector, [2] suggest that a peri-operative insult and likely triggering of innate immunity may result in a later inflammatory response that results in a decrease in transgene protein production, and [3] provide evidence that a short course of immunosuppression does not alter liver transduction by AAV vectors.

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