Evidence for Contribution of CD4+CD25+ Regulatory T Cells in Maintaining Immune Tolerance to Human Factor IX following Perinatal Adenovirus Vector Delivery

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequently observed; however the underlying mechanisms remain largely undefined. In this study fetal and neonatal BALB/c mice received adenovirus vector to deliver human factor IX (hFIX) cDNA. The long-term tolerance of hFIX was robust in the face of immune challenge with hFIX protein and adjuvant but was eliminated by simultaneous administration of anti-CD25+ antibody. Naive irradiated BALB/c mice which had received lymphocytes from donors immunised with hFIX developed anti-hFIX antibodies upon immune challenge. Cotransplantation with CD4+CD25+ cells isolated from neonatally tolerized donors decreased the antibody response. In contrast, cotransplantation with CD4+CD25− cells isolated from the same donors increased the antibody response. These data provide evidence that immune tolerance following perinatal gene transfer is maintained by a CD4+CD25+ regulatory population.

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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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Mapping of genes that control the antibody response to human factor IX in mice

Blood ◽

10.1182/blood-2004-03-1126 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1029-1035 ◽

Cited By ~ 23

Author(s):

Jay N. Lozier ◽

Nahid Tayebi ◽

Pei Zhang

Keyword(s):

Antibody Response ◽

Human Factor ◽

Inbred Mouse ◽

Interleukin 10 ◽

Factor Ix ◽

Control Factor ◽

Mouse Strains ◽

Inbred Mouse Strains ◽

Adenoviral Gene Transfer ◽

Human Factor Ix

AbstractWe tested the hypothesis that the antibody response to human factor IX in mice is controlled by genetic factors, especially histocompatibility antigens. Seven inbred mouse strains were immunized against human factor IX by adenoviral gene transfer or serial injections of human factor IX protein. A/J mice had the highest antibody response and 2 C57 mouse strains had the lowest response. We used the adenovirus vector to immunize 26 recombinant inbred mouse strains (AXB and BXA) derived from A/J and C57BL/6J mice and observed highly significant linkage (logarithmic odds [LOD] scores ∼4.8) for the polymorphic D17Mit62 marker that is 1 centimorgan (∼300 000 base pair [bp]) from the mouse major histocompatibility complex (MHC) locus (H-2). Experiments in mice with chimeric MHC genes indicated that class IaK or class II H-2 (or both) genes were critical, but other genes contributed to the antibody response. Polymorphic markers from chromosomes 1 and 10 that are near important immunoregulatory genes such as interleukin 10 and the interferon-γ gene show suggestive linkage (LOD scores of ∼2.3-2.6) to the factor IX antibody response. This study confirms the hypothesis that H-2 (and other) genes control factor IX antibody development in mice and suggests their potential importance for factor IX antibody development in humans with hemophilia B.

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In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor

Blood ◽

10.1182/blood-2002-03-0779 ◽

2003 ◽

Vol 101 (4) ◽

pp. 1359-1366 ◽

Cited By ~ 82

Author(s):

Simon N. Waddington ◽

Suzanne M. K. Buckley ◽

Megha Nivsarkar ◽

Sarah Jezzard ◽

Holm Schneider ◽

...

Keyword(s):

Immune Tolerance ◽

Human Factor ◽

Adult Life ◽

Factor Ix ◽

Clotting Factor ◽

Target Cells ◽

Proliferating Cells ◽

Disease Manifestation ◽

Adult Mice ◽

Human Factor Ix

The fundamental hypotheses behind fetal gene therapy are that it may be possible (1) to achieve immune tolerance of transgene product and, perhaps, vector; (2) to target cells and tissues that are inaccessible in adult life; (3) to transduce a high percentage of rapidly proliferating cells, and in particular stem cells, with relatively low absolute virus doses leading to clonal transgene amplification by integrating vectors; and (4) to prevent early disease manifestation of genetic diseases. This study provides evidence vindicating the first hypothesis; namely, that intravascular prenatal administration of an adenoviral vector carrying the human factor IX (hFIX) transgene can induce immune tolerance of the transgenic protein. Following repeated hFIX protein injection into adult mice, after prenatal vector injection, we found persistence of blood hFIX and absence of hFIX antibodies in 5 of 9 mice. Furthermore, there was substantial hFIX expression after each of 2 reinjections of vector without detection of hFIX antibodies. In contrast, all adult mice that had not been treated prenatally showed a rapid loss of the injected hFIX and the development of high hFIX antibody levels, both clear manifestations of a strong immune reaction.

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Persistent Systemic Production of Human Factor IX in Mice by Skeletal Myoblast-Mediated Gene Transfer: Feasibility of Repeat Application to Obtain Therapeutic Levels

Blood ◽

10.1182/blood.v90.3.1075 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1075-1082 ◽

Cited By ~ 34

Author(s):

Jian-Min Wang ◽

Hong Zheng ◽

Mila Blaivas ◽

Kotoku Kurachi

Keyword(s):

Gene Transfer ◽

Human Factor ◽

Plasmid Vector ◽

Systemic Circulation ◽

Scid Mice ◽

Factor Ix ◽

Therapeutic Level ◽

Muscle Creatine Kinase ◽

Human Factor Ix

Abstract Myoblast-mediated gene transfer and its repeated applications were tested for achieving a long-term stable systemic production of human factor IX (hFIX) at a therapeutic level in SCID mice. Primary skeletal myoblasts were stably transfected with a hFIX expression plasmid vector, pdLMe4βAhIXm1, which contains a hFIX minigene under the control of a β-actin promoter with muscle creatine kinase enhancers. Myotubes derived from the myoblasts produced 1,750 ng hFIX/106 cells/24 hours in culture. hFIX secretion by the myoblasts and thereof derived myotubes were equally efficient, and myotubes were shown to have a sufficient secretory capacity to handle a substantially elevated production of hFIX. After intramuscular injection of 5, 10, and 20 × 106 myoblasts, SCID mice stably produced hFIX into the systemic circulation proportional to the number of implanted cells, and the expression levels were maintained for at least up to 10 months (end of the experiment). Additional cell injections administered to animals that originally received 10 × 106 cells approximately 2 months later elevated the systemic hFIX levels to an average of 182 ± 21 ng/mL, a therapeutic level, which persisted for at least 8 months (end of the experiment). These results indicate that long-term, stable systemic production of hFIX at therapeutic levels can be achieved by repeated application of myoblast-mediated gene transfer.

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