Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-12-04
2001-12-04
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C514S04400A, C435S320100, C424S093600
Reexamination Certificate
active
06325998
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to DNA delivery methods. More particularly, the invention relates to the use of recombinant adeno-associated virus (AAV) virions for in vitro and in vivo delivery of erythropoietin (EPO) to muscle cells and tissue for the treatment of anemia. The method provides for sustained, high-level expression of EPO.
BACKGROUND OF THE INVENTION
The production of red blood cells in mammals, erythropoiesis, is under the control of the hormone erythropoietin (EPO). EPO is normally present in low concentrations in plasma, where it is sufficient to maintain equilibrium between normal blood cell loss (i.e., through aging) and red blood cell production.
Anemia is a decrease in red blood cell mass caused by decreased production or increased destruction of red blood cells. EPO is currently used for treatment of the anemias associated with end-stage renal failure and acquired immunodeficiency syndrome (AIDS), particularly in subjects who are being treated with zidovudine (AZT). EPO is also used for amelioration of the anemia associated with cancer chemotherapy.
Another group of anemic disorders, each of which results from an inherited abnormality in globin production, is termed the hemoglobinopathies. Hemoglobinopathies include a spectrum of disorders that can be classified broadly into two types. The first type are those that result from an inherited structural alteration in one of the globin chains, for example sickle cell anemia. These disorders give rise to the production of abnormal hemoglobin molecules. The second major subdivision of hemoglobinopathies, the thalassemias, results from inherited defects in the rate of synthesis of one or more of the globin chains. This causes ineffective erythropoiesis, hemolysis, and varying degrees of anemia due to the inadequate production of red blood cells. Accordingly, EPO can be used in the treatment of anemias, for example, hemoglobinopathies which are characterized by low or defective red blood cell production and/or increased red blood cell destruction.
&bgr;-thalassemia is a common single gene disorder arising from approximately 100 different mutations of the beta globin gene locus. Weatherall, D. J. (1994) in
The Molecular Basis of Blood Diseases
, Second Edition, Stamatoyannopoulos et al. eds., pages 157-205: WB Saunders, Philadelphia, Pa. The condition affects more than one million people worldwide. Current clinical management of severely anemic thalassemic patients generally involves transfusion with normal-matched donor blood, splenectomy, and other measures that help prolong circulating red blood cell survival. Schwartz et al. (1991) “The thalassemia syndromes,” in
Hematology: Basic Principals and Practice
, First Edition, Hoffman et al. eds., pages 368-392, Churchill Livingstone, New York, N.Y.
Sickle cell anemia is a condition resulting from the production of abnormal &bgr;-globin molecules due to an inherited mutation in the &bgr;-globin gene. The mutation results in the substitution of valine for glutamic acid in the &bgr;6 position of the molecule. Individuals homozygous for the abnormal &bgr;-globin gene (&bgr;
S
) are symptomatic. Additionally, individuals heterozygous for &bgr;
S
and heterozygous for other abnormal &bgr;-globin genes, for example, the &bgr;
C
gene or for &bgr;-thalassemia, may also have symptoms of sickle cell anemia. Manifestations of the disease are due to the fact that, under certain physiologic conditions, the abnormal hemoglobin molecule (HbS) polymerizes, resulting in “sickling” of red blood cells. The consequences of sickling of red blood cells are increased cell destruction, resulting in anemia, and blockage in capillary beds which can result in damage to a number of organs such as the kidney, spleen, lung, bones and eyes.
Recent advances in molecular biology have led to an understanding of the molecular interactions between the various globin chains and, based on this knowledge, pharmacological treatment using butyrate, chotrimazole, or hydroxyurea (De Franceschi et al. (1996)
Blood
87:1188-1195; Sauvage et al. (1993)
Br. J. Haematol
. 84:492) to increase globin concentration has been used in short-term clinical trials in small numbers of thalassemic patients to varying degrees of success. Olivieri et al. (1992)
Blood
80:3258; Rachmilewitz et al. (1991)
Blood
78:1145. The use of these pharmacological agents can mitigate the chain imbalance in beta-thalassemia and the precipitation of sickle hemoglobin in patients with sickle cell anemia. In a much smaller population of thalassemic patients, bone marrow transplantation of matched marrow has been shown to be curative (Thomas et al. (1982)
Lancet
2:227-229; Lucarelli et al. (1990)
N. Engl. J. Med
. 322:417-421), however, this approach is only available to a limited number of patients. Since each of these approaches benefit only a few patients and carry the risk of substantial side effects, including death, novel therapeutic strategies are needed.
One new therapeutic approach for the treatment of thalassemia involves the delivery of high levels of systemic erythropoietin (EPO) to patients in order to increase red blood cell mass and survival. Administration of human EPO has been shown to transiently increase levels of erythropoieis and circulating red blood cell mass in rodents, normal primates, and in a small number of experimental studies with human patients. Al-Khatti et al. (1988)
Trans. Assoc. Am. Physicians
101:54-61; Nagel et al. (1993)
Blood
81:9-14; Olivieri et al. (1992)
Blood
80:3258; Rachmilewitz et al. (1991)
Blood
78:1145; Leroy-Viard et al. (1991)
Blood
78:1596; Al-Khatti et al. (1987)
N. Engl. J. Med
. 317:415.
Administration of high doses of human EPO has shown efficacy in the treatment of sickle cell disease. For example, EPO has been shown to stimulate fetal hemoglobin formation in sickle cell anemia (Al-Khatti et al. (1988)
Trans. Assoc. Am. Physicians
101:54-61), and some subjects with sickle cell disease who were treated with high doses of EPO responded with increased percentages of F-reticulocytes in peripheral blood (nagel et al. (1993)
Blood
81:9-14). Further, high doses of EPO with adjunctive hydroxyurea has been shown to improve anemia in several sickle cell patients. Rodgers et al. (1993)
New Engl. J. Med
. 328:73.
Another new therapeutic strategy for the treatment of anemia entails gene therapy of hematopoietic stem cells, wherein transduced stem cells are used as a reservoir of gene-corrected cells. Williams et al. (1984)
Nature
310:476-480; Lemischka et al. (1986)
Cell
45:917-927. In particular, investigators have shown that murine bone marrow, transduced ex vivo by a retroviral vector containing the EPO gene, and then transplanted into lethally-irradiated thalassemic mice, can provide high levels of EPO secretion and improvement in the murine &bgr;-thalassemic phenotype in a portion of the reconstituted animals. Villeval et al. (1994)
Blood
84:928-933. However, several fundamental obstacles prevent gene therapy of hematopoietic stem cells from being realized. These obstacles include: unresolved problems with efficient delivery and integration into quiescent stem cells (Mulligan, R. C. (1993)
Science
260:926-932); vector design and stability (Takekoshi et al. (1995)
Proc. Natl. Acad. Sci. USA
92:3014-3018); transgene silencing (Challita et al. (1994)
Proc. Natl. Acad. Sci. USA
91:2567-2571) and efficient engraftment and repopulation of gene-modified stem cells (Kohn et al. (1995)
Nat. Med
. 1:1017-1023).
Other researchers have reported the transduction of myoblast cells with DNA encoding EPO in vitro without using viral vectors, and the subsequent transplantation of the transduced cells into a murine host. International Patent Publication WO 95/13376, published May 18, 1995. Yet further research has explored ex vivo transduction of cells using adenovirus vectors. The transduced cells are then used to form a matrix (an “organoid”) which is surgically implanted in the peritoneal cavity to secrete EPO. Naffakh et al. (1995)
Proc. Natl.
Kurtzman Gary J.
Podsakoff Gregory M.
Avigen, Inc.
Crouch Deborah
Robins & Pasternak LLP
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