Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-10-16
2002-04-02
Brusca, John S. (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023500
Reexamination Certificate
active
06365575
ABSTRACT:
INTRODUCTION
The present invention provides novel methods for gene delivery and expression in areas that are currently inaccessible through the use of conventional direct protein delivery techniques. In particular, the methods and related products provided herein can be used in the treatment of pulmonary disorders and delivery of anti-viral proteins.
BACKGROUND OF THE INVENTION
The following review of the background of the invention is merely provided to aid in the understanding of the present invention and neither it nor any of the references cited within it are admitted to be prior art to the present invention.
There are presently several approaches being studied for delivering genes to humans. These approaches have either: (i) removed somatic cells, permanently transformed them in vitro using retrovirus vectors, and reinfused the transformed cells; or (ii) used viruses to deliver the gene. Such therapy has been designed for use with patients having inherited deficiency of gene products, such as proteins, due to abnormalities of the gene during development. Such therapies have also been used with disorders such as emphysema, wherein it is thought that the disease process is a result of a relative deficiency of an antiprotease over a long period of time. Further, diseases such as acute lung injury resulting in the adult respiratory distress syndrome (ARDS) are thought to involve a relative deficiency of antiprotease activity. In addition, cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians (Boat, T. F. et al.,
The Metabolic Basis of Inherited Disease
2649-2680, 1989). Even though CF can affect several organ systems, almost all patients develop chronic obstructive pulmonary disease and chronic pulmonary infections with resultant respiratory failure and early death.
Normally, the lung contains sufficient quantities of serine antiproteases, principally &agr;
1
antitrypsin (&agr;
1
AT), to combat the effects of toxic substances involved in such diseases, such as neutrophil elastase (NE). However, in CF and other neutrophil-dominated inflammatory lung diseases, the antiprotease defense system fails to prevent proteolytic damage to lung tissue (Boat, T. F. et al.,
The Metabolic Basis of Inherited Disease
2649-2680, 1989; Richman-Eisenstat, J. B. Y. et al.,
Am. J. Phys
. 264: L413-L418, 1993).
One attempt at solving some of the above problems is described in International Patent Publication WO 92/19730 (hereby incorporated in its entirety, including any drawings) which describes means for the delivery of a gene encoding human &agr;
1
antitrypsin to the lungs for expression of the human &agr;
1
antitrypsin capable of alleviating the enzyme deficiency. Further advances regarding cationic liposome mediated antiprotease gene transfer to reduce the infectivity of RSV in cultured cells is reported in M. Persmark et al.,
J. Investig. Med
. 43 S:220, 1995 Other attempts to deliver particular genes to cells of the lung or airway are described in International Patent Applications with publication numbers WO 93/12756 and WO 93/12240, both of which are incorporated herein by reference in their entirety including any drawings.
Despite the progress and success that has been achieved by such attempts, there still remains a need for a general method to provide gene delivery and expression to areas currently inaccessible to direct protein delivery. See R. C. Hubbard, et al. P.N.A.S. 86:680-684, 1989 (describing attempt to deliver protein via an aerosol and stating “In the present study, ≈{fraction (1/500)}th of the administered dose was recoverable in lung lymph, a value likely too low to provide adequate protection for &agr;
1
-antitrypsin deficiency”). Such obstacles have previously prevented the successful in vivo treatment of alpha antitrypsin (hereinafter “AAT”) related disorders such as congenital AAT deficiency, as well as other disorders related to other proteins (for example, prostaglandin (hereinafter “PG”) synthase see U.S. patent application Ser. No. 08/459,493, filed Jun. 2, 1995 incorporated herein by reference in its entirety including any drawings) that could not previously be treated by direct protein delivery to the desired target area using conventional methods of protein delivery.
SUMMARY OF THE INVENTION
The present invention is based in part on the surprising discovery that certain genes (preferred are genes encoding antiproteases such as AAT) can be delivered and expressed in vivo to certain target areas in animals (preferably mammals, more preferably humans) which have previously been inaccessible (i.e., an insufficient amount or inappropriate form of the protein is able to be provided to give a therapeutic response) to direct protein delivery.
A significant and unexpected advantage of the present invention is the ability of the delivered gene to provide a therapeutic response in situations where direct delivery of the protein (even when delivered at several fold higher serum concentrations) does not produce a therapeutic effect. In particular, delivery of the AAT gene has been found to essentially eliminate endotoxin induced increase in pulmonary vascular resistance, even when serum protein levels are approximately 20 fold lower than produced by protein delivery, which, (in contrast) produces no discernable effect on endotoxin response. In addition, delivery of the AAT gene is able to prevent respiratory syncytial virus infection of lung epithelial cells, where direct delivery of the encoded protein does not. Thus, such methods provide a method for successfully treating disorders caused by a deficiency of the product encoded by the gene of interest in the target area in the particular organism suffering from such a disorder.
The &agr;
1
-antitrypsin protein is the major antiprotease in the lungs of humans. It is believed that both acute lung injury associated with inflammation and emphysema are a consequence of protease/antiprotease imbalance and increasing the antiprotease activity in the lungs is one possible approach to prevention and therapy of these conditions. In addition, there is a genetic form of &agr;
1
-antitrypsin deficiency where patients develop emphysema at an early age.
Patients with &agr;
1
-antitrypsin deficiency are now being treated with intravenous administration of the &agr;-antitrypsin protein. This intervention is expensive, requires relatively frequent intravenous infusions, can cause reactions in the patient and is of unproven efficacy. In addition, since the protein is derived from human blood products, a risk of infection by contaminating viruses, such as HIV is present. Gene therapy by delivery of the DNA encoding &agr;
1
-antitrypsin to the airway cells could provide a less invasive, safer, cheaper and more effective therapy.
Thus, in one aspect the invention provides a method for delivering a nucleic acid molecule to a location in an animal that is inaccessible to direct protein delivery. Those skilled in the art will understand that several conventional methods for directly delivering proteins exist and are commonly used in the art. The method involves the step of administering a positively charged liposome to the animal. The positively charged liposome is associated with the nucleic acid molecule, wherein said nucleic acid molecule is in operable association with a promoter. Those skilled in the art will recognize that a wide variety of promoters may be used to assist in targeting the desired location.
In preferred embodiments, the nucleic acid molecule encodes human &agr;
1
antitrypsin, the location is selected from the group consisting of an endothelial lung cell, a smooth muscle cells adjacent to the endothelial lung cell, and lung parenchyma, or the location is selected from the group consisting of a liver cell, a muscle cell, an osteogenic cell, synoviocyte, and a lung cell. Other preferred locations and genes are shown in Table I below.
TABLE I
Clinical Indication
Gene
Target
Musculoskeletal
Muscle reconstruction and
IGF-1
muscle
rehabilitation
Cachexia (muscle wasting)
IGF-1, GH1 a HEH
muscle, liv
Brigham Kenneth
Canonico Angelo
Meyrick Barbara
Stecenko Arlene
Brusca John S.
Needle & Rosenberg P.C.
Vanderbilt University
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