Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-13
2003-03-04
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C530S324000, C514S04400A
Reexamination Certificate
active
06528287
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to biological structures designed to induce apoptosis in HIV-1 infected cells, and more specifically to recombinant human serum transferrins designed to introduce apoptosis-inducing peptides into HIV-1 infected cells.
2. Description of the Prior Art
The preservation of equilibrium in a multicellular organism, the process of homeostasis, requires a delicate balance between cell proliferation and cell death. In a well developed, adult organism the maintenance and renewal of many specialized cell lines is a matter of continuing importance. Therefore, cell proliferation and in particular its tight checks and balances necessary for optimal adult health has been a process of keen interest to those involved in biological research. Accordingly, the study of infectious diseases and more recently the focused inspection of mechanisms associated with cancer have followed these adult self-preservation paths, expanding our knowledge of the many factors involved in cell proliferation—where some of the more recent research has elucidated the importance of growth factors, proto-oncogenes, and tumor suppressors such as p53.
Over the whole life cycle the viability of an organism is maintained by a continuous balance between cell proliferation and cell death by necrosis or apoptosis. While the necessity of cell suicide in an organized biological system has been well appreciated, it is only recently that it was given substantial research attention as a therapeutic mechanism.
For example, the study of Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV) has revealed that uninfected CD4+ T cells are induced to undergo apoptosis by adjacent HIV-infected cells. It appears that the survival strategy of this virus depends in part on causing mass suicide in the ranks of its primary immunological opponent, the T lymphocyte, while remaining concealed in the suicide inducer, the infected cell. See, e.g., Meyaard, L. et al. (1992)
Science
257:217; Groux, H. et al. (1992)
J. Exp. Med.
175:331; Banda, N. K. et al. (1992)
J. Exp. Med.
176:1099. Since apoptosis, unlike necrotic cell death, does not normally evoke inflammation or a strong immune response, this viral stratagem is particularly successful; the disease remains essentially symptom free until opportunistic infections are allowed to take hold as a result of depleted T cell ranks.
The opposing therapeutic stratagems need equal brilliance. Following the clandestine path of this virus, the use of apoptosis induction as a therapeutic tool is attractive as it can avoid potentially harmful inflammatory and immune system responses. The insidious nature of the HIV process, its current prevalence, and its essentially asymptomatic onset are best opposed by equally asymptomatic response mechanisms in any therapeutic regimen. Accordingly, therapeutic mechanisms that induce apoptosis in HIV infected cells are universally desired, and it is one such mechanism that is disclosed herein.
Those in the field will appreciate that recent (1999) estimates by the World Health Organization (WHO) indicate that there are no less than 33.6 million people living with HIV/AIDS. Recently it has been observed and reported that the combination of simultaneous protease and reverse transcriptase inhibitors does not confer resistance to HIV. See Michael, N. and Moore, J. (1999)
Nature Medicine
5:7, 740-741. The infective nature of this disease offers only somber future statistics. Accordingly, various new drugs to combat this epidemic have been proposed, including those based on cleavage by HIV-1 protease, such as the toxic pro-drugs activated inside the infected cell (the Trojan Horse strategy), peptide drugs and others.
At the same time the study of natural transport proteins has revealed the unique attributes of the human serum transferrin as a delivery mechanism, and its concurrent convenience for recombinant manipulation. For example, U.S. Pat. No. 5,986,067 to Funk et al. (1999) teaches the usefulness of recombinant transferrin proteins as vehicles for metal chelation while Ali, S. A. et al. (1999)
J. Biol. Chem.
274:34, 24066-24073 describe the insertion of a peptide sequence into the N-terminal lobe of human serum transferrin via cassette mutagenesis and PCR-ligation-PCR mutagenesis techniques.
When Ali et al. inserted a peptide into the N-terminal lobe of human serum transferrin (HST), they demonstrated that the recombinant HST retained its native functions, including mediation of iron transport and uptake into cells. Human serum transferrin is a monomeric glycoprotein that binds tightly and reversibly to two ferric ions together with two bicarbonate co-ions. The roles of HST are 1) the regulation of the availability of free iron in body fluids and 2) mediation of the transport and uptake of iron into cells via receptor-mediated endocytosis. Even though all living cells need iron, only those cells which have high iron requirement (i.e. HIV-infected cells and cancer cells) express large numbers of transferrin receptors. See lacopetta et al. (1982)
Biochem. Biophys. Acta.
687:204-210. Significantly, HIV-1 replication has been shown to induce up-regulation of HST receptor expression, and therefore HIV-1 infected cells are particularly susceptible to an HST-directed therapy. The use of recombinant human serum transferrin is therefore uniquely suitable for the process disclosed herein.
An essential step in the life cycle of human immunodeficiency virus type-1 (HIV-1) is the processing of the Gag and Gag-Pol polyproteins which is performed by the virus encoded HIV-1 protease. The cleavage of viral polyproteins by HIV-1 protease results in structural and catalytic proteins required for both viral maturation and infection, and therefore HIV-1 protease has been a target of anti-AIDS drugs. See Friedler, A. et al. (1999)
J. Mol. Biol.
287:93-101. One novel approach that has been recently examined is the use of toxic prodrugs which are cleaved selectively in cells infected by HIV-1—often called the “Trojan Horse” strategy. A chimeric Vpr protein (an auxiliary HIV-1 gene product) which contained at its C-terminus nine HIV-1 protease cleavage sites was shown to completely abolish viral infectivity as cleavage of the chimeric protein interfered with the processing of viral precursor proteins—leading to the production of incompletely processed noninfectious virus particles. See Serio, D. et al. (1997)
Proc. Natl. Acad. Sci. USA
94:3346-3351. The current invention described herein combines the Trojan Horse strategy with recent knowledge of the existence of peptides which cause cell suicide. The proposed recombinant human serum transferrins would inhibit HIV-1 by acting as competitive substrates for HIV-1 protease and in so doing would also selectively release “cell suicide”
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peptides in the HIV-1 infected cell. Therefore, the proposed invention endeavors to not only inhibit the virulence of HIV-1 but also to eradicate those cells infected by HIV-1.
SUMMARY OF THE INVENTION
Accordingly it is the general purpose and objective of the present invention to design a recombinant mammalian protein directed at seizing the normal process of apoptosis or “cell suicide” as a therapeutic stratagem.
Further objects of the invention are to create various recombinant human serum transferrin proteins containing one of several peptides flanked by two HIV-1 protease cleavage sites and possessing an apoptosis-inducing motif.
Other objectives of the invention are to provide a modification of a protein structure generally functional in basic biological processes whereby the protein modification includes apoptotic peptide segments inserted between cleavage sites selected for a targeted infection.
Yet further objectives of the invention are to create recombinant modifications of a natural transport protein so as to conform it to induce cell apoptosis in an HIV-1 infected cell.
Additional objectives of the invention are to insert an apoptotic mammalian peptide which may be r
Bak-Boychuk Michael
Park Hankyel T.
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