Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-02-10
2002-06-11
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S456000, C435S457000
Reexamination Certificate
active
06403370
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of adenoviral vectors and their use in treating disease.
BACKGROUND OF THE INVENTION
Basic Adenoviral Vector Technology
Adenoviruses (Ad) consist of nonenveloped icosahedral (20 facets and 12 vertices) protein capsids with a diameter of 60-90 nm and inner DNA/protein cores (Horwitz, 1990). The outer capsid is composed of 252 capsomers arranged geometrically to form 240 hexons (12 hexons per facet) and 12 penton bases; the latter are located at each vertex from which protrude the antennalike fibers. This structure is responsible for attachment of Ad to cells during infection. Wild-type Ad contain 87% protein and 13% DNA and have a density of 1.34% g/ml in CsCl.
The double-stranded linear DNA genome of Ad is approximately 36 kb, and is conventionally divided into 100 map units (mu). Each end of the viral genome has a 100-150 bp repeated DNA sequence, called the inverted terminal repeats (ITR). The left end (194-385 bp) contains the signal for encapsidation (packaging signal). Both the ITRs and the packaging signal are cis-acting elements necessary for adenoviral DNA replication and packaging (Sussenbach, 1984; Philipson, 1984).
A simplified map of the adenovirus type 5 (Ad5) genome with a few key landmarks is diagrammed in
FIG. 1
(Stratford-Perricaudet and Perricaudet, 1991; Graham and Prevec, 1991). The early (E) and late (L) regions of the genome contain several transcription units and are divided according to the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome as well as a few cellular genes (Nevins, 1993). The expression of the E2 region (E2A and E2B) leads to the synthesis of the proteins needed for viral DNA replication (Pettersson and Roberts, 1986). The proteins from the E3 region prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Wold and Gooding, 1991). The E4 proteins are involved in DNA replication, late gene expression and splicing, and host cell shut-off (Halbert et al, 1985). The products of the late genes, including the majority of the viral capsid proteins, are expressed after processing of a 20-kb primary transcript driven by the major late promoter (MLP) (Shaw and Ziff, 1980). The MLP (located at 16.8 mu) is particularly efficient during the late phase of infection, and the mRNAs issued from this promoter possess a 5′ tripartite leader (TL) sequence, which increases the preference of the host cell for such transcripts as opposed to host cell mRNAs.
The use of Ad as vectors for expression of heterologous genes began soon after the observation of hybrids between Ad and simian virus 40 (SV40) during the 1960s. Since then, Ad vectors have gradually developed into one of the major viral vectors in the current field of gene therapy, because: (a) Ad have been widely studied and well characterized as a model system for eukaryotic gene regulation, which served as a solid base for vector development; (b) The vectors are easy to generate and manipulate; (c) Ad exhibits a broad host range in vitro and in vivo with high infectivity, including non-dividing cells; (d) Ad particles are relative stable and can be obtained in high titers, e.g., 10
10
-10
12
plaque-forming unit (PFU)/ml; (e) The life cycle of adenovirus does not require integration into the host cell genome, and, therefore, the foreign genes delivered by Ad vectors are expressed episomally, thus having low genotoxicity if applied in vivo; (f) Side effects have not been reported following vaccination of U. S. recruits with wild-type Ad, demonstrating their safety for in vivo gene transfer. Ad vectors have been successfully used in eukaryotic gene expression (Levrero et al, 1991; Ghosh-Choudhury, 186), vaccine development (Granhaus and Horwitz, 1992; Graham and Prevec, 1992), and gene transfer in animal models (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1992; Rich et al, 1993). Experimental routes for administrating recombinant Ad to different tissues in vivo have included intratracheal instillation (Rosenfeld et al, 1992), muscle injection (Quantin et al, 1992), peripheral intravenous injection (Herz and Gerard, 1993), and stereotactic inoculation to brain (LaSalle et al, 1993). The initial Ad-mediated gene therapy trial in humans was the transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to lung tissues (Crystal et al, 1994).
Gene-Transfer Mediated Anticancer Immunity
One of the most effective current approaches to cancer gene therapy involves alteration of the tumor-host relationship and facilitation of recognition and destruction of malignant cells by the host immune system. In the tumor-bearing individual, a lack of an effective immune response may be due in part to either weak tumor cell antigenicity, lack of immune co-stimulation, or a tumor-specific immunosuppressive environment. Gene transfer of cytokines to tumor cells provides a strategy for augmentation of an effective anti-tumor immune response (Miller et al, 1994). In recent years, a number of cytokine genes have been isolated, cloned and characterized. Systemic administration of certain of these immunomodulators, such as IL-2, has resulted in an anti-tumor response. However, significant toxicity has accompanied the use of many of these biologics owing to the high concentrations needed to generate clinical effects. The combination of significant undesired effects and marginal therapeutic outcomes from systemic administration has stimulated efforts to genetically engineer tumor cells to produce the cytokines themselves (Rosenberg et al, 1989).
In animal models, gene-modified tumor cells have been used as vaccines to stimulate anti-tumor response (Miller et al, 1994; Dranoff and Mulligan, 1995). The appeal of tumor directed cytokine gene transfer is that the cytokine, produced locally, is immunologically more efficient and does not cause systemic toxicity. Tumor antigens expressed on neoplastic cells in combination with high local concentrations of cytokine(s), creates an immunological micro-environment virtually impossible to reproduce with exogenous cytokine administration. This immunological micro-environment created by such cytokine-producing tumor cells has been shown to result in generation of cytotoxic T lymphocytes. In a number of different animal models, cytokine-producing tumor cells have been shown to be effective in decreasing the tumorgenicity and increasing the expression of immunologically important molecules (Miller et al, 1994; Dranoff and Mulligan, 1995). The initial antitumor rejection appears to be accompanied by a nonspecific inflammatory response. However, rejection of cytokine secreting tumor cells has in most instances led to the generation of systemic, tumor specific immunity that is T cell-dependent.
In addition, new evidence indicates that co-stimulation of T cells by the co-stimulatory molecule B7 has both a positive and negative effect on T cell activation (Leach et al, 1996). Other co-stimulatory molecules for T cells such as ICAM-I, LFA-3 and VCAM-I have also been implicated in the induction of an anti-tumor response (Springer, 1990). The most powerful of these co-stimulatory signals is provided by the interaction of CD28 on a T cell with either or both of its primary ligands, B7-1 (CD80) and B7-2 (CD86) on the surface of an antigen presenting cell (Lenschow et al, 1996). In a variety of model systems, tumor cells transfected with the B7 cDNA induced potent antitumor responses against both modified and unmodified tumor cells (Townsend and Allison, 1993)
It has long been known that both Class I and Class II MHC molecules are involved in the tumor antigen presentation, although different pathways are utilized by the two classes of molecules. Class I MHC has been shown to activate tumor-specific CTL in vitro. Early work on tumor vaccination included transfection of MHC class I genes and resulted in suppression of the tumor cells in tumorigenicity and/or metastasis in mouse models (Hui et al, 19
Alemany Ramon
Fang Xiangming
Zhang Wei-Wei
GenStar Therapeutics Corporation
Guzo David
McDonnell & Boehnen Hulbert & Berghoff
LandOfFree
Oncolytic/immunogenic complementary-adenoviral vector system does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Oncolytic/immunogenic complementary-adenoviral vector system, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oncolytic/immunogenic complementary-adenoviral vector system will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2923913