Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-06-15
2003-11-25
Wehbé, Anne M. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093200, C435S320100, C435S325000, C435S455000, C514S04400A
Reexamination Certificate
active
06652850
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to improved methods and compositions for transfecting cells, particularly dendritic cells and other antigen presenting cells, through the use of cationic liposomes to facilitate transfection with adeno-associated viral (AAV) plasmids. Transfected cells expressing genes of interest, such as tumor-associated or viral antigens, are used for immunization and therapy.
2. Description of the Background Art
Transfection of eukaryotic cells has become an increasingly important technique for the study and development of gene therapy. Advances in gene therapy depend in large part upon the development of delivery systems capable of efficiently introducing DNA into a target cell. A number of methods have been developed for the stable or transient expression of heterologous genes in cultured cell types. These include transduction techniques which use a carrier molecule or virus.
Most gene therapy strategies have relied on transduction by transgene insertion into retroviral or DNA virus vectors. However, adenovirus and other DNA viral vectors can produce infectious sequelae, can be immunogenic after repeated administrations, and can only package a limited amount of insert DNA.
Of the viral vector systems, the recombinant adeno-associated viral (AAV) transduction system has proven to be one of the most efficient vector systems for stably and efficiently carrying genes into a variety of mammalian cell types (Lebkowski, J. S. et al.,
Mol. Cell. Biol
. (1988) 8:3988-3996). It has been well-documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through inverted terminal repeats (ITRs) of the viral DNA, and that the physical structure of integrated AAV genomes suggest that viral insertions usually appear as multiple copies with a tandem head to tail orientation via the AAV terminal repeats (Kotin, R. M. et al.,
Proc. Natl. Acad. Sci. USA
(1990) 87:2211-2215). Thus, the AAV terminal repeats (ITRs) are an essential part of the AAV transduction system.
Although recombinant adeno-associated viral (AAV) vectors differ from adenoviral vectors, the transgene DNA size limitation and packaging properties are the same as with any other DNA viral vectors.
AAV is a linear single stranded DNA parvovirus, and requires co-infection by a second unrelated virus in order to achieve productive infection. AAV carries two sets of functional genes: rep genes, which are necessary for viral replication, and structural capsid protein genes (Hermonat, P. L. et al.,
J. Virol
. (1984) 51:329-339). The rep and capsid genes of AAV can be replaced by a desired DNA fragment to generate AAV plasmid DNA. Transcomplementation of rep and capsid genes are required to create a recombinant virus stock. Upon transduction using such virus stock, one recombinant virus uncoats in the nucleus and integrates into the host genome by its molecular ends.
Although extensive progress has been made, transduction techniques suffer from variable efficiency, significant concern about possible recombination with endogenous virus, cellular toxicity and host immune reactions. Thus, there is a need for non-viral DNA transfection procedures.
Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (Faller, D. V. et al.,
J. Virol
. (1984) 49:269-272).
Preformed liposomes that contain synthetic cationic lipids have been shown to form stable complexes with polyanionic DNA (Felgner, P. L. et al.,
Proc. Natl. Acad. Sci. USA
(1987) 84:7413-7417). Cationic liposomes, liposomes comprising some cationic lipid, that contain a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-bromide (DDAB) have efficiently transferred heterologous genes into eukaryotic cells (Rose, J. K. et al.,
Biotechniques
(1991) 10:520-525). Cationic liposomes can mediate high level cellular expression of transgenes, or mRNA, by delivering them into a variety of cultured cell lines (Malone, R. et al.,
Proc. Natl. Acad. Sci. USA
(1989) 86:6077-6081).
Ecotropic and amphotropic packaged retroviral vectors have been shown to infect cultured cells in the presence of cationic liposomes, such as Lipofectin (BRL, Gaithersburg, Md.), and in the absence of specific receptors (Innes, C. L. et al.,
J. Virol
. (1990) 64:957-961).
Even though non-viral techniques have overcome some of the problems of the viral systems, the need still remains for improved transfection efficiency in non-viral systems, increased range of cell types that are transfectable, increased duration of expression in transfected cells, and increased levels of expression following transfection. Some improved efficiency is attained by the use of promoter enhancer elements in the plasmid DNA constructs (Philip, R., et al.,
J. Biol. Chem
. (1993) 268:16087-16090).
Immune Destruction of Tumor Cells
Interleukin-2 (IL-2) has been used to treat neoplasms such as metastatic renal cell carcinoma, as one approach to the immune-mediated destruction of human cancer. Although durable complete remissions have been achieved, the overall response rate has been low.
Testing of recombinant IL-2 (rIL-2) (Chiron Corp., Emeryville, Calif.) on cancer patients has revealed dose-limiting toxicity which was dependent upon the route and schedule of IL-2 administration. High dose bolus IL-2 administration was associated with significant toxicity involving nearly every organ system. Moreover, a 4% mortality rate in ECOG 0 performance status patients has been found with high dose IL-2. For an overview of ECOG performance status, see, e.g., Oken, Am.
J. Clin. Oncol
. (CCT) 5:649-655 (1982), Table 2, at p. 654).
As distinguished from bolus administration, use of lower dose (1-7×10
6
Cetus units/M
2
/day) continuous intravenous infusion of IL-2 has demonstrated clinical efficacy and lowered toxicity, suggesting an improved safety profile in adoptive immunotherapy of advanced cancer (West, W. H. et al., (1987)
N. Engl. J. Med
. 316:898).
Cell populations which potentially mediate or promote the immune destruction of tumors when combined with IL-2 include lymphokine activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) in particular tumor-infiltrating lymphocytes (TIL). TIL are primarily T lymphocytes found in close apposition to a tumor mass which can be isolated, expanded, and activated in vitro. TIL are of interest in the treatment of neoplasia because of their affinity and presumably their specificity for tumor cells as well as their cytotoxic action. TILs have been reinfused into patients along with exogenous IL-2 (see, e.g., U.S. Pat. No. 5,126,132, Rosenberg, Jun. 30, 1992) which, in some instances, resulted in durable complete remissions of advanced malignancies.
Dendritic Cells
Many attempts have been made to elicit immune responses in subjects that would lead to destruction and removal of unwanted cells, tissues or microorganisms, in particular, tumors or oncogenic viruses such as Epstein-Barr virus (EBV). However, success has been limited. One difficulty is in adequately presenting tumor-associated antigens to the immune system to evoke a cellular immune response. Dendritic cells (“DC”; plural is “DCs”) are known to be highly potent antigen-presenting cells (“APC; plural is (APCs”). For a review of DCs and their role in immunogenicity, see Steinman, R.,
Annu. Rev. Immunol
. 9:271-296 (1991), which reference is incorporated by reference in its entirety. The present invention employs such cells as a means to present tumor antigens to elicit a specific immune response in vitro or in vivo.
Antigen-specific CTL have been a subject of active investigation for their potential immunotherapeutic utility in the treatment of cancer or virus infection. Tumor-associated antigens (TAA), identified in a number of different types of tumors, including carcinomas can be used for in vitro generation of CTL with lytic activity against cells of the tumor. CEA (carcinoembryonic antigen) is a wel
Lebkowski Jane S.
Philip Ramila
Aventis Pharmaceuticals Inc.
Synnestvedt & Lechner LLP
Wehbe Anne M.
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