pCAR and its uses

Details pCAR and its uses pCAR and its uses

2001-07-05

2004-09-21

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C536S023500, C536S023100, C536S023200, C536S024100, C800S018000, C800S003000, C800S021000

Reexamination Certificate

active

06794186

ABSTRACT:

The invention provides improvements in the field of animal models for testing effects of genes introduced into animal cells or tissue by adenoviral gene transfer.
Adenoviruses infect cells using two cell surface receptors, the “Coxsackie B and adenovirus 2 and 5 receptor” (hereinafter referred to as CAR; Bergelson J. M., et al, Science 275, 1320-23, 1997) and the integrin receptors (&agr;&ngr;&bgr;3 or &agr;&ngr;&bgr;5; Wickham, T. J. et al, Cell 73, 309-19, 1993) the contents thereof being incorporated herein by reference. Adenoviral based vectors are widely used in gene therapy, as they represent one of the most efficient ways to deliver genes to target cells. They are of particular interest for in vivo gene therapy proof-of concept experiments in rodent models. However, rodent tissues are not well transducible with adenoviral vectors.
In its broad aspect the invention is concerned with genetic modification of target cells which are normally refractory to adenoviral transduction. More particularly the invention provides a plasmid construct that expresses a porcine adenovirus receptor (PCAR) and transgenic animals that show expression of pCAR.
Organ transplants of liver, kidney, lung and heart are now regularly performed as treatment for endstage organ disease. Despite the use of modern immunosuppressive drugs acute and chronic graft (tissue or organ) rejection still remain major factors in graft loss. There is, therefore, a continued need for means to inhibit acute and chronic graft rejection and increase graft acceptance, e.g. through induction of peripheral tolerance without causing serious toxic side effects typically associated with conventional immunosuppressant therapy. When considering cell transplantation, e.g. bone marrow derived cells, islet cells, neuronal cells etc. one is faced with similar problems of rejection. Making organs or cells less immunogenic through genetic modification is seen as an alternative or add on to conventional immunosuppression.
Rodent animal models are of crucial importance for testing the immunomodulatory effects of new gene products. However in the case of using adenovirus as gene delivery vehicle rodent models have so far proven to be of limited value, as many rodent organs or cell types are refractory to adenoviral transduction. This may be due to the fact that either the adenoviral receptor CAR is not expressed or only weakly expressed on the cell surface of the cells of interest.
Accordingly, the invention provides a plasmid or vector construct that comprises a DNA molecule which expresses porcine CAR (SEQ ID NO:4 hereinafter referred to as pCAR) or a biologically active fragment or derivative thereof, for example a C-terminally truncated porcine CAR (SEQ ID NO:2hereinafter referred to as &Dgr;pCAR), that retains full functionality as adenoviral receptor.
pCAR comprises an intracellular domain, a transmembrane domain and a an extracellular domain that binds to the adenoviral fibre proteins, i.e. a total sequence of 365 amino-acids. It will be understood that any nucleic acid sequence encoding a porcine CAR homologue is a candidate for utilization in the present invention. For example, it may include a pCAR sequence with a modified, mutated or truncated region thereof, that retains the activity of mediating adenoviral transduction. It will be further understood by the skilled person that any nucleic acid sequence which encodes a biologically active form of pCAR, including but not limited to a genomic or cDNA sequence or functionally equivalent variant or mutant thereof or a fragment thereof which encodes a biologically active protein fragment or derivative which mediates adenoviral transduction, may be utilized in the present invention. For example, &Dgr;pCAR may comprise the leader sequence of 19 amino-acids, the extracellular domain of 216 amino-acids, the transmembrane domain of 24 amino-acids and a truncated cytoplasmic domain, e.g. limited to 3 amino-acids. Two potential sites for N-glycosylation are located at Asn 106 and Asn 201. Amino-acids present in the sequence which are not essential to the activity may be changed by mutation, e.g. amino-acid 258 may be changed from Val to Ile; amino-acid 262 may be changed from His to Arg.
Preferred nucleic acid sequence for use in the invention is e.g. as disclosed in SEQ ID NO: 1 from nucleotide 3229 to nucleotide 4014. The corresponding amino acid sequence encoded by such DNA sequence is indicated in SEQ ID NO:2.
Any known expression vector or plasmid that is capable of expression upon transfection of a specified eukaryotic target cell may be utilized to pratice the invention. “Plasmid” and “vector” can be used interchangeably in the present specification as the plasmid is the most commonly used form of vector. An expression vector is a vector capable of directing the expression of genes to which they are operatively linked. An operable linkage as used herein refers to the position, orientation and linkage between a structural gene and expression control element(s) such that the structural gene can be expressed in any host cell. The term “expression control element” includes promoters, enhancers, ribosome binding sites etc. Any eukaryotic promoter and/or enhancer sequences available to the skilled person which are known to control expression of the nucleic acid of interest may be used in plasmid vector constructs, including but not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma (RVS) promoter, a Murine Leukemia (MLV) promoter, a herpes simplex virus (HVS) promoter, such as HSV-tk, a &bgr;-actin promoter, e.g. chicken &bgr;-actin, as well as any additional tissue specific or signal specific regulatory sequence that induces expression in the target cell or tissue of interest. A preferred expression vector or plasmid according to the invention is e.g. an eukaryotic expression vectors, e.g. a p&bgr;-actin-p16PL vector such as p(chicken)&bgr;-actin-p16PL.
In one such embodiment, a DNA sequence encoding pCAR is subcloned into the DNA plasmid expression vector, e.g. p&bgr;-actin-p16PL, resulting in p&bgr;-actin-pCAR-p16PL. p16PL is a standard mammalian expression vector, containing a gene that encodes a selectable marker, e.g. an antibiotic resistance gene, and a &bgr;-actin promoter active in mammalian cells (K. M. Marsden et al, J. Neurosc., May 15, 1996, 16(10): 3265-3273). Such a construct, which may be constructed by one of ordinary skill with components available from numerous sources, will drive expression of a pCAR DNA fragment ligated downstream of the &bgr;-actin promoter subsequent to transfection of the target cell. More specifically, pCAR is cloned from pig liver RNA using a PCR based approach. The PCR fragment is inserted into the expression vector pSport (Life Technologies). This plasmid serves as template to create the truncated version of &Dgr;pCAR. Preferably p&bgr;-actin is p&bgr;-(chicken) actin.
The invention further provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic or eukaryotic cell, e.g. bacterial such as
E. Coli
, yeast or mammalian cells, e.g. CHO or COS cells.
The host cells of the invention may preferably be used to produce nonhuman transgenic animals, preferably a mammal, more preferably a rodent such as a rat or mouse, or a pig.
For example, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a pCAR-coding sequence has been introduced. A transgenic animal of the invention, more preferably a mammal, most preferably a rodent or a pig, may be created by introducing a pCAR expression construct into the male pronuclei of a fertilized oocyte, e.g. by microinjection, or into embryonic stem cells, e.g. by electroporation. Methods for generating transgenic rodents have become conventional in the art and are described e.g. in U.S. Pat. Nos. 4,736,866, 4,870,009, 4,873,191, or in Manipulating the Mouse Embryo, B. Hogan, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). For example the expression construct may be intro

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