Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1995-08-15
2001-03-20
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C424S093200
Reexamination Certificate
active
06204052
ABSTRACT:
The invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy.
In particular the invention relates to novel vectors, especially for use in gene therapy, although they can be used for other recombinant expression purposes such as in providing transgenic animals.
Gene therapy is a recently developed concept for which a wide range of applications can be and have been envisaged.
In gene therapy a molecule carrying genetic information is introduced in some or all cells of a host, whereby the genetic information is added to the genetic information of the host in a functional format.
The genetic information added may be a gene or a derivative of a gene, such as a cDNA, which encodes a protein. In this case the functional format means that the protein can be expressed by the machinery of the host cell.
The genetic information can also be a sequence of nucleotides complementary to a sequence of nucleotides (be it DNA or RNA) present in the host cell.
The functional format in this case is that the added DNA (nucleic acid) molecule or copies made thereof in situ are capable of base pairing with the complementary sequence present in the host cell. Alternatively the added DNA or copies thereof in situ could interact with proteins present in the cells
Applications include the treatment of genetic disorders by supplementing a protein or other substance which is, through said genetic disorder, not present or at least present in insufficient amounts in the host, the treatment of tumors and (other) acquired diseases such as (auto)immune diseases or infections, etc.
As may be clear from the above, there are basically two different approaches in gene therapy, one directed towards compensating a deficiency present in a (mammalian) host and the other directed towards the removal or elimination of unwanted substances (organisms or cells).
The invention provides vectors which are suitable for both kinds of gene therapy.
A problem associated with the introduction of any foreign material into mammalian hosts, especially via systemic routes, is that there is always a risk of inducing an immune response. This is also true in gene therapy. If the genetic information is provided through a medium which may lead to an immune-response, the result will be that such genetic information will never be incorporated into the target cells, or that it will be incorporated, but that the cells will be eliminated by the immune system.
In both cases neither kind of gene therapy will be efficacious, since the new genetic information will only be available for a very short period of time. Moreover, repeated treatments will be impossible.
For the purpose of gene therapy, adenoviruses carrying deletions have been proposed as suitable vehicles.
Adenoviruses are non-enveloped DNA viruses. The genome consists of a linear, double stranded DNA molecule of about 36 kb
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. Recombinant adenovirus vectors have been generated for gene transfer purposes. All currently used adenovirus vectors have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant Virus replication defective. It was demonstrated that recombinant adenoviruses are able to efficiently transfer recombinant genes to airway epithelium of rhesus monkeys (1,2). In addition, we have observed a very efficient adenovirus mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animals models (lung tumors, glioma) and human xenografts (lung tumors) in immunodeficient mice in vivo.
In contrast to for instance retroviruses, adenoviruses a) do not integrate into the host cell genome; b) are able to infect non-dividing cells and c) are able to transfer recombinant genes in vivo extremely efficiently. Those features make adenoviruses attractive candidates for in vivo gene transfer of for instance suicide and/or cytokine genes into tumor cells. Recently, in vitro adenovirus mediated gene transfer of Il-2 was reported (3).
As disclosed in for instance W093/19191, the E3 region of the virus, which is not essential for growth of the virus in vitro, nor for infection in vivo, has also been deleted from the viral vector.
For a better understanding of the present invention a brief description of the E3 region is given below (reviewed in (4)).
Nine mRNAs from the E3 region are identified in group C adenoviruses (group C=Ad2 and Ad5 commonly cause cold-like respiratory infections)(5). From some of the mRNAs, the corresponding proteins have been identified. Proteins encoded by this area have molecular weights of 19, 14.7, 11.6, 10.4 and 6.7 kDa). Group B adenoviruses apparently encode two E3 proteins (20.1 and 20.4 kD) that are not found in group C adenoviruses. None of the E3 proteins is required for adenovirus replication in cultured cells or in acute infections of the lungs of hamsters or cotton rats. Despite this, E3 is always maintained in natural isolates of adenoviruses (4). A short description of the function of some E3 proteins is presented (4-6):
A function of the 19 kDa protein, called gp 19K because it is a glycoprotein, is to protect adenovirus infected cells against MHC class-I restricted cytotoxic T-cell lysis. This glycoprotein complexes intracellularly with Class I histocompatibility antigens, thereby reducing recognition of the infected cell by the cellular immune system.
Another protein of the E3 region (14.7 kDa) is responsible for suppression of cytolysis induced by TNF. TNF is secreted by activated macrophages and lymphocytes and is cytotoxic or cytostatic to certain tumor cells (see for review (6)). TNF also lyses cells infected with certain viruses and is released during infections by influenza virus. It has been shown that mouse C3HA fibroblasts are lysed by TNF when infected by adenovirus mutants that lack region E3. Uninfected cells are not lysed by TNF, nor are cells infected by wild-type adenovirus. Mutant recombinant adeoviruses that do not express E3-10.4K, E3-14.5K and E3-14.7K induced increased infiltration of neutrophils. Vaccinia virus vectors have been generated that express E3-14.7K, TNF or both proteins. The vectors expressing TNF were less virulent in mice than control vectors, whereas E3-14.7K increased the virulence of the TNF expressing vectors. This led to the conclusion that E3-14.7K counteracts the antiviral effect of TNF in vivo.
The 10.4 kDa and 14.5 kDa proteins encoded by the E3 region function in concert to down-regulate the EGF receptor (tyrosine-kinase) in adenovirus-infected cells. Stimulation of the kinase activity of EGF-R results in the activation of cellular metabolism and eventually in the induction of DNA synthesis and mitosis.
The biological significance of EGF-R downregulation is unknown. It has been suggested that 10.4K/14.5K mimic EGF in activating the kinase activity of EGF-R which could be a mechanism by which adenovirus activates quiescent cells. Alternatively, perhaps the purpose of 10.4/14.5 K is to eliminate EGF-R, so that it cannot signal. Elimination of these receptors should preclude an inflammatory response induced by EGF (6).
Other functions than those known so far might be provided by E3. It is of interest that it has been demonstrated that deletion of the E3 region leads to a more rapid replication and increased toxicity as compared to recombinant adenoviruses in which the E3 has been replaced by other genes, that do not have E3 like properties. It should be noted that both strands of adenovirus contain protein coding sequences. The sequences at the opposite of E3 on the 1 strand space the E4 and E2 regions. Although no known transcripts are derived from this area, it does not exclude that deletion has consequences for virus replication.
Mutants that have an intact E1 region, but whose E3 region is largely deleted, were found to replicate like wild-type virus in the cotton rats lungs, but the lymphocyte and macrophage/monocyte inflammatory response was markedly increased. Onset of viral multiplication, which reached maximum titers 2-4 days after infection was soon foll
Bout Abraham
Valerio Domenico
Van Bekkum Dirk W.
Crouch Deborah
IntroGene B.V.
Trask & Britt
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