Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-07-19
2001-07-24
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S069100, C435S320100, C435S235100, C424S093210, C536S023100
Reexamination Certificate
active
06265212
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the field of recombinant DNA technology, more, in particular, to the field of gene therapy. Specifically, the present invention relates to gene therapy using materials derived from adenovirus, in particular, human recombinant adenovirus, and relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses. Furthermore, this invention also pertains to the screening of replication-competent and revertant E1 adenoviruses from recombinant adenoviruses used in gene therapy.
STATE OF THE ART
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 specific genetic information is introduced into some or all cells of a host. This results in the specific genetic information being added to the host in a functional format. The specific genetic information added may be a gene or a derivative of a gene, such as a cDNA (which encodes a protein), or the like. In the case where cDNA is added, the encoded 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. With this functional format, the added DNA molecule or copies made thereof in situ are capable of base pairing with the complementary sequence present in the host cell.
Applications of such gene therapy techniques include, but are not limited to, the treatment of genetic disorders by supplementing a protein or other substance which is, through the genetic disorder, not present or at least present in insufficient amounts in the host, the treatment of tumors or other such non-acquired diseases, and the treatment of acquired diseases such as immune diseases, autoimmune diseases, infections, and the like.
As may be clear from the above, there are basically three different approaches in gene therapy. The first approach is directed toward compensating for a deficiency present in a host (such as a mammalian host). The second approach is directed toward the removal or elimination of unwanted substances (organisms or cells). The third approach is directed toward the application of a recombinant vaccine (e.g., directed against tumors or foreign micro-organisms).
Adenoviruses carrying deletions have been proposed as suitable vehicles for the purpose of gene therapy. Adenoviruses are essentially non-enveloped DNA viruses. Gene-transfer vectors derived from such adenoviruses (known as “adenoviral vectors”) have several features that make them particularly useful for gene transfer. These features include, but are not limited to: 1) the fact that the biology of the adenoviruses is characterized in detail, 2) that the adenovirus is not associated with severe human pathology, 3) that the adenovirus is extremely efficient in introducing its DNA into the host cell, 4) that the adenovirus can infect a wide variety of cells and has a broad host-range, 5) that the adenovirus can be produced in large quantities with relative ease, and 6) that the adenovirus can be rendered replication defective by deletions in the early-region 1 (“E1”) of the viral genome, thus providing an important safety feature.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36,000 base pairs with the 55 kiloDalton (“kD”) terminal protein covalently bound to the 5′ terminus of each strand. The adenovirus DNA contains identical Inverted Terminal Repeats (ITR) of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are located within the ITRs at the genome ends. The synthesis of the DNA occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is single stranded and can form a structure known as a “panhandle” intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may proceed from both ends of the genome simultaneously, obviating the need to form the panhandle intermediate structure. The replication is summarized in
FIG. 14
(adapted from Lechner, R. L. and Kelly Jr., T. J., “The Structure of Replicating Adenovirus 2 DNA Molecules.
J. Mol. Biol
. 174, pp. 493-510 (1977), hereby incorporated herein by reference).
During the productive infection cycle, the viral genes are expressed in two phases: an early phase and a late phase. The early phase is the period up to viral DNA replication, and the late phase is the period which coincides with the initiation of viral DNA replication. During the early phase only the early gene products, encoded by regions E1, E2, E3 and E4, are expressed, which carry out a number of functions that prepare the cell for synthesis of viral structural proteins (see Berk, A. J.,
Ann. Rev. Genet
. 20, pp. 45-79 (1986), hereby incorporated herein by reference). During the late phase, the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (see Tooze, J.,
DNA Tumor Viruses
(revised), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1981), hereby incorporated herein by reference).
The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes. Both the E1A and E1B are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are:
i) to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and
ii) to transcriptionally activate the E1B gene and the other early regions (E2, E3, E4).
Transfection of primary cells with the E1A gene alone can induce unlimited proliferation (known as “immortalization”), but does not result in complete transformation. However, the expression of E1A, in most cases, results in the induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (see Jochemsen, et al.,
EMBO J
. 6, pp. 3399-3405 (1987), hereby incorporated herein by reference). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level expression of E1A can cause complete transformation in the absence of E1B (see Roberts et al.,
J. Virol
. 56, pp. 404-413 (1985), hereby incorporated herein by reference).
The E1B encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33 kD proteins, which form a complex that is essentially localized in the nucleus, function to inhibit the synthesis of host proteins and to facilitate the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomittantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene-product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect (cyt-phenotype) (see Telling et al., “Absence of an Essential Regulatory Influence of the Adenovirus E1B 19-kiloDalton Protein on Viral Growth and Early Gene Expression in Human Diploid W138, HeLa, and A549 cells,”
J. Virol
. 68, pp.541-547 (1994), hereby incorporated herein by reference). The deg and cyt phenotypes are suppressed when the E1A gene is mutated, thus indicating that these phenotypes are a function of E1A. (see White et al.,
J. Virol
. 6
Bout Abraham
Fallaux Frits J.
Hoeben Robert C.
Schouten Govert
Valerio Domenico
Clark Deborah J. R.
IntroGene B.V.
Trask & Britt
Wilson Michael C.
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