Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-23
2002-10-22
Nguyen, Dave T. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S455000, C435S325000
Reexamination Certificate
active
06468771
ABSTRACT:
BACKGROUND
1. Field of the Invention
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 methods of delivering DNA to target cells and the subsequent integration of that DNA into the target cell genome.
2. State of the Art
In the field of gene therapy, many different methods have been developed to introduce new genetic information into target cells. Currently, the most efficient means of introducing DNA into target cells is by employing modified viruses, so-called recombinant viral vectors. The most frequently used viral vector systems are based on retroviruses, adenoviruses, herpes viruses or the adeno-associated viruses (AAV). All systems have their specific advantages and disadvantages. Some of the vector systems possess the capacity to integrate their DNA into the host cell genome, whereas others do not. From some vector systems the viral genes can be completely removed from the vector while in other systems this is not yet possible. Some vector systems have very good in vivo delivery properties, while others do not. Some vector types are very easy to produce in large amounts, while others are very difficult to produce.
The present invention combines functional components of two vector systems, thereby combining the favorable properties of both vector systems. The present invention was made during research involving adenovirus and adeno-associated virus. The invention typically provides DNA having a packaging signal which allows it to be encapsidated into virus particles of viruses which allow for encapsidation of large nucleic acids, such as adenovirus particles, which DNA (at least a part thereof) has the capacity to integrate into the host cell genome. The invention also provides for methods to ensure the absence of harmful viral genes from the encapsidated DNA. Absence of viral genes from the vector is the best way to avoid expression of viral gene products in target cells and thus the best way to circumvent immune responses to viral gene products expressed by transduced target cells.
The present invention can convey the above properties onto adenovirus vectors but also to other viruses, such as herpes or polyomaviruses.
The invention will, however, be explained in more detail based on adenovirus and adeno-associated virus vectors. Currently, adenovirus vectors attract a lot of attention and it is expected that the first registered gene therapy medicine will carry the foreign gene into the diseased cells of the patient through adenovirus vector mediated gene transfer. An important problem regarding adenovirus vectors is that they do not integrate into the host cell genome. In rapidly dividing tissue, such as the hemopoietic system, the vector is rapidly lost. Another problem with the current generation of adenovirus vectors is that they are immunogenic. In vivo, vector infected cells are cleared from the body by a potent immune reaction involving both a cellular and a humoral immune component.
For the purpose of gene therapy, adenoviruses carrying deletions have been proposed as suitable vehicles. Gene-transfer vectors derived from adenoviruses (so-called adenoviral vectors) have a number of features that make them particularly useful for gene transfer for such purposes. E.g. the biology of the adenoviruses is characterized in detail, the adenovirus is not associated with severe human pathology, the virus is extremely efficient in introducing its DNA into the host cell, the virus can infect a wide variety of cells and has a broad host-range, the virus can be produced in large quantities with relative ease, and the virus can be rendered replication defective by functional deletion of the early-region 1 (E1) of the viral genome.
During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication, and the late phase, 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 (Berk, 1986). During the late phase the late viral gene products are expressed and 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 (Tooze, 1981).
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, which both 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 (immortalization), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (Jochemsen et al., 1987). 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 (Roberts et al., 1985). 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 in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomitantly 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) (Telling et al., 1994). The deg and cyt phenotypes are suppressed when, in addition, the E1A gene is mutated, indicating that these phenotypes are a function of E1A (White et al., 1988). Furthermore, the E1B 21 kD protein slows down the rate by which E1 A switches on the other viral genes. It is not yet known through which mechanism(s) the E1B 21 kD protein quenches these E1A dependent functions.
Vectors derived from human adenoviruses, in which at least the E1 region has been deleted and replaced by a gene-of-interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36,000 base pairs with the 55 kD terminal protein covalently bound to the 5′ terminus of each strand. The Ad DNA contains identical Inverted Terminal Repeats (TR) of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are within the TRs exactly at the genome ends. DNA synthesis occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand can form a so-called “panhandle” intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may go from both ends of the genome simultaneously, obliterating the requirement to form the panhandle structure. The replication is summarized in
FIG. 1
adapted from (Lechner and Jr., 1977).
As
Einerhand Markus Peter
Schouten Govert Johan
Valerio Domenico
Introgene
Nguyen Dave T.
TraskBritt
Whiteman Brian
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