Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-08-14
2003-10-21
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S171000, C435S173300, C435S242000, C435S242000, C536S023100, C536S023500, C536S024100, C536S023720, C424S185100, C424S188100, C424S192100, C424S199100, C424S204100, C424S205100, C424S230100, C424S231100, C424S232100, C424S233100
Reexamination Certificate
active
06635472
ABSTRACT:
FIELD OF INVENTION
This invention relates to the fields of genetic engineering, virus replication, and gene transfer.
DESCRIPTION OF RELATED ART
Retroviruses and retroviral vectors are potentially powerful tools for the transfer of genes into animals and humans, for the creation of transgenic animals, and for vaccines. However, the use of the retroviruses has certain disadvantages. Most vexatious of them is the obligatory integration of the DNA form of the viral genome for its expression and replication (Cannon et al., 1996; Englund et al., 1995). The integration may have unforeseen and undesired consequences. It could lead to the transcriptional activation through the juxtaposition of the retroviral promoter/enhancer. It could also result in insertional mutagenesis. Both events could result in the production of tumors (Katzir et al., 1985; Neel et al., 1981; Ott et al., 1992), the former by activation of a dominant oncogene, the latter by inactivating a tumor suppressor gene. Other unwanted sequelae may be induced by similar mechanisms (Wu et al., 1993).
Several practitioners in the art have made attempts to circumvent the undesirable consequences of random integration into the host chromosome. For example, they have exploited the idea of tethering the viral integrase protein to the site-specific DNA binding proteins. Such hybrid proteins should recognize specific DNA sequences on the chromosome and integrate the provirus specifically into those regions. However, it is not easy to map all the potential targets in the genome and the resulting hybrid proteins also have not been very efficient (Bushman, 1994; Goulaouic and Chow, 1996). Other strategies, though not entirely successful or simple, were proposed as well.
For example, U.S. Pat. No. 5,118,627 discloses a microbial shuttle vector independently replicative in bacterial and mammalian cells which includes in its DNA sequence bacterial plasmid sequences allowing replication in bacteria, and an SV40 viral ori and SV40 promoters and terminators.
U.S. Pat. No. 5,324,645 discloses a highly retrovirus producing DNA construct having a gene encoding retrovirus, which does not include a retrovirus long terminal repeat sequence. This construct is incorporated into a vector for gene amplification.
U.S. Pat. No. 5,338,674 discloses a live non-pathogenic RNA tumor virus having an altered genome that encodes the antigenic determinants of a pathogen but has no genes coding for pathogenicity.
U.S. Pat. No. 5,420,026 discloses self-assembled, replication defective, hybrid, and virus-like particles having capsid and membrane glycoproteins from at least two different virus types.
Facing the disadvantages associated with retroviral replication involving the obligatory integration step, the instant inventor came forward with a new invention that overcomes these drawbacks. In this application the inventor discloses the construction and testing of novel retroviruses and vectors derived from them that can replicate episomally in vertebrate cells without integration into the chromosomal DNA of the host, therefore rendering them safer for use in gene transfer, in gene therapy, and for vaccines.
GENERAL ASPECTS OF THE INVENTION
This invention is based on a surprising finding that a recombinant hybrid murine leukemia virus (MLV) can replicate in certain mouse cells without integration.
The infectious viral agent such as retrovirus is usually packaged as a virus particle or a virion. It consists of a capsid built of capsid proteins, virus-encoded enzymes, e.g., DNA polymerase, integrase, and protease, a viral RNA genome, which can carry a foreign nucleic acid of interest, and an envelope, in some cases comprising glycoproteins, spanning through the lipid membrane surrounding the particle. Following fusion of the virus envelope with the cellular membrane or endocytosis and virus release into the cytoplasm of the cell, the initial step in the replication involves a reverse transcription step. In this step the RNA genome, which in the case of retroviruses is flanked by short terminal repeats (termed R), is converted to a double-stranded linear DNA form, by virus-encoded reverse transcriptase (RT) or viral DNA polymerase. In this process of reverse transcription, the long terminal repeat (LTR) sequence is produced at each end of the double-stranded DNA. It is this two-LTR configuration that is integrated into the host cell genome by the virus-encoded integrase (IN). Integration occurs at random, or nearly random, sites on host chromosomal DNA. This integrated viral DNA is called a provirus.
Upon retroviral infection almost half of retroviral DNA (Cannon et al., 1996; Leavitt et al., 1996; Yang et al., 1980), goes through an abortive process whereupon the cellular ligase joins both viral ends forming one- or two-LTR circular molecules (Hong et al., 1991; Kulkosky et al., 1990; Pauza, 1990; Smith et al., 1990; Whitcomb and Hughes, 1991; Whitcomb et al., 1990). Under normal circumstances, both one-LTR and two-LTR molecules are rapidly lost from the cell and do not give rise to a new infectious viruses (Englund et al., 1995; Sakai et al., 1993; Stevenson et al., 1990). Thus, the prior art is unambiguous that retroviruses cannot replicate episomally or extrachromosomally unless they integrate into chromosomal DNA of a host cell.
However, this default in retroviral replication, as discovered by this inventor can be overridden. The solution to this problem is originally derived from a replication strategy of a small DNA virus.
One of the best-studied small DNA viruses is the simian virus 40 or SV40 (Shah, 1996). Other small DNA viruses replicate their DNA by a mechanism that closely resembles that of SV40 (Cole, 1996). One skilled in the art would recognize the similarity between the replication strategy described for SV40 and other similar small DNA viruses. For example, bovine papilloma virus or BPV uses so-called E1 and E2 proteins that are similar to analogous counterparts in SV40 (Piirsoo et al., 1996; Lehman and Botchan, 1998).
The instant invention discloses a polynucleotide construct encoding a recombinant retrovirus or vector that is capable, following the conversion of the viral RNA into DNA, of amplification and maintenance of the viral DNA as an extrachromosomal or episomal molecule. As used hereinafter the term episomal replication means that a virus such as retrovirus is able to replicate without integration into host's chromosomal DNA and without gradual loss from a dividing host cell also meaning that said virus replicates extrachromosomally or episomally. The retrovirus or vector is originally engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a small DNA virus such as SV40. The term origin of DNA replication or ori as used herein means generally a regulatory genetic element found on an episomal DNA molecule allowing the replication of said DNA molecule so that these DNA molecules are not eventually lost upon division of a host cell. This mechanism assures stable extrachromosomal or episomal replication. Following infection, the double-stranded retroviral DNA is generated by means of reverse transcription and circularized upon entry into the host cell. The retrovirus carries ori and the host cell provides the cognate replication protein of small DNA virus, which is used for amplification and replication of the circular DNA retroviral genome. A replication protein can be so-called large T antigen of SV40 or it can be E1 or E2 of BPV. This is the first known hybrid retrovirus that does not have to integrate into the host genome in order to replicate efficiently in the host cells.
The instant invention also discloses a recombinant retrovirus or vector that is capable of amplification and maintenance of the viral DNA as an extrachromosomal molecule with the aid of minichromosome maintenance element (MME)—a regulatory element from bovine papilloma virus (BPV), which allows stable replication of episomal constructs without gradual loss from the host cell (Piirsoo et al., 1996).
Furthermore, the instant inve
Housel James
Li Bao Qun
Rubicon Laboratory, Inc.
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