Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-12-09
2001-08-21
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C536S023400
Reexamination Certificate
active
06277601
ABSTRACT:
The foamy viruses CM subgroup of retroid viruses has attracted scientific interest because of Their unique replication strategy and because of their potential use as gene transfer vectors (35). It has been proposed that FVs may be ideal tools for the development of a gene delivery System due to specific properties of this virus group, such as the absence of FV antibodies in the human population, the benign course of natural FV infections, their very broad host cell range, and an extended packaging limit due to the size of the FV genome (4, 30, 32). However, limited knowledge of the molecular biology of this virus group has so far not allowed the development of safe packaging cell lines and vectors, such as those that have been derived for murine retroviruses, among others (27). For instance, the FV is a DNA virus with a complex genome organization In addition to LTRs (Long Terminal Repeat), a packaging region and gag, pol, env genes, it also comprises several genes such as bell, bel2, bel3, bet, beo and bes located between env and 3′LTR The env gene encodes a 130 kDa glycosylated precursor that is cleaved giving rise to the surface (SU) and transmembrane (TM) subunits (see FIGS.
1
and
4
). The TM subunit includes in its 3′ part a tansmembrane anchor domain (A in
FIG. 4
) composed of hydrophobic residues which is followed by a cytopltnic tail. Furthermore, FVs express their Pol protein from a spliced MRNA independently of the Gag protein, and the mechanism of FV genome packaging and particle assembly, as well as the significance of high amounts of reverse transcribed DNA in the extra-cellular particle are largely unknown (10, 18, 39). Other unique features include the nuclear localization of the Gag precursor protein (31, 40) and the predominant budding into intracytoplasmic vesicles which may be a consequence of the retention of the Env precursor protein in the ER (13).
Moloney retrovirus-based gene transfer vectors are currently the main vehicles for high efficiency stable gene transfer into a wide variety of cell types (20). Major limitations of this vector system are the restricted host cell range and the inefficient infectivity for some human cells (reviewed in (1)). Recently, several methods using the pseudotyping with foreign envelope proteins, such as the vesicular stomatitis virus (VSV) G glycoprotein (6, 38) or the gibbon ape leukemia virus (GALV) envelope protein (2, 34) have been shown to overcome these disadvantages.
However, the expression of VSV-G for example is highly toxic for the producer cells and has prevented the generation of stable VSV-G packaging cells line (8, 22, 37).
The invention concerns constructs for the expression of a protein comprising at least a modified FV envelope protein.
The preferred FV according to the present invention is the human foamy virus (HFV), but others may be used (e. g. Simian FV).
The modification may consist in at least a mutation, deletion, substitution and/or addition of one or several amino acid (aa) of said modified FV envelope (env) protein or a combination thereof Such modification(s) is preferably located into the cytoplasmic tail. Advantageously, a modified FV envelope protein is truncated at aa 975 or, more preferably, 981. The truncation may extend up to the stop codon or alternatively comprise before the stop codon one or several residues optionally from the original FV env protein.
Furthermore, a construct of the invention may express a mature modified FV envelope protein or a precursor thereof or a chimeric protein resulting from the fusion of sequences of various origins. In a particularly preferred embodiment, the modified FV env protein in use in the present invention is a fusion protein which furthermore comprises all or preferably a part of a non-FV envelope protein. Examples of suitable non-FV viruses include avian retroviruses, bovine retroviruses, feline retroviruses, murine retroviruses such as Mine Leukemia Virus (MuLV) and particularly Moloney MuLV (MoMuLV), Friend Murine Leukemia Virus (FrMuLV) especially strain FB 29, Murine Sarcome Virus (MSV), primate retroviruses such as GaLV, VSV or lentiviruses such as HIV (Human Immunodeficiency Virus) or SIV (Simian Immunodeficiency Virus).
The fusion betwen FV and non-FV env proteins can be made at different locations. Fusions within the TM subunit are advantageous. According to a first alternative, the fusion is within the tansmembrane anchor domain of said FV and non-FV envelope proteins. A preferedexample is a protein that comprises the extracellular domain and the 5′ part of the transmembrane anchor domain of the HFV envelope protein and the 3′ part of the transmembrane anchor domain and the cytoplasmic domain of the non-FV envelope protein, particularly of the SIV envelope protein.
A second alternative is that the fusion is within the cleavage site of said FV and non-FV envelope proteins. A preferedexample is a protein that comprises the SU domain and all or part of the cleavage site of the HFV envelope protein and all or part of the cleavage site and the TM domain, comprising the transmembrane anchor domain and the cytoplasmic domain of the non-FV envelope protein, particularly of the SIV envelope protein. The replacement of the cleavage site of the FV envelope protein by its equivalent from the non-FV envelope protein is also envisageable.
Another alternative is that the fusion is at the junction between the transmembrane anchor domain and the cytoplasmic domain or within the cytoplasmic domains of said FV and non-FV envelope proteins. A prefered example is a protein that comprises the extracellular domain, the transmembrane anchor domain and all or part of the cytoplasmic domain of the HFV envelope protein and all or part of the cytoplasmic domain of the non-FV envelope protein, particularly of the SIV envelope protein.
In a particularly preferred embodiment, a protein according to the invention consists in HFV protein envelope which all or part of the cytoplasmic domain is replaced by all or part of a cytoplasmic domain of a non-FV viral envelope protein, especially of a MuLV envelope protein. Advantageousy, the fusion protein consists in the fusion of a MuLV cytoplasmic domain to a modified HFV envelope protein. The MuLV cytoplasmic domain in use in the present invention may be processed or unprocessed. <<Processed>> means that it contains the cleavage site normally recognized by the corresponding retroviral protease and <<unprocessed>> that it does not contain it or that it is not functional (mutation, deletion or truncation).
The preferred construct of the invention is the one allowing expression of the fusion protein designated hereinafter HFV &Dgr;2 MuLV.
Alternatively, a prefered protein according to the invention consists in a HFV envelope protein in which all or part of the cytoplasmic tail is replaced by all or part of a cytoplasmic domain of a SIV envelope protein. There exists two versions of the SIV envelope protein : a long form having a cytoplasmic tail of 164 amino acids which is present in SIV particles replicating in the natural host, the rhesus monkey (Macaca mulatta) and a short form containing only 18 amino acids. This short form is selected for when virus isolated from the monkey are cultured on human cell lines (42).
It is also possible that the construct of the invention is mutated in the donor and/or acceptor splicing sites naturally present in the FV env protein encoding sequence.
The construct of the invention may include regulatory elements to allow transcription and translation of the sequence coding for the modified FV env protein. In particular, a suitable promoter may be linked upstream from the FV env encoding sequence in an operative way by conventional recombinant techniques. Such a promoter may be of prokaryote, eukaryote or viral origin and may be constitutive or regulated. Such regulatory elements are well known in the art.
It is also in the scope of the invention that the construct of the invention may additionally comprise a selection gene enabling detection
Lindemann Dirk
Rethwilm Axel
Winter Arend Jan
Burns Doane Swecker & Mathis L.L.P.
Park Hankyel T.
Transgene S.A.
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