Equine infectious anemia virus vectors

Details Equine infectious anemia virus vectors Equine infectious anemia virus vectors

1999-01-27

2001-11-06

Nguyen, Dave Trong (Department: 1633)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093100, C424S093210, C424S093600, C435S069100, C435S320100, C435S325000, C435S455000, C514S04400A, C536S023100

Reexamination Certificate

active

06312683

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
FIELD OF INVENTION
This invention relates to a retroviral vector. In particular, but not exclusively, it relates to retroviral vectors capable of transferring genetic material to non-dividing or slowly-dividing cells derived from non-primate lentiviruses.
BACKGROUND OF THE INVENTION
There has been considerable interest, for some time, in the development of retroviral vector systems based on lentiviruses, a small subgroup of the retroviruses. This interest arises firstly from the notion of using HIV-based vectors to target anti-HIV therapeutic genes to HIV susceptible cells and secondly from the prediction that, because lentiviruses are able to infect non-dividing cells (Lewis & Emerman 1993 J.Virol. 68, 510), vector systems based on these viruses would be able to transduce non-dividing cells (e.g. Vile & Russel 1995 Brit. Med. Bull. 51, 12). Vector systems based on HIV have been produced (Buchschacher & Panganiban 1992 J.Virol. 66, 2731) and they have been used to transduce CD4+ cells and, as anticipated, non-dividing cells (Naldini et al, 1996 Science 272, 263). In addition lentiviral vectors enable very stable long-term expression of the gene of interest. This has been shown to be at least three months for transduced rat neuronal cells. The MLV based vectors were only able to express the gene of interest for six weeks.
HIV-based vectors produced to date result in an integrated provirus in the transduced cell that has HIV LTRs at its ends. This limits the use of these vectors as the LTRs have to be used as expression signals for any inserted gene unless an internal promoter is used. The use of internal promoters has significant disadvantages. The unpredictable outcome of placing additional promoters within the retroviral LTR transcription unit is well documented (Bowtell et al, 1988 J.Virol. 62, 2464; Correll et al, 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al, 1991 Mol.Cell.Biol. 11, 5848; Hantzopoulos et al, 1989 PNAS 86, 3519; Hatzoglou et al, 1991 J.Biol.Chem 266, 8416; Hatzoglou et al, 1988 J.Biol.Chem 263, 17798; Li et al, 1992 Hum.Gen.Ther. 3, 381; McLachlin et al, 1993 Virol. 195, 1; Overell et al, 1988 Mol.Cell Biol. 8, 1803; Scharfman et al, 1991 PNAS 88, 4626; Vile et al, 1994 Gene Ther 1, 307; Xu et al, 1989 Virol. 171, 331; Yee et al, 1987 PNAS 84, 5197). The factors involved appear to include the relative position and orientation of the two promoters, the nature of the promoters and the expressed genes and any selection procedures that may be adopted. The presence of internal promoters can affect both the transduction titers attainable from a packaging cell line and the stability of the integrated vector.
HIV and other lentiviral LTRs have virus-specific requirements for gene expression. For example, the HIV LTR is not active in the: absence of the viral Tat protein (Cullen 1995 AIDS 9, S19). It is desirable, therefore, to modify the LTRs in such a way as to change the requirements for gene expression. In particular tissue specific gene expression signals may be required for some gene therapy applications.
HIV vectors have a number of significant disadvantages which may limit their therapeutic application to certain diseases. HIV-1 has the disadvantage of being a human pathogen carrying potentially oncogenic proteins and sequences. There is the risk that introduction of vector particles produced in packaging cells which express HIV gag-pol will introduce these proteins into the patient leading to seroconversion. For these reasons, there is a need to develop lentiviral-based vectors which do not introduce HIV proteins into patients.
SUMMARY OF THE INVENTION
We have now found it possible to provide an improved lentiviral vector which overcomes the limitations of HIV-based vectors. It is important in the development of any retroviral vector system to remove sequences from the retroviral genome which may inhibit the capacity of the vector to transfer heterologous genes or which may transfer disadavantageous protein coding sequences to the target cell. Retroviruses are limited in the length of RNA sequences which can be packaged efficiently and so the existence of long regions of the retroviral genome severely limits the coding capacity of the vector for heterologous coding RNA.
We have also found it possible to provide a lentiviral vector based on a non-primate lentivirus which has a high coding capacity for heterologous coding sequences and which has a reduced capacity to transfer retroviral components to the target cell.
It has surprisingly been found that the amount of vector genomic sequence required from a non-primate lentivirus to produce an efficient cloning vector is substantially less than has been described for an HIV-based vector.
The sequence requirements for packaging HIV vector genomes are complex. The HIV-1 packaging signal encompasses the splice donor site and contains a portion of the 5′-untranslated region of the gag gene which has a putative secondary structure containing 4 short stem-loops. Additional sequences elsewhere in the genome are also known to be important for efficient encapsidation of HIV. For example the first 350 bps of the gag protein coding sequence may contribute to efficient packaging and ill defined regions of env are also implicated. For the construction of HIV-vectors capable of expressing heterologous genes, a packaging signal extending to 350 bps of the gag protein-coding region has been used.
We have surprisingly found that the structure of the packaging signal in non-primate lentiviruses is entirely different from that of HIV. Instead of a short sequence of 4 stem loops followed by an ill defined region of gag and env sequences, we have discovered that a shorter region of the gag gene suffices for efficient packaging. Indeed deletion of larger regions of the gag gene in EIAV vectors is advantageous and leads to higher titre viral vector being produced. This information can be used to provide improved vectors constructed from non-primate lentivirus sequences which have high titre and advantageous features compared to HIV vectors.
In a first aspect of the invention, there is provided a retroviral vector genome containing a deleted gag gene from a non-primate lentivirus wherein the deletion in gag removes one or more nucleotides downstream of nucleotide 350 of the gag coding sequence. Preferably the deletion extends from nucleotide 350 to at least the C-terminus of the gagpol coding region. More preferably the deletion additionally removes nucleotide 300 of the gag coding region and most preferably the deletion retains only the first 150 nucleotides of the gag coding region. However even larger deletions of gag can also be used, for example the gag coding region contains the first 109 nucleotides of the gag coding region. It may also be possible for the gag coding region to contain only the first 2 nucleotides of the gag coding region.
Additional features of the lentiviral genome are included in the vector genome which are necessary for transduction of the target cell; replication; reverse transcription and integration. These are, at least, a portion of an LTR containing sequence from the R-region and U5 region, sequences from the 3′ LTR which contain a polypurine tract (PPT) and a 3′ LTR from the non-primate lentivirus or a hybrid LTR containing sequences from the non-primate lentivirus and other elements. Optionally, the retroviral genome may contain accessory genes derived from a retrovirus, such as, but not limited to, a rev gene, a tat gene, a vif gene, a nef gene, a vpr gene or an S2 gene. Additional components may be added such as introns, splice-donor sites, a rev responsive element (RRE), cloning sites and selectable marker genes.
Moreover, we have now surprisingly demonstrated that a non-primate lentivirus minimal vector system can be constructed which requires neither S2, Tat, env nor dUTPase for either vector production or for transduction of dividing and non-dividing cells.
Thus acco

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