Lentiviral vectors

Details Lentiviral vectors Lentiviral vectors

1999-04-05

2001-05-22

Clark, Deborah J. R. (Department: 1633)

Chemistry: molecular biology and microbiology

Vector, per se

C435S235100, C435S455000, C435S456000, C526S085000

Reexamination Certificate

active

06235522

ABSTRACT:

This invention relates to retroviral vector particles and to DNA constructs encoding RNA genomes for retroviral vectors. In particular it relates to retroviral vectors capable of transferring genetic material to non-dividing or slowly-dividing cells.
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-diving cells (Naldini et al, 1996 Science 272, 263). However, in general gene transfer efficiencies are not as high as with comparable murine retrovirus vector systems.
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 (see later). 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 or signals that respond to exogenous signals may be necessary. In murine retroviruses this is often achieved simply by replacing the enhancer-like elements in the U3 region of the MLV LTR by enhancers that respond to the desired signals. This is not feasible with viruses such as HIV because within the U3 and R regions of their LTRs are sequences, known as IST and TAR, which may inhibit gene expression and may or may not be responsive to Tat protein when heterologous, perhaps tissue specific, control sequences are inserted in the U3 region (Cullen 1995 AIDS 9, S19; Alonso et al, 1994 J. Virol. 68, 6505; Ratnasabapathy et al, 1990 4, 2061;Sengupta et al, 1990 PNAS 87, 7492; Parkin et al, 1988 EMBO.J 7, 2831)). Even if the signals are responsive it is undesirable to have to supply Tat as it further complicates the system and Tat has some properties of oncoproteins (Vogel et al, 1988 Nature 335, 606). Overall, these considerations mean that the R region of HIV and other lentivirus vectors must be removed if effective expression from non-lentiviral sequences in the LTR is to be achieved.
We have described previously in PCT/GB96/01230 a method for replacing both the U3 and R regions of retroviral vector genomes. The observation that R regions could be replaced was surprising as it was previously believed that these were specific to the virus that is providing the reverse transcriptase for the conversion of the RNA viral genome to the preintegrated form of the proviral DNA. PCT/GB 96/01230 describes in particular retrovirus vectors for delivering therapeutic genes whose expression in the target cell is HIV-dependent. Delivery to non-dividing or slowly-dividing cells is not addressed, and application of the invention to HIV or any other lentivirus-based vectors is not addressed. The general approach described in PCT/GB 96/01230 now provides a means of producing an HIV-based vector with the U3 enhancer and R regions replaced by any sequence of choice providing that appropriate polyadenylation and transcription termination regions are included in the R region.
The present invention provides in one aspect a retroviral vector particle based on a first retrovirus, said retroviral vector particle capable of infecting and transducing non-dividing mammalian target cells, said retroviral vector particle comprising a packagable RNA genome capable of being inserted into a target cell genome when in the form of a DNA provirus, said RNA genome comprising sequences which provide in the DNA provirus:
a) a non-lentiviral expression control element located in the 5′ long terminal repeat (LTR) of the provirus in place of the promoter function of the first retrovirus; and
b) a selected gene or genes under transcriptional control of the non-lentiviral expression control element in a), the selected gene or genes located between the LTRs.
In another aspect, the invention provides a DNA construct encoding the packagable RNA genome for the retroviral vector particle described herein, operably linked to a promoter. In the DNA construct, the selected gene or genes may be present, or be absent in which case the construct has an insertion site e.g. a restriction enzyme site, at which the selected gene or genes may be inserted.
In a further aspect, the invention provides a retroviral vector particle production system comprising a host cell transfected or transduced with a DNA construct as described herein, said system capable of producing retroviral vector particles as described herein.
In yet another aspect, the invention provides a retroviral vector particle production system comprising a set of nucleic acid sequences encoding the components of a retroviral vector particle as described herein.
In still further aspects, the invention provides the use of the retroviral vector particles described herein for gene therapy and in the preparation of a medicament for use in gene therapy; and a method of performing gene therapy on a target cell which method comprises infecting and transducing the target cell using a retroviral vector particle as described herein. The invention further provides transduced target cells resulting from these uses and methods. The invention thus provides a gene delivery system for use in medicine.
That the vector particle according to the invention is “based on” a first retrovirus means that it is derived from that retrovirus. The genome of the vector particle comprises components from that retrovirus as a backbone. The vector particle as a whole contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include gag and pot proteins derived from the first retrovirus.
Preferably, the first retrovirus is a lentivirus which provides the ability to infect and transduce non-dividing cells. During the infection process, lentiviruses form a pre-integration complex in the target cell cytoplasm containing integrase, core proteins and the proviral DNA. The complex is able to pass across the nuclear membrane of the target cell, by is means of signal sequences in the proteins. Other retroviruses either lack the proteins, or have the proteins but without the appropriate signal sequences. It is therefore expected to be possible in principle to introduce into retroviruses other than lentiviruses the ability to infect non-dividing cells.
Examples of lentiviruses are HIV, SIV, FIV, BLV, EIAV, CEV and visna virus. Of these, HIV and SIV are presently best understood. However, preferred for use in gene therapy would be a non-immunodeficiency lentivirus because the immunodeficiency viruses inevitably bring with them safety considerations and prejudices.
The non-lentiviral expression control element will usually be a promoter which term includes known promoters, in part or in their entirety, which may be constitutively acting or it may be a regulated promoter inducible only under certain conditions e.g. in the presence of a regulatory protein. This enables expression of the selected gene or genes to be restricted e.g. to particular cell types or to cells in which a particular exogenous signal is present. For example, heavy metal induction of a

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