Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-04-08
2002-08-06
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C424S188100, C424S208100
Reexamination Certificate
active
06429009
ABSTRACT:
The present invention relates to a composition and to a method of imparting resistivity to superinfection with HIV.
More in particular the invention concerns a composition and a method to confer resistance to human cells, in particular T-cells, to the superinfection with a retrovirus, specifically the human immunodeficiency virus (HIV).
Several experimental strategies aimed at blocking HIV replication in vivo have been proposed. So far, most have been based on immunotherapeutic or chemotherapeutic approaches, in which the anti-HIV mechanism of action is well known. However, none of the immunotherapeutic and chemotherapeutic approaches proposed thus far has been demonstrated to effect a resolutive anti-HIV therapy.
The goal of a more recently developed alternative anti-HIV experimental design was to render the target cell resistant to HIV replication through the induction of intracellular immunization (Baltimore, Nature 335: 395-396 (1988)). Inhibition of the superinfecting HIV was, in fact, demonstrated in cells expressing HIV-trans-dominant proteins (Malim et al., J. Exp. Med. 176: 1197-1201 (1992); Green et al., Cell 58:215-223 (1989); Modesti et al., New Biol. 3:759-768 (1991); Trono et al., Cell 59:113-120 (1989), Lisziewicz et al., Annual Meeting, Laboratory of Tumor Cell Biology, Gene Therapy (1993); Buchschacher et al., Hum. Gene Ther. 3:391-397 (1992); Stevenson et al., Cell 83:483-486 (1989); and Liu et al., Gene Therapy 1:32-37 (1994)), HIV genome-directed ribozymes (Yu et al., Gene Therapy 1:13-26 (1994); Lorentzen et al., Virus Genes 5:17-23 (1991); Sioud et al., PNAS USA 88:7303-7307 (1991); Weerasinghe et al., J. Virol. 65:5531-5534 (1991); and Yamada et al., Gene Therapy 1:38-45 (1994)), tat/rev decoys (Sullenger et al., Cell 63:601608 (1990); Sullenger et al., J. Virol. 65:6811-6816 (1991); and Smith et al., UCLA/UCI AIDS Symposium: Gene Therapy Approaches to Treatment of HIV Infection (1993)), or antisense RNA (Rhodes et al., J. Gen. Virol. 71:1965-1974 (1990); Rhodes et al., AIDS 5: 145-151 (1991), Sczakiel et al., J. Virol. 65:468-472 (1991); Joshi et al., J Virol. 65:5524-5530 (1991), and Chatterjee et al., Science 258:1485-1488 (1992)). Moreover, an effective anti-HIV intracellular immunization was achieved by transfecting HIV-susceptible cells with DNA coding for either an anti-gpl60 single chain antibody (Marasco et al., PNAS USA 90:7889-7893 (1993)) or a monoclonal anti-rev single chain variable region (Duan et al., Abstract from the 1994 Annual Meeting, Laboratory of Tumor Cell Biology, Sep. 25-Oct. 1, 1994, MD USA).
The prior art methods, however, suffer from the following disadvantages. Many of the above mentioned methods do not work properly in practice because of the fact that anti-HIV compounds able to target a single step of the HIV replication could easily induce the emergence of HIV resistant mutants. This is a very common biological event, considering the extraordinary ability of HIV to mutate as demonstrated, for example, by the HIV strains resistant to antiretroviral chemical compounds (i.e., AZT, ddI) isolated from AIDS patients treated with such drugs. Consequently, many investigators are attempting to synthesize anti-HIV reagents able to target different steps of the HIV life cycle. In addition, some of the described methods have been demonstrated to be either deleterious for the host cells, or difficult to be effectively applicable in clinical protocols.
The present invention seeks to overcome the disadvantages of the above-described methods. The authors of the invention have set up a method to confer resistitivity to human cells, in particular to T-cells, to HIV superinfection, following to a stable integration of an HIV non-infective non-producer genomic variant into at least some of cell nuclear genomes. In order to confer resistivity to human cells to HIV superinfection, the instant invention provides a composition to be used for HIV intracellular immunization, during the AIDS therapy. the invention has several advantages, including an easy way of administration, the lacking of HIV spontaneous reversion of non defective variants from a non producer to a producer phenotype, and the ability to confer a good resistance to superinfection.
Evidence obtained by in vitro experiments indicates that F12-HIV non defective non producer variant expression could inhibit different steps of the wild type HIV superinfecting life cycle. In fact, the authors of the invention have shown a block of the wild-type HIV replication, eithr before or after its own tetrotrascrption, depending on the F12-HIV expressing cells tested. Moreover, no modifications or impairments of the physiologic cell functions were demonstrated in any cells harbouring the F12-HIV genome.
“HIV” is used herein to encompass all designations past and present assigned to those viruses implicated as causative agents of acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC), such as HIV, e.g., HIV-1 and HIV-2, and HTLV, e.g., HTLV-III. By “superinfection” is meant that which is known to and understood by those of ordinary skill in the art, i.e., the ability of a given virus to infect a cell already infected by a virus. By “resistivity” is meant the capacity to resist superinfection by a virus, in particular a retrovirus, specifically HIV. By “nondefective” is meant that the genome is complete, whereas by “nonproducer” is meant that viral particles are not produced.
The instant invention providees a composition comprising a retroviral vector comprising a non-defective, non-producer, HIV variant genome, in a sufficient amount to get a stable integration of said non-defective, non-producer, HIV variant genome, in at least some of nuclear genomes of target cells, and the expression of said non-defective, non-producer, HIV variant genome in said target cells, in order to confer resistivity to HIV superinfection to cells, in particular to T cells.
Preferably, the non-defective, non-producer, HIV variant genome integrates in at least the 1% of cell nuclear genomes. More preferably the non-defective, non-producer, HIV variant genome integrates in from about 1% to about 10% of cell nuclear genomes. Preferably the non-defective, non-producer, HIV variant genome is the HIV F12 genome. It is further preferred that the 3′ LTR of the F12-HIV genome be deleted prior to insertion into the retroviral vector and that the genome be inserted into the retroviral vector in antisense orientation. A retroviral vector derived from Moloney murine leukemia virus is preferred for the composition of the invention.
A further aspect of the invention provides the use of a retroviral vector comprising a non-defective, non-producer, HIV variant genome in sufficient amount to get a stable integration of the non-defective, non-producer, HIV variant genome into at least some of the nuclear genomes of the cells get in contact with said vector and the expression into said cells of said nondefective, nonproducer HIV variant genome.
Preferably, the non-defective, non-producer, HIV variant genome integrates in at least the 1% of cell nuclear genomes. More preferably the non-defective, non-producer, HIV variant genome integrates in from about 1% to about 10% of cell nuclear genomes. Preferably the non-defective, non-producer, HIV variant genome is the HIV F12 genome. It is preferred that the 3′ LTR of the F12-HIV genome be deleted prior to insertion into the retroviral vector and that the genome be inserted into the retroviral vector in antisense orientation. A retroviral vector derived from Moloney murine leukemia virus is preferred for the composition of the invention.
Alternatively the instant invention provides the use of a composition comprising a retroviral vector a non-defective, non-producer, HIV variant genome to be utilised for the production of a AIDS therapy medicament.
An applicative method of the invention is of imparting resistivity to HIV superinfection to human cells, by means of removing cells from an human, contacting the removed-cells with the composition of the invention in suff
Federico Maurizio
Ferrari Giuliana
Mavilio Fulvio
Verani Paola
Istituto Superiore Disanita′
Parkin Jeffrey S.
Scheiner Laurie
Smith , Gambrell & Russell, LLP
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