Title:  Inhibition of HIV-1 replication by antisense RNA expression

United States Patent:  6,776,986

Issued:  August 17, 2004

Inventors:  Boehnlein; Ernst (Randolph, NJ); Escaich; Sonia (Paris, FR); Ilves; Heini (Palo Alto, CA); Veres; Gabor (Palo Alto, CA)

Assignee:  Novartis AG (Basel, CH)

Appl. No.:  194300

Filed:  November 23, 1998

PCT Filed:  June 6, 1997

PCT NO:  PCT/EP97/02952

PCT PUB.NO.:  WO97/46673

PCT PUB. Date:  December 11, 1997

Abstract

Novel antisense sequences to the unspliced or single spliced portions of mRNA transcript from HIV-1 provirus, optionally co-expressed with an inhibitory transdominant mutant HIV-1 protein, are found to be useful in the treatment of HIV-1 infection.

SUMMARY OF THE INVENTION

Hereinafter we present the results of the antiviral activity of sequences complementary to the pol, vif, env genes and 3'LTR in HIV-1 infection experiments using a human CD4⁺ T cell line (CEM-SS) and peripheral CD4⁺ T lymphocytes (PBLs). Retroviral vectors are constructed expressing chimeric RNAs containing 1,100-1,400 nt long complementary HIV-1 sequences. The most efficient inhibition of HIV-1 replication is observed with an antisense sequence complementary to the HIV-1 env gene both in the CEM-SS cell line and in PBLs. This strong antiviral effect is further demonstrated in high inoculation dose infection experiments where reduction of the HIV-1 mRNAs correlates with low level of Gag and Tat protein production indicating that antisense RNA acts early during HIV-1 replication. Comparing the anti-HIV-1 efficacy of the antisense RNAs to the well documented (Bevec, D.,et al. 1992. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative rev trans-activator. Proc. Natl. Acad. Sci. USA 89:9870-9874, Escaich, S., et al 1995. RevM10-mediated inhibition of HIV-1 replication in chronically infected T cells. Hum. Gene Ther. 6:625-634, Malim, M. H., et al. 1992. Stable expression of transdominant rev protein in human T cells inhibits human immunodeficiency virus replication. J. Exp. Med. 176:1197-1201 and Nabel, G. J., et al. 1995. A molecular genetic intervention for AIDS--effects of a transdominant negative form of Rev. Hum. Gene Ther. 5:79-92) trans-dominant RevM10 protein demonstrates the potency of the antisense mediated inhibition of HIV-1 replication.

It has further been discovered that antisense sequences to the gag, env, and pol, especially the env and pol portions of the full length transcript are particularly effective.

The above mentioned antisense constructs are particularly useful for providing gene therapy to patients suffering from HIV-1 infection, e.g., by transducing the HIV-1-susceptable cells of such patients, e.g., CD4+ cells or cells which are progenitors of CD4+ cells, e.g., hematopoietic stem cells (for example CD34+/Thy-1+cells), with the antisense constructs of the invention, so that the transduced cells and their progeny are resistant to HIV-1 infection.

The antisense constructs of the invention are suitably prepared by incorporating a wild-type HIV-1 gene or gene fragment into a vector in reverse orientation with respect to its promotor so that when the gene is incorporated into the genome of the host cell and transcribed, the opposite strand of the DNA is transcribed, producing a messenger RNA transcript which is complementary to the mRNA from the wild-type gene or gene fragment and will anneal with it to form an inactive RNA-RNA duplex, which is subject to degradation by cellular RNases.

Transduction of the HIV-1 susceptable cells using the antisense vectors can be carried out in vivo or ex vivo, but is suitably carried out ex vivo, by removing blood from the patient, selecting the target cells, inoculating them with a vector containing the antisense construct of the invention, and reintroducing the transduced cells into the body. By natural selection, the transduced HIV-1 resistant cells will replace the native HIV-1 susceptible cells, thereby enabling the patient to overcome the infection and regain immunocompetence. Alternatively, the patient receives non-autologous CD4+ cells or progenitors of CD4+ cells from a compatable donor which cells have been transduced with the antisense construct of the invention.

The invention thus provides:

1. A nucleic acid sequence which, when stably integrated into a human cell, is capable of generating mRNA which anneals e.g., under in vivo conditions, with a mRNA transcript from an HIV-1 provirus encoding env, env and pol or env, pol and gag and which is at least 0.6 kb, preferably at least 1 kb in length, most preferably 1-2 kb, e.g. from 1.1 to 1.5 kb; and which is selected from:

(i) a sequence which is antisense to the 1.4 kb fragment from the Apa1 cleavage site at ca. base 2004 of an HIV-1 provirus to the Pflm 1 cleavage site ca. base 3400 of an HIV-1 provirus, e.g. which is antisense to the sequence in FIG. 1 (SEQ. ID. NO. 1);

(ii) a sequence which is antisense to the 1.2 kb fragment from the Pflim 1 cleavage site ca. base 3400 of an HIV-1 provirus to the EcoR1 cleavage site ca. base 4646 of an HIV-1 provirus, e.g. which is antisense to the sequence in FIG. 2 (SEQ. ID. NO. 2);

(iii) a sequence which is antisense to the 1.3 kb fragment from the ApaL1 cleavage site ca. base 6615 of an HIV-1 provirus to the Bsm1 cleavage site ca. base 8053 of an HIV-1 provirus, e.g., which is antisense to the sequence in FIG. 3 (SEQ. ID. NO.3); and

(iv) a sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99%, homologous to a sequence according to (i), (ii), or (iii) and which is capable of generating mRNA which annealss to the same mRNA transcript as that hybridizing to mRNA generated by (i), (ii), or (iii).

It is understood that the nucleic acid described in 1 above will be in RNA for when in a retroviral vector and will be converted to DNA upon incorporation of the provirus into the target cell. It is intended that both the RNA and DNA forms of the constructs are included within the scope of the invention.

The invention further provides

2. A vector comprising an antisense sequence according to 1 above.

The vector may be any vector capable of transducing a human hematopoietic cell, for example, an ecotropic, xenotropic, amphotropic or pseudotyped retroviral vector, an adeno-associated virus (AAV) vector, or an adenovirus (AV) vector. Preferably, the vector is a retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), or murine embryonic stem cell virus (MESV), or for example, a vector from the pLN series described in Miller and Rosman (1989) BioTechniques 7, pp. 980-986. The antisense sequence replaces the retroviral gag, pol and/or env sequences. The promotor controlling expression of the antisense may be a strong viral promoter, for example MoMLV LTR.

The range of host cells that may be infected by a retrovirus or retroviral vector is generally determined by the viral env protein. The recombinant virus generated from a packaging cell can be used to infect virtually any cell type recognized by the env protein provided by the packaging cell. Infection results in the integration of the viral genome into the transduced cell and the consequent stable expression of the foreign gene product. The efficiency of infection is also related to the level of expression of the receptor on the target cell. In general, murine ecotropic env of MoMLV allows infection of rodent cells, whereas amphotropic env allows infection of rodent, avian and some primate cells, including human cells. Xenotropic vector systems utilize murine xenotropic env, and also allow infection of human cells. The host range of retroviral vectors may be altered by substituting the env protein of the base virus with that of a second virus. The resulting, "pseudotyped" virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line. For example, the G-glycoprotein from vesicular stomatitis virus (VSV-G) may be substituted for the MMLV env protein, thereby broadening the host range. Preferably the vector and packaging cell line of the present invention are adapted to be suitable for transduction of human cells. Preferably, the vector is an amphotropic retroviral vector, for example, a vector as described in the examples below.

Optionally, the vector may contain more than one antisense sequence according to 1 above, e.g., two different antisense sequences, for example to pol and env, as described in the examples below.

Preferably, the construct lacks the retroviral gag, pol and/or env sequences, so that the gag, pol and env functions must be provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce replication-defective or transducing virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but generally will not produce infectious viral particles since it is lacking essential viral sequences. The packaging cell line is preferably transfected with separate plasmids encoding gag-pol and env, so that multiple recombination events are necessary before a replication-competent retrovirus (RCR) can be produced. Suitable retroviral vector packaging cell lines include those based on the murine NIH/3T3 cell line and include PA317 (Miller & Buttimore (1986) Mol. Cell Biol. 6:2895; Miller & Rosman (1989) Bio Techniques 7:980), CRIP (Danos & Mulligan (1988) Proc. Natl Acad Sci USA 85:6460), and gp+am12 (Markowitz et al. (1988) Virology 167:400); and also cell lines based on human 293 cells or monkey COS cells, for example ProPak A packaging cells, e.g., as described in Pear et al. (1993) Proc. Natl. Acad. Sci. USA 90:8392-8396; Rigg et al., (1996) Virology 218; Finer, et al. (1994) Blood 83:43-50; Landau, et al. (1992) J. Virol. 66:5110-5113. Retroviral vector DNA can be introduced into packaging cells either by stable or transient transfection to produce retroviral vector particles.

The antisense constructs of the invention have the further advantage that they will not interfere with expression of HIV inhibitory proteins, e.g., transdominant mutant proteins corresponding to the early phase short MRNA transcripts, for example mutants of tat or rev. Expression of such transdominant mutant proteins is useful in treating HIV infection because the mutant proteins interfere with the function of the wild-type HIV proteins and so inhibit HIV replication. A transdominant mutant protein of particular interest is RevM10, the use of which is described e.g., in Escaich, et al. Hum. Gene Ther. (1995) 6:625-634, and in WO 90/14427. Previously, co-expression of HIV antisense and transdominant mutant proteins was considered impractical because it was expected that the antisense would interfere with expression of the mutant protein. Using the antisense constructs of the invention, co-expression of the antisense with the transdominant mutant protein is not only feasible but provides a synergistic inhibition of the HIV by interfering with the virus at different stages of its replication cycle.

Thus the invention provides in a further embodiment:

3. A retroviral vector according to 2 above (i.e., comprising an antisense sequence according to 1 above) and further comprising a gene for an HIV-1 inhibitory protein, e.g., a gene for a transdominant mutant form of tat or rev, especially the gene for RevM10.

Packaging cell lines comprising the vectors according to 2 or 3 above, e.g, as described above, are also within the scope of the invention.

The invention also provides in a further embodiment:

4. A cellular composition comprising at least one human hematopoietic cell (e.g. CD4+ cell or progenitor of CD4+ cells, e.g., a stem cell, e.g., a CD34+/Thy-1+ cell) stably transduced with an antisense sequence according to 1 above and optionally additionally transduced with a gene for a transdominant mutant form of tat or rev, especially RevM10, e.g., transduced with a vector according to 2 or 3, supra, e.g., for use in a method according to 5 below;

The invention also provides in a further embodiment:

5. A method for treatment of HIV-1 infection in a subject in need thereof comprising

isolating hematopoietic cells (e.g. CD4+ cells or progenitors of CD4+ cells, e.g., stem cells, e.g., CD34+/Thy-l+ cells) from said patient;

transducing said cells with an antisense sequence according to 1 above, and optionally additionally or simultaneously transducing said cells with a gene for an HIV-1 inhibiting transdominant mutant form of tat or rev, especially RevM10, e.g., transducing said cells with a vector according to 2 or 3, supra; and

reintroducing the transduced cells into the patient.

The invention also provides in a further embodiment:

6. The use of an antisense sequence according to 1 above or a vector according to 2 or 3 above in the manufacture of a cellular composition according to 4 above or in a method of treatment according to 5 above.

Claim 1 of 30 Claims

What is claimed is:

1. A nucleic acid sequence which, when stably integrated into a human cell, is capable of generating mRNA which anneals with a mRNA transcript from an HIV-1 provirus encoding env and pol or env, pol and gag, wherein the nucleic acid sequence is 1.1 to 1.5 kb in length and is antisense to the 1.4 kb fragment from the Apa1 cleavage site at ca. base 2004 of an HIV-1 provirus to the Pflm1 cleavage site ca. base 3400 of an HIV-1 provirus.

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