WO1995027783A1 - Inhibition of hiv-1 multiplication in mammalian cells - Google Patents

Inhibition of hiv-1 multiplication in mammalian cells Download PDF

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WO1995027783A1
WO1995027783A1 PCT/CA1995/000190 CA9500190W WO9527783A1 WO 1995027783 A1 WO1995027783 A1 WO 1995027783A1 CA 9500190 W CA9500190 W CA 9500190W WO 9527783 A1 WO9527783 A1 WO 9527783A1
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htv
cells
signal
rna molecules
antisense orientation
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PCT/CA1995/000190
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French (fr)
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Sadna Joshi-Sukhwal
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Joshi Sukhwal Sadna
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV

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  • This invention relates to human immunodeficiency virus type 1 (HIV-1) and to inhibition of multiplication thereof in mammalian cells expressing chimeric RNA molecules containing HTV-l packaging ⁇ signal and Gag coding sequences in antisense orientation; to said cells and therapeutic compositions comprising said cells; and retroviral vectors expressing said chimeric RNA molecules.
  • HSV-1 human immunodeficiency virus type 1
  • AIDS Acquired immunodeficiency syndrome
  • HTV- 1 a retrovirus, called HTV- 1 , which mainly infects T-lymphocytes and monocytes/macrophages derived from haematopoietic stem cells.
  • Tat-TAR, Rev-RRE, and gag/i * signal interactions are crucial for rr ⁇ /w-activation, late gene expression, and virion RNA packaging, respectively. Interference during these processes may take place by providing the cell with interfering RNA or protein molecule(s); e.g. the sense (decoys of viral protein binding sites) or antisense RNAs to TAR, RRE, signal, and Tat, Rev, or Gag open reading frames or trans-dominant mutants of HTV-l Tat, Rev, or Gag proteins.
  • the Tat protein of HTV-l allows tians-activation of HTV-l gene expression, while Rev protein of HTV-l allows the switch from early to late gene expression. While both of these genes overlap with each other, they are translated in different reading frames.
  • Antisense RNA to HTV-l Tat/Rev mRNA has been shown to confer resistance to HTV-l infection in mammalian cell lines (1, 2, 3, 4).
  • Antisense RNA complementary to a specific portion of HTV-l RNA molecule, upon hybridization with target RNA sequences, disrupt reverse transcription, processing, translation, and/or transport of this RNA.
  • Antisense RNAs have been shown to alter specific gene expression in several cell systems, including bacteria, Xenopus oocytes, Drosophila embryos, plants, and mammalian cells (5, 6). The degree of inhibition obtained in these studies was variable and depended upon many factors, including size, hybridization location, secondary structure, and level of expression of both the antisense RNA and the target mRNA whose expression was being modulated. Synthetic oligodeoxynucleotides, when added to the culture medium, have also been shown to inhibit HTV-l multiplication.
  • a sense RNA approach has been used to block replication of the genome of a plant RNA virus by employing the origin of replication located at the 3' end of the genome as a competitive inhibitor for viral replicase (7).
  • RNA-RNA and RNA-protein interactions are crucial for HTV-l replication, trans-activation, transcription, transport, translation, and packaging, and the HTV-l RNA sequences involved in these interactions are known.
  • Non-HTV-1 RNAs containing TAR sequence in a sense orientation have been shown to compete with HTV-l mRNAs for binding to RNA and/or protein and to result in inhibition of HTV-l multiplication.
  • the cw-acting TAR element is a 59 nucleotide-long RNA stem-loop structure present at the 5' end of all HTV-l transcripts (8).
  • the Tat protein binds to a bulge region present within this structure.
  • Tat binding in itself is not sufficient (9) and a number of specific TAR RNA-binding cellular proteins are required for HTV-l r/r ⁇ w-activation.
  • Retroviral vectors expressing HTV-l TAR RNA decoys (10, 11, 12, 13) have been shown to confer HTV-l resistance.
  • Retroviral vectors expressing antisense RNA to the HTV-l tat gene have also been shown to confer HTV-l resistance (1, 2, 3, 4, 14).
  • HTV-l Tat protein when expressed from retroviral vectors in either Tat- or Tat- and Rev-inducible manner, failed to protect cells against HTV-l infection (16, 17).
  • Vectors expressing antisense RNA targeted to the Gag mRNA 5' leader region (18, 19) have also been shown to inhibit HTV-l multiplication.
  • the Gag (p55) and Gag-Pol (pi 60) polyproteins are translated from the 9.4 kilobase (kb) genomic mRNA.
  • the pol gene is expressed as a result of frameshift near the end of the gag reading frame.
  • the viral protease cleaves the Gag polyprotein (p55) into the pl7, p24, p7, and p9 proteins, and the Gag-Pol polyprotein (pl60) into the p6, pll (protease), p51, p64 (2 subunits of reverse transcriptase, RT), pl5 (RNase H), and p34 (integrase) proteins.
  • RRE is a 234 nucleotide-long RNA sequence located within the env reading frame (20, 21). RRE has been predicted to form a highly complex secondary structure containing a central stem I surrounded by stem-loops ⁇ , LI, TV and V (21). The 66 nucleotide-long stem-loop II has been found to contain the primary Rev binding site and is also sufficient for Rev response in vivo (22, 23). In the absence of Rev, the translation of unspliced and singly spliced mRNA into protein is prevented by cw-acting repressor sequences (CRS) present in the HTV-l gag, pol, and env open reading frames (20, 24, 25).
  • CRS cw-acting repressor sequences
  • Rev-RRE interaction is sufficient to override the inhibitory action of the CRS such that these mRNAs can now reach the cytoplasm and become translated.
  • Plasmids expressing one, three, and six copies of RRE have been shown to interfere with the HTV-l Rev protein activity in a transient co-transfection experiment performed in HeLa cells (26); over expression of RRE decoys has also been shown to inhibit HTV-l multiplication in CEM cells (27).
  • retroviral vectors allowing constitutive or Tat-inducible expression of taz/w-dominant mutants of either Rev (16) or Tat and Rev (15, 17, 28) were shown to confer resistance to HTV-l infection.
  • the HTV-l signal is required in cis for specific recognition and packaging of the viral genomic RNA; two copies of the HTV-l genomic RNA are encapsidated per virus particle. Nucleotides located between the major splice donor site and the Gag initiation codon are essential for HTV-l RNA packaging (29, 30, 31); this region has been shown to fold into a stable secondary structure involving four stem-loops (32). The precise length of the HTV-l signal required for packaging is not known but it can be inferred from studies performed using Moloney murine leukemia virus (MoMuLV) (33) that it would be contained within 1000 nucleotides downstream of the primer binding site.
  • MoMuLV Moloney murine leukemia virus
  • the HTV-l ⁇ signal is recognized in cis by the zinc finger motif within the nucleocapsid domain and by. one other domain of the HTV-l Gag polyprotein precursor (34, 35). Cells allowing constitutive expression of trans dominant mutant
  • Gag proteins have been shown to repress HTV-l replication (36).
  • the HTV-l Rev rr ⁇ /w-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, pp.
  • HIV-1 structural gene expression requires binding of the Rev fr ⁇ /w-activator to its RNA target sequence.
  • RNA sequences in the gag region of HTV-l decrease RNA stability and inhibit expression in the absence of Rev protein. J. Virol. 66, pp. 150-159.
  • RNA packaging signal of HTV-l Virology 188, pp. 590-599. 32.
  • the HTV-l packaging signal and major splice donor region have a conserved stable secondary structure. J.
  • HTV-l gag mutants can dominantly interfere with the replication of the WT virus. Cell 59, pp. 113-120.
  • HIV-1 gene(s) to infect patients' bone marrow (BM) stem cells or peripheral blood lympocytes (PBLs) which, upon transplantation and differentiation, would potentially give rise to an HTV-l resistant immune system.
  • BM bone marrow
  • PBLs peripheral blood lympocytes
  • the invention provides a gene therapeutic method of inhibiting HTV-l multiplication in a mammal comprising treating said mammal with an effective amount of mammalian cells expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the mammalian cells are human bone-marrow cells, and more preferably, blood cells.
  • the invention provides mammalian cells harboring proviral vector DNA expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the invention provides a retroviral vector expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the retroviral vector is derived from the Moloney murine leukemia virus (MoMuLV).
  • MoMuLV Moloney murine leukemia virus
  • MoMuLV-derived retroviral vectors were engineered to express HTV-l ⁇ signal and Gag coding sequences in anti-sense orientation in chimeric RNAs. These sequences were expressed under control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Both, ⁇ signal and Gag coding sequences were expressed as part of the 3' untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD 4 + lymphocyte-derived MT4 cell line.
  • HSV herpes simplex virus
  • tk thymidine kinase
  • the invention provides a therapeutic composition for confering HTV-l resistance to a mammal comprising cells as hereinbefore defined in association with a pharmaceutically acceptable carrier, diluent or adjuvant therefor. It will be readily understood by the person skilled in the art that the cells should be present in an effective therapeutic amount.
  • PBLs bone marrow
  • BM cells contain stem cells which are capable of both self-renewal and differentiation into lymphocytes, macrophages, and other hematopoietic cells such as erythrocytes, granulocytes, and megakaryocytes.
  • stem cells represent only 1 per 10,000 nucleated cells present within the bone marrow, a large number of bone marrow cells are infected by retroviral vectors to ensure transformation of these rare stem cells.
  • Gene therapy for the treatment of AIDS consists of using retroviral vectors to deliver the anti-HTV-1 RNA molecules to human PBLs and BM stem cells.
  • Transformants selected in vitro are transplanted back to the patient. Following differentiation, these transformed cells lead to the development of an immune system in which various blood cells (including CD4 lymphocytes and macrophages) express the anti-HTV-1 RNA molecules and are therefore resistant to HTV-l.
  • Preferred methods of in vitro stimulation and culture for gene transfer into mammalian cells, particularly stem cells, with a gene transfer vector, particularly, a retrovious vector are disclosed in Canadian Patent Application No. 2086844, published July 8, 1994 - Dube et al, which disclosure is included herein by reference. Following appropriate stimulation and culture in vitro.
  • PBLs and BM cells are infected by cocultivation with packaging cell lines producing retroviral vector particles. Transformants are then selected in vitro for growth in medium containing appropriate cytokines and antibiotics. These transformants are then transplanted back to the patient. Following transplantation and differentiation, blood samples are tested for anti-HTV-1 gene expression and for the ability of these cells to resist HTV-l infection. Other disease symptoms, viral load and emergence of resistant HTV-l isolates are examined as well.
  • Fig.1 represents LTR-LTR sequences present in the proviral DNA integrated in the target cell line as part of the map of retroviral vectors expressing antisense RNA to HTV-l ⁇ signal and Gag coding sequence; and Fig. 2 shows the results of HTV-l infections on a pool of stable MT4 transformants expressing HTV-l ⁇ signal and Gag coding sequence-containing RNAs.
  • the retroviral vector, pUCMoTN (39), used in this study, is derived from MoMuLV. This vector allow neo gene expression (conferring G418 R ) under control of HSV tk. The ⁇ signal and Gag sequences were cloned in this vector as part of the 3' untranslated region of the neo mRNA.
  • the pUCMoTN- ⁇ Gag " and pUCMoTN- ⁇ + Gag + vectors were constructed as follows.
  • a 4.0 EcoRI fragment from pBKBHIOS (NIH#182) was cloned into the EcoRI site of the pUC18 vector (Pharmacia).
  • a 1440 bp BamHJ-BglR fragment containing HTV-l ⁇ signal and Gag coding sequences was isolated from this pUC- ⁇ vector.
  • This fragment (containing Sstl-BglR sequences of HTV-l strain HXB2) was cloned into the unique Bam ⁇ I site in the pUCMoTN-Rzl vector (40).
  • the resulting clones were characterized by restriction enzyme analysis and clones containing a single copy of the ⁇ signal and Gag coding sequences in antisense (pUCMoTN- ⁇ 'Gag") and sense (pUCMoTN- ⁇ Gag " ) orientations, with respect to the vector were selected.
  • ecotropic Psi-2 (41) and amphotropic PA317 (ATCC cat# CRL0978) (42) packaging cell lines were cultured in ⁇ -MEM medium supplemented with 2mM
  • L-Gln 0.1 volume of antibiotics/antimycotic solution (penicillin, 1000 units/ml; streptomycin, 1000 ⁇ g/ml; Fungizone R , 2.5 ⁇ g/ml), and 10% FBS (Hyclone) at 37°C in a humidified atmosphere with 5% CO 2 .
  • the human CD 4 + lymphocyte-derived MT4 suspension cell line, NTH Cat #120 was cultured in RPMI 1640 medium also supplemented with Gin, antibiotics/antimicotic agents, and FBS (GIBCO) as above and were incubated at 37°C in a humidified atmosphere with 5% CO 2 .
  • the selective media was prepared as above except that it also contained G418 (200 ⁇ g/ml for Psi-2 and PA317 cell lines; and 400 ⁇ g/ml for MT4 cell line).
  • Psi-2 cells were transfected as follows using the Calcium phosphate co- precipitation technique (using the CellPhect Transfection Kit from Pharmacia): a 120 ⁇ l retroviral DNA solution (3 ⁇ g) was mixed with 120 ⁇ l Buffer A (0.5 M CaCl 2 , 0.1 M HEPES) and incubated at 22°C for 10 min. An equal volume (240 ⁇ l) of Buffer B (0.28 M NaCl, 0.05 M HEPES, 0.75 mM NaH 2 PO 4 , 0.75 mM Na 2 HPO 4 ) was added, mixed immediately by vortexing and incubated for 15 min. The mixture was then added drop wise to the cell culture (50% confluent in 60 mm plates containing 3 ml fresh medium).
  • Buffer A 0.5 M CaCl 2 , 0.1 M HEPES
  • the cells were incubated under normal growth conditions for 6 hrs, then washed twice with fresh medium.
  • the cells were subjected to glycerol shock with 1.5 ml 15% glycerol in 10 mM HEPES pH 7.5, 150 mM NaCl for 3 min at 22°C, then washed once with fresh medium.
  • Fresh medium (5 ml) was added and the cells were grown under normal conditions for 2 days.
  • the transfected cells were washed once with phosphate-buffered saline (PBS) containing antibiotics/antimycotic agents, trypsinized with 0.05% trypsin, 0.53 mM EDTA-4Na (GIBCO), transferred to 100 mm plates and grown in selective medium containing 200 ⁇ g/ml G418. The medium was changed every 3-4 days until selection was complete (15-20 days). The number of resistant colonies was then determined. The cells were washed with PBS, trypsinized and re-seeded.
  • PBS phosphate-buffered saline
  • G418 0.53 mM EDTA-4Na
  • Vector particles released from the transformed Psi-2 cells were obtained by filtering culture medium from cells at 50-100% confluency through a 0.22 ⁇ m filter. These particles were used to infect PA317 cells as described previously (43). Essentially 2 x 10 s cells were seeded for 6 hours in 60 mm tissue culture dishes in 4 ml medium, after which this medium was replaced by 1 ml medium containing 8 ⁇ g/ml polybrene and 100 ⁇ l vector particles. After a 2 hour incubation at 37°C, 3 ml medium was added and the incubation continued for 16 more hours. Cells were then trypsinized and transferred to 100 mm tissue culture dishes in the presence of selective medium containing 200 ⁇ g/ml G418. The selective medium was changed every 4-5 days and the number of colonies counted after 14 days. Vector particles released from the PA317 cells (50-100% confluent) were then collected and used to infect MT 4 cells.
  • the MT4 cells (3 x 10 5 ) were pelleted and resuspended in 0.5 ml RPMI 1640 medium containing 16 ⁇ g/ml polybrene. Vector particles (0.2 ml) and RPMI 1640 medium (0.3 ml) were then added and gently mixed to the cells. Cells were transferred to 60 mm petri dishes, and incubated under normal growth conditions for 2 hrs. Four ml of fresh RPMI 1640 medium were then added and the cells were grown overnight. The infected cells were then centrifuged, resuspended in selective medium containing 400 ⁇ g/ml G418 and transferred to 100 mm petri dishes. Every 3-4 day, half of the cell suspension was removed and replaced with fresh selective medium. By day 20, all of the uninfected cells had died and the remaining stably transformed cells were frozen and were used in the following experiments.
  • PCR Polymerase chain reaction
  • RT reverse transcription
  • Genomic DNA isolated (37) from the MT4 cells stably transformed with MoTN-t '" Gag " vector particles was used in PCR as follows. PCR reaction (100 ⁇ l) was performed in the presence of MgCl 2 (1.5 mM), oligonucleotides (20 mM each; amplification buffer (1 x concentration; Promega), dNTPs (10 mM each), genomic DNA (1 ⁇ g), and Taq polymerase (2 units, Promega).
  • the samples were overlaid with 100 ⁇ l mineral oil and amplified using Perkin-Elmer Cetus Instruments DNA Thermal Cycler by using three linked files as follows: File 1, STEP-CYCLE 1 min at 95 °C; File 2, STEP-CYCLE 1 min at 55 °C; File 3, STEP-CYCLE 1 min at 72°C; with a total of 45 cycles.
  • PCR products (10 ⁇ l aliquots) were then analyzed by electrophoresis on a 3% agarose gel.
  • RNA isolated from the MT4 cells stably transformed with MoTN- ⁇ " Gag " vector particles were grown for 48 hrs and then the total RNA was extracted using the Guanidium thiocyanate-Phenol-Chloroform procedure (44) .
  • Reverse transcription was performed as follows: total RNA (5 ⁇ g) was incubated with oligo dT (20 mM) for 10 min at 65°C in a total volume of 20 ⁇ l. The reaction mixture was chilled on ice for 2 min and RNA guard (75 units; Pharmacia), reverse transcription buffer (1 x concentration; BRL), DTT (5 mM), dNTPs (12.5 mM each), and
  • HTV-l Actively dividing various MT4 transformants (1 x 10° cells/ml) were each infected with HTV-l as follows: a 2 ml cell culture was incubated with 20 ⁇ l HTV-l strain NL -3, NIH Cat #78, (10 64 TdD 50 /ml) for 2 hrs at 37°C. The cells were then pelleted, washed 3 times with PBS, resuspended in 2 ml medium, transferred to 35 mm dishes and allowed to grow at 37 °C Every 3 days for up to day 30, a 1 ml sample containing cells and medium from each infected cell culture was removed and frozen at -70°C One ml of complete medium was added back to the culture each time.
  • MoMuLV-derived retroviral vector pUCMoTN was modified to express
  • HTV-l ⁇ signal and Gag coding sequences in antisense orientation This molecule was expressed as part of the 3' untranslated region of the neo mRNA in between the stop codon and the poly (A) site (Fig. 1).
  • the pUCMoTN- ⁇ - " Gag " vector expressed a single copy of antisense RNA to both of the HTV-l ⁇ signal and Gag coding region (Fig. 1).
  • Gag coding region-containing RNAs in these vectors is under the control of the MoMuLV 5' LTR and HSV tk promoters.
  • the aforementioned retroviral vectors were first used to transfect an ecotropic packaging cell line Psi-2; (41), and the vector particles released from this cell line were used to infect an amphotropic packaging cell line (PA317; 42).
  • the resulting amphotropic pseudotyped retroviral vector particles capable of infecting human cells were then used to infect a human CD4 + lymphoid (MT4) cell line and the G418 R stable transformants were selected.
  • the presence of the anti-HTV-1 gene and the level of therapeutic RNA/protein produced in these cells were then monitored as described below.
  • MoTN- ⁇ " Gag" vector particle The presence of HTV-l ⁇ signal and Gag coding sequences within their genome was confirmed by PCR analysis. The presence of ⁇ signal and Gag coding sequence-containing RNAs was confirmed by RT-PCR. As expected, a 315 bp PCR or RT-PCR product was visible in both cases.
  • MT4 cells stably transformed with MoTN- ⁇ " Gag " vector particles expressing antisense RNA to HIV-1 ⁇ signal and Gag coding sequences were challenged with HTV-l.
  • MT4 cells transformed with the parental retroviral vectors lacking test DNA sequences served as control.
  • Virus production was monitored by measuring the level of p24 antigen (HTV-l gag gene product) in the cell culture supernatant every 3 days for up to 30 days post-infection.
  • the MT4 transformants expressing antisense RNA to the ⁇ signal and Gag coding sequences delayed virus production for up to 30 days (Fig. 2).
  • HTV-l resistance of sense RNA-expressing cells was monitored as follows. MT4 cells stably transformed with MoTN- ⁇ + Gag + vector particles were subjected to challenge by HTV-l and virus production in the culture supernatant was measured every 3 days post-infection. MT4 cells expressing sense RNA to the HTV-l ⁇ sequence and Gag coding sequences failed to prevent HTV-l multiplication (Fig. 2).
  • retroviral vectors were, thus, engineered that expressed ⁇ signal and Gag coding sequences in antisense orientation. The retroviral vector particles were used to infect the human CD 4 + lymphocyte-derived MT4 cells and stable G418 R transformants were selected. The pool of these transformants was then infected with HTV-l and virus production measured for up to 30 days post-infection.
  • HTV-l ⁇ signal and Gag coding sequences in sense orientation HTV- 1 production began even earlier than in the control cells (Fig. 2). If the HTV-l ⁇ signal and Gag coding sequence-containing retroviral vector RNA was also packaged by HTV-
  • the infectivity of these chimeric RNA-containing virus particles should have been reduced.
  • the lack of resistance observed with the HTV-l ⁇ signal and Gag coding sequences expressed in the sense orientation may be explained by the fact that the length of the HTV-l ⁇ signal used in the present experiments was not sufficient to allow packaging of non-viral mRNA.

Abstract

A method of inhibiting human immunodeficiency virus type 1 (HIV-1) in a mammal using mammalian cells, particularly, human CD4 containing lymphocytes, which express chimeric RNA molecules containing HIV-1 γ signal and/or Gag coding sequences in antisense orientation. HIV-1 production was delayed up to 30 days when compared with control cells lacking the test DNA sequences. Retroviral vectors expressing the chimeric RNA molecules are provided.

Description

INHTOΓΠQN OF HIV-I MULTIPLICATION IN MAMMALIAN CELLS
FIELD OF INVENTION
This invention relates to human immunodeficiency virus type 1 (HIV-1) and to inhibition of multiplication thereof in mammalian cells expressing chimeric RNA molecules containing HTV-l packaging ψ signal and Gag coding sequences in antisense orientation; to said cells and therapeutic compositions comprising said cells; and retroviral vectors expressing said chimeric RNA molecules.
BACKGROUND TO THE INVENTION
Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus, called HTV- 1 , which mainly infects T-lymphocytes and monocytes/macrophages derived from haematopoietic stem cells.
An ideal step at which to inhibit virus multiplication would be during an early stage in the virus life cycle. However, if later stages during the virus life cycle are blocked, virus production will be inhibited, which in turn will prevent new rounds of infection. In this case, resistance will be confined to the second and subsequent rounds of infection. During the HTV-l life cycle, Tat-TAR, Rev-RRE, and gag/i * signal interactions are crucial for rrα/w-activation, late gene expression, and virion RNA packaging, respectively. Interference during these processes may take place by providing the cell with interfering RNA or protein molecule(s); e.g. the sense (decoys of viral protein binding sites) or antisense RNAs to TAR, RRE, signal, and Tat, Rev, or Gag open reading frames or trans-dominant mutants of HTV-l Tat, Rev, or Gag proteins.
The Tat protein of HTV-l allows tians-activation of HTV-l gene expression, while Rev protein of HTV-l allows the switch from early to late gene expression. While both of these genes overlap with each other, they are translated in different reading frames. Antisense RNA to HTV-l Tat/Rev mRNA has been shown to confer resistance to HTV-l infection in mammalian cell lines (1, 2, 3, 4). Antisense RNA, complementary to a specific portion of HTV-l RNA molecule, upon hybridization with target RNA sequences, disrupt reverse transcription, processing, translation, and/or transport of this RNA. Antisense RNAs have been shown to alter specific gene expression in several cell systems, including bacteria, Xenopus oocytes, Drosophila embryos, plants, and mammalian cells (5, 6). The degree of inhibition obtained in these studies was variable and depended upon many factors, including size, hybridization location, secondary structure, and level of expression of both the antisense RNA and the target mRNA whose expression was being modulated. Synthetic oligodeoxynucleotides, when added to the culture medium, have also been shown to inhibit HTV-l multiplication.
A sense RNA approach has been used to block replication of the genome of a plant RNA virus by employing the origin of replication located at the 3' end of the genome as a competitive inhibitor for viral replicase (7). RNA-RNA and RNA-protein interactions are crucial for HTV-l replication, trans-activation, transcription, transport, translation, and packaging, and the HTV-l RNA sequences involved in these interactions are known. Non-HTV-1 RNAs containing TAR sequence in a sense orientation have been shown to compete with HTV-l mRNAs for binding to RNA and/or protein and to result in inhibition of HTV-l multiplication.
The cw-acting TAR element is a 59 nucleotide-long RNA stem-loop structure present at the 5' end of all HTV-l transcripts (8). The Tat protein binds to a bulge region present within this structure. However, Tat binding in itself is not sufficient (9) and a number of specific TAR RNA-binding cellular proteins are required for HTV-l r/røw-activation. Retroviral vectors expressing HTV-l TAR RNA decoys (10, 11, 12, 13) have been shown to confer HTV-l resistance. Retroviral vectors expressing antisense RNA to the HTV-l tat gene have also been shown to confer HTV-l resistance (1, 2, 3, 4, 14). However f/røw-dominant mutant of HTV-l Tat protein (15), when expressed from retroviral vectors in either Tat- or Tat- and Rev-inducible manner, failed to protect cells against HTV-l infection (16, 17). Vectors expressing antisense RNA targeted to the Gag mRNA 5' leader region (18, 19) have also been shown to inhibit HTV-l multiplication.
The Gag (p55) and Gag-Pol (pi 60) polyproteins are translated from the 9.4 kilobase (kb) genomic mRNA. The pol gene is expressed as a result of frameshift near the end of the gag reading frame. Following budding of virus particles, the viral protease cleaves the Gag polyprotein (p55) into the pl7, p24, p7, and p9 proteins, and the Gag-Pol polyprotein (pl60) into the p6, pll (protease), p51, p64 (2 subunits of reverse transcriptase, RT), pl5 (RNase H), and p34 (integrase) proteins. RRE is a 234 nucleotide-long RNA sequence located within the env reading frame (20, 21). RRE has been predicted to form a highly complex secondary structure containing a central stem I surrounded by stem-loops π, LI, TV and V (21). The 66 nucleotide-long stem-loop II has been found to contain the primary Rev binding site and is also sufficient for Rev response in vivo (22, 23). In the absence of Rev, the translation of unspliced and singly spliced mRNA into protein is prevented by cw-acting repressor sequences (CRS) present in the HTV-l gag, pol, and env open reading frames (20, 24, 25). Rev-RRE interaction is sufficient to override the inhibitory action of the CRS such that these mRNAs can now reach the cytoplasm and become translated. Plasmids expressing one, three, and six copies of RRE have been shown to interfere with the HTV-l Rev protein activity in a transient co-transfection experiment performed in HeLa cells (26); over expression of RRE decoys has also been shown to inhibit HTV-l multiplication in CEM cells (27). As well, retroviral vectors allowing constitutive or Tat-inducible expression of taz/w-dominant mutants of either Rev (16) or Tat and Rev (15, 17, 28) were shown to confer resistance to HTV-l infection. The HTV-l
Figure imgf000005_0001
signal is required in cis for specific recognition and packaging of the viral genomic RNA; two copies of the HTV-l genomic RNA are encapsidated per virus particle. Nucleotides located between the major splice donor site and the Gag initiation codon are essential for HTV-l RNA packaging (29, 30, 31); this region has been shown to fold into a stable secondary structure involving four stem-loops (32). The precise length of the HTV-l signal required for packaging is not known but it can be inferred from studies performed using Moloney murine leukemia virus (MoMuLV) (33) that it would be contained within 1000 nucleotides downstream of the primer binding site. The HTV-l φ signal is recognized in cis by the zinc finger motif within the nucleocapsid domain and by. one other domain of the HTV-l Gag polyprotein precursor (34, 35). Cells allowing constitutive expression of trans dominant mutant
Gag proteins have been shown to repress HTV-l replication (36).
Vectors expressing antisense RNAs targeted to the Gag mRNA 5' leader region (18, 19) have been shown to inhibit HTV-l multiplication.
REFERENCE LIST
The present specification refers to the following publications, each of which is expressly incorporated herein by reference.
1. RHODES, A. and JAMES, W. (1991). Inhibition of heterologous strains of HTV by antisense RNA. AIDS 5, pp. 145-151.
2. RITTNER, K. and SCZAKIEL, G (1991). Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1.
Nucl. Acids Research. 19, pp. 1421-1426.
3. SCZAKIEL, G. , OPPENLANDER, M. , RITTNER, K. , AND PAWLITA, M. (1992). Tat- and Rev- directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis. J. Virol. 66, pp. 5576-5581.
4. LO, STEVE K.M., BIASOLO, M.A., DEHNI, G., PALU, G. and HASELTINE W.A. (1992). Inhibition of replication of HTV-l by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA. Virology, 190, pp. 176-183. 5. GREEN, PJ. , PINES, O. and INOUYE, M (1986). The role of antisense RNA in gene regulation. Ann. Rev. Biochem. 55, pp. 569-97.
6. TAKAYAMA, K.M. and INOUYE, M (1990). Anti-sense RNA. Critical reviews in Biochem. & Mol. Biol. 25: pp. 155-185.
7. MORCH, M.D., JOSHI, R.L. and HAENNI, A.L. (1987). A new "sense" RNA approach to block viral RNA replication in vitro. Anucl. Acids Res. 15, pp. 4123-4130.
8. SELBY M.J., BAIN, E.S., LUCIW, P.A., and PETERLIN, B.M. (1989). Structure, sequence, and position of the stem-loop in TAR determine transcriptional elongation by Tat through the HTV-l long terminal repeat. Genes Dev. 3, pp. 547-558.
9. ROY, S., DELLESfG, U., CHEN, C.H., ROSEN, C.A., and SONENBERG, N. (1990). A bulge structure in HTV-l TAR RNA is required for Tat binding and Tat-mediated trα^s-activation. Genes Dev. 4, pp. 1365-1373. 10. GRAHAM, G.J., and MAIO, J.J. (1990). RNA transcripts of the HTV trans activation response element can inhibit action of the viral trans-activator. Proc. Natl. Acad. Sci. USA, 87, pp. 5817-5821. 11. SULLENGER, B.A., GALLARDO, H.F., UNGERS, G.E., and GILBOA, E.
(1990). Overexpression of TAR sequences renders cells resistant the HTV replication. Cell 63, 601-608 and Analysis of trans-acting response decoy RNA-mediated inhibition of HTV-l trans-activation. J. Virol. 65, pp. 6811- 6816. 12. JOSHI, S., VAN BRUNSCHOT, A., ASAD, S., VAN DER ELST, I., READ,
S.E., and BERNSTEIN, A. (1991). Inhibition of HIV-1 multiplication by antisense and sense RNA expression. J. Virol. 65, pp. 5524-5530.
13. LISZIEWICZ, J., RAPPAORT, J. and DHAR, R. (1991). Tat-regulated production of multimerized TAR RNA inhibits HTV-l gene expression. New Biol. 3, pp. 82-89.
14. RHODES, A. and JAMES, W. (1990). Inhibition of HTV replication in cell culture by endogenously synthesized antisense RNA. J. Gen. Virol. 71, pp. 1965-1974.
15. GREEN, M., ISHINO, M., and LOEWENSTEIN, P.M. (1989). Mutational analysis of HTV-l tat minimal domain peptides: Identification of rrα/zj-dominant mutants that suppress HTV-LTR-driven gene expression. Cell 58, pp. 215-223.
16. BAHNER, I., ZHOU, C, YU, X.J., HAO, Q.L., GUATELLI, J.C. and KOHN, D.B. (1993). Comparison of taz/w-dominant inhibitory mutant HTV-l genes expressed by retroviral vectors in human T lymphocytes. J. Virol. 67, pp. 3199-3207.
17. LIEM, S.E. , LI. XIAOYI, and JOSHI, S. (1993) The development of retroviral vectors allowing either Tat- or Tat- and Rev-inducible expression of trαns- dominant mutants of HTV-l Tat and Rev proteins to confer anti-HTV-1 resistance. Human Gene Therapy 4, pp. 625-634. 18. SCZAKIEL, G., PAWLITA M. and KLEINHEINZ A. (1990). Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribved in sense and antisense orientation from the 5 'leader/gag region. Biochemical and Biophysical Research Communications Vol. 169, pp. 643-651. 19. SCZAKIEL, G. and PAWLITA, M. (1991). Inhibition of Human Immunodeficiency Virus Type 1 Replication in Human T Cells Stably Expressing Antisense RNA. J. Virol. 65, pp. 468-472. 20. ROSEN, C.A., TERWILLIGER, E., DAYTON, A., SODROSKI, J.G., and
HASELTINE, W.A. (1988). Intragenic cw-acting art gene-responsive sequences of the HTV. Proc. Natl. Acad Sci. U.S.A. 85, pp. 2071-2075.
21. MALIM, M.H., HAUBER, J., LE, S.Y., MAIZEL, J.V., and CULLEN, B.R. (1989). The HTV-l Rev rrα/w-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, pp.
254-257.
22. MALIM, M.H., TELEY, L.S., MCCARN, D.F., RUSCHE, J.R., HAUBER, J., and CULLEN, B.R. (1990). HIV-1 structural gene expression requires binding of the Rev frα/w-activator to its RNA target sequence. Cell 60, pp. 675-683.
23. OLSEN, H.S., NELBOCK, P., COCHRANE, A.W., And ROSEN, CA. (1990). Secondary structure is the major determinant for interaction of HTV Rev protein with RNA. Science 247, pp. 845-848.
24. COCHRANE, A.W., JONES, K.S., BEIDAS, S., DILLON, P.G., SKALKA, A.M., and ROSEN, CA. (1991). Identification and characterization of intragenic sequences which repress HTV structural gene expression. J. Virol. 65, pp. 5305-5313.
25. SCHWARTZ, S. , FELBER, B.K. , and PAVLAKIS, J.N. (1992). Distinct RNA sequences in the gag region of HTV-l decrease RNA stability and inhibit expression in the absence of Rev protein. J. Virol. 66, pp. 150-159.
26. ZIMMERMANN, K., WEBER, S., DOBROVNIK, M., HAUBER, J., and BOHNLEN, E. (1992). Expression of chimeric neo-Rev responsive element sequences interferes with Rev-dependent HIV-1 gag expression. Human Gene Therapy 3, pp. 155-161. 27. LEE, T.C, SULLENGER, B.A., GALLARDO, H.F., UNGERS, G.E., and
GTLBOA, E. (1992). Over expression of RRE-derived sequences inhibits HTV-l replication in CEM cells. New Biol. 4, pp. 66-74. 28. MALIM, M.H., BOHNLEIN, S., HAUBER, J., and CULLEN, B.R. (1989). Functional dissection of the HTV-l Rev trans-activator — Derivation of a trans- dominant repressor of rev function. Cell 58, pp. 205-214.
29. LEVER, A., GOTTLINGER, H., HASELTINE, W., and SODROSKI, J. (1989). Identification of a sequence required for efficient packaging of HTV-l
RNA into virions. J. Virol. 63, pp. 4085-4087.
30. ALDOVESfl, A. and YOUNG, R.A.(1990). Mutations of RNA and protein sequences involved in HTV-l packaging result in production of noninfectious virus. J. Virol. 64, pp. 1920-1926. 31. HAYASHI, T., SHIAOA, T., IWAKURA, Y., and SHffiUTA, H. (1992).
RNA packaging signal of HTV-l. Virology 188, pp. 590-599. 32. HARRISON, G.P. and LEVER, A.M.L. (1992). The HTV-l packaging signal and major splice donor region have a conserved stable secondary structure. J.
Virol. 66, pp. 4144-4153. 33. BENDER, M.A., PALMER, T.D., GELINAS, R.E., and MILLER, A.D.
(1987). Evidence that the packaging signal of Moloney murine leukemia virus extends into gag region. J. Virol. 61, pp. 1639-1646.
34. GORELICK, R.J., NIGIDA, S.M. JR., BESS, J.W. JR., ARTHUR, L.O., HENDERSON, L.E., and REIN, A. (1990). Noninfectious HTV-l mutants deficient in genomic RNA. J. Virol. 64, pp. 3207-3211.
35. LUBAN, J. and GOFF, S.P. (1991). Binding of HTV-l RNA to recombinant HTV-l Gag polyprotein. J. Virol. 65, pp. 3203-3212.
36. TRONO, D., FFJNBERG, M.B., and BALTIMORE, D. (1989). HTV-l gag mutants can dominantly interfere with the replication of the WT virus. Cell 59, pp. 113-120.
37. SAMBROOK, J., FRITSCH, E.F., and MANIATIS, T. (1989). Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York.
38. BERGER, S.L., and KIMMEL, A.R. (1987). Guide to molecular cloning techniques. Methods Enzymol. 152, pp. 469-481.
39. MAGLI, M.C, DICK, J.E., HUSZAR, D., BERNSTEIN, A., and PHILLIPS, R.A. (1987). Modulation of gene expression in multiple haematopoietic cell lineages following retroviral vector gene transfer. Proc. Natl. Acad Sci. U.S.A. 84, pp. 789-793.
40. WEERASINGHE, M., LTEM, S.E., ASAD, S., READ, S.E., and JOSHI, S. (1991). Resistance to HIV-1 infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HTV-l RNA-specific
Ribozyme. J. Virol. 65, pp. 5531-5534.
41. MANN, R., MULLIGAN, R.C, and BALTIMORE, D. (1983). Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33, pp. 153-159. 42. MILLER, A.D. and BUTTTMORE, C (1986). Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell Biol. 6, pp. 2895-2902.
43. JOSHI, S., VAN BRUNSCHOT, A., ROBSON, I., and BERNSTEIN, A. (1990). Efficient replication, integration, and packaging of retroviral vectors with modified long terminal repeats containing the packaging signal. Nucleic
Acids Res. 18, pp. 4223-4226.
44. CHOMCZYNSKI, P. and SACCHI, N. (1987). Single step method of RNA isolation by acid guanidium triocyanate-phenol-chloroform extract. Anal. Biochem. 162: pp. 156-159. It would be of much value to provide retroviral vectors expressing anti-
HIV-1 gene(s) to infect patients' bone marrow (BM) stem cells or peripheral blood lympocytes (PBLs) which, upon transplantation and differentiation, would potentially give rise to an HTV-l resistant immune system.
As a result of extensive investigations, I have discovered methods of inhibiting the multiplication of HTV-l in a mammalian cell.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method of inhibiting HTV-l multiplication in a mammalian cell.
It is a further object of the present invention to provide cells of use in said method. It is a yet further object of the present invention to provide therapeutic compositions comprising said cells.
It is a still yet further object of the present invention to provide vectors, particularly, retroviral vectors of use in the preparation of said cells. Accordingly, in one aspect the invention provides a gene therapeutic method of inhibiting HTV-l multiplication in a mammal comprising treating said mammal with an effective amount of mammalian cells expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
Preferably, the mammalian cells are human bone-marrow cells, and more preferably, blood cells.
In a further aspect the invention provides mammalian cells harboring proviral vector DNA expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
In a yet further aspect the invention provides a retroviral vector expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
Preferably, the retroviral vector is derived from the Moloney murine leukemia virus (MoMuLV).
Thus, in the development of the present invention, MoMuLV-derived retroviral vectors were engineered to express HTV-l φ signal and Gag coding sequences in anti-sense orientation in chimeric RNAs. These sequences were expressed under control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Both, φ signal and Gag coding sequences were expressed as part of the 3' untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD4 + lymphocyte-derived MT4 cell line. The stable MT4 transformants harbouring proviral vector DNA expressing φ signal and Gag coding sequences in antisense orientation, were each tested for their susceptibility to HTV-l infection. The stable MT4 transformants were then tested for interfering RNA (containing φ signal and Gag coding sequences) production by reverse transcription- polymerase chain reaction (RT-PCR) analysis as well as for their susceptibility to HTV-
1 infection. Compared to the results obtained with the control cells lacking any of the test DNA sequence, the rate of HTV-l production was delayed by up to 30 days in the antisense RNA-expressing cells. These results indicate that retroviral vectors expressing the HTV-l φ signal and Gag coding sequences in antisense orientation can be used to confer HTV-l resistance. In a further aspect, the invention provides a therapeutic composition for confering HTV-l resistance to a mammal comprising cells as hereinbefore defined in association with a pharmaceutically acceptable carrier, diluent or adjuvant therefor. It will be readily understood by the person skilled in the art that the cells should be present in an effective therapeutic amount. In gene therapy in patients using human peripheral blood lymphocytes
(PBLs) and bone marrow (BM) cells, the source of CD4+ lymphocytes and macrophages - major targets of HTV-l infection - are the peripheral blood or bone marrow cells. PBLs are easy to access. However, these fully differentiated cells have a limited life span and therefore will only provide short term resistance. PBL gene therapy will therefore have to be repeated after a certain interval. BM cells contain stem cells which are capable of both self-renewal and differentiation into lymphocytes, macrophages, and other hematopoietic cells such as erythrocytes, granulocytes, and megakaryocytes. For gene therapy to have a sustained effect, it should be performed at the level of such self-renewing, pluripotent haematopoietic stem cells allowing continued production of progeny cells containing the therapeutic gene. As stem cells represent only 1 per 10,000 nucleated cells present within the bone marrow, a large number of bone marrow cells are infected by retroviral vectors to ensure transformation of these rare stem cells.
Gene therapy for the treatment of AIDS provided herein consists of using retroviral vectors to deliver the anti-HTV-1 RNA molecules to human PBLs and BM stem cells. Transformants selected in vitro are transplanted back to the patient. Following differentiation, these transformed cells lead to the development of an immune system in which various blood cells (including CD4 lymphocytes and macrophages) express the anti-HTV-1 RNA molecules and are therefore resistant to HTV-l. Preferred methods of in vitro stimulation and culture for gene transfer into mammalian cells, particularly stem cells, with a gene transfer vector, particularly, a retrovious vector are disclosed in Canadian Patent Application No. 2086844, published July 8, 1994 - Dube et al, which disclosure is included herein by reference. Following appropriate stimulation and culture in vitro. PBLs and BM cells are infected by cocultivation with packaging cell lines producing retroviral vector particles. Transformants are then selected in vitro for growth in medium containing appropriate cytokines and antibiotics. These transformants are then transplanted back to the patient. Following transplantation and differentiation, blood samples are tested for anti-HTV-1 gene expression and for the ability of these cells to resist HTV-l infection. Other disease symptoms, viral load and emergence of resistant HTV-l isolates are examined as well.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the invention may be better understood, a preferred embodiment will now be described by way of example only, with reference to the accompanying drawings, wherein:
Fig.1 represents LTR-LTR sequences present in the proviral DNA integrated in the target cell line as part of the map of retroviral vectors expressing antisense RNA to HTV-l φ signal and Gag coding sequence; and Fig. 2 shows the results of HTV-l infections on a pool of stable MT4 transformants expressing HTV-l φ signal and Gag coding sequence-containing RNAs.
MATERIALS AND METHODS Materials
All restriction enzymes were purchased from GIBCO BRL Ontario, Canada. T4 DNA ligase was obtained from Pharmacia, Quebec, Canada, and Calf intestinal phosphatase was obtained from Boehringer Mannheim, Quebec, Canada. Fetal bovine serum (FBS) was obtained from Hyclone and GIBCO BRL. Geneticin (G418), a mixture of antibiotic-antimycotic agents (containing penicillin, streptomycin, and FungizoneR), L-Gln, Eagle's minimal essential medium, α-modification (α-MEM), and RPMI 1640 medium were purchased from GIBCO BRL. Plasmid Construction
Unless otherwise stated, all recombinant DNA techniques were performed as described in (37, 38). The retroviral vector, pUCMoTN (39), used in this study, is derived from MoMuLV. This vector allow neo gene expression (conferring G418R) under control of HSV tk. The φ signal and Gag sequences were cloned in this vector as part of the 3' untranslated region of the neo mRNA.
The pUCMoTN-^Gag" and pUCMoTN-^+Gag+ vectors were constructed as follows.
A 4.0 EcoRI fragment from pBKBHIOS (NIH#182) was cloned into the EcoRI site of the pUC18 vector (Pharmacia). A 1440 bp BamHJ-BglR fragment containing HTV-l φ signal and Gag coding sequences was isolated from this pUC-^ vector. This fragment (containing Sstl-BglR sequences of HTV-l strain HXB2) was cloned into the unique BamΗI site in the pUCMoTN-Rzl vector (40). The resulting clones were characterized by restriction enzyme analysis and clones containing a single copy of the φ signal and Gag coding sequences in antisense (pUCMoTN-^'Gag") and sense (pUCMoTN-^ Gag") orientations, with respect to the vector were selected.
Mammalian Cell lines
The ecotropic Psi-2 (41) and amphotropic PA317 (ATCC cat# CRL0978) (42) packaging cell lines were cultured in α-MEM medium supplemented with 2mM
L-Gln, 0.1 volume of antibiotics/antimycotic solution (penicillin, 1000 units/ml; streptomycin, 1000 μg/ml; FungizoneR, 2.5 μg/ml), and 10% FBS (Hyclone) at 37°C in a humidified atmosphere with 5% CO2. The human CD4 + lymphocyte-derived MT4 suspension cell line, NTH Cat #120, was cultured in RPMI 1640 medium also supplemented with Gin, antibiotics/antimicotic agents, and FBS (GIBCO) as above and were incubated at 37°C in a humidified atmosphere with 5% CO2. The selective media was prepared as above except that it also contained G418 (200 μg/ml for Psi-2 and PA317 cell lines; and 400 μg/ml for MT4 cell line).
Transfection and Infection of Mammalian Cell Lines
Psi-2 cells were transfected as follows using the Calcium phosphate co- precipitation technique (using the CellPhect Transfection Kit from Pharmacia): a 120 μl retroviral DNA solution (3 μg) was mixed with 120 μl Buffer A (0.5 M CaCl2, 0.1 M HEPES) and incubated at 22°C for 10 min. An equal volume (240 μl) of Buffer B (0.28 M NaCl, 0.05 M HEPES, 0.75 mM NaH2PO4, 0.75 mM Na2HPO4) was added, mixed immediately by vortexing and incubated for 15 min. The mixture was then added drop wise to the cell culture (50% confluent in 60 mm plates containing 3 ml fresh medium). The cells were incubated under normal growth conditions for 6 hrs, then washed twice with fresh medium. The cells were subjected to glycerol shock with 1.5 ml 15% glycerol in 10 mM HEPES pH 7.5, 150 mM NaCl for 3 min at 22°C, then washed once with fresh medium. Fresh medium (5 ml) was added and the cells were grown under normal conditions for 2 days. On day 3, the transfected cells were washed once with phosphate-buffered saline (PBS) containing antibiotics/antimycotic agents, trypsinized with 0.05% trypsin, 0.53 mM EDTA-4Na (GIBCO), transferred to 100 mm plates and grown in selective medium containing 200 μg/ml G418. The medium was changed every 3-4 days until selection was complete (15-20 days). The number of resistant colonies was then determined. The cells were washed with PBS, trypsinized and re-seeded.
Vector particles released from the transformed Psi-2 cells were obtained by filtering culture medium from cells at 50-100% confluency through a 0.22 μm filter. These particles were used to infect PA317 cells as described previously (43). Essentially 2 x 10s cells were seeded for 6 hours in 60 mm tissue culture dishes in 4 ml medium, after which this medium was replaced by 1 ml medium containing 8 μg/ml polybrene and 100 μl vector particles. After a 2 hour incubation at 37°C, 3 ml medium was added and the incubation continued for 16 more hours. Cells were then trypsinized and transferred to 100 mm tissue culture dishes in the presence of selective medium containing 200 μg/ml G418. The selective medium was changed every 4-5 days and the number of colonies counted after 14 days. Vector particles released from the PA317 cells (50-100% confluent) were then collected and used to infect MT4 cells.
The MT4 cells (3 x 105) were pelleted and resuspended in 0.5 ml RPMI 1640 medium containing 16 μg/ml polybrene. Vector particles (0.2 ml) and RPMI 1640 medium (0.3 ml) were then added and gently mixed to the cells. Cells were transferred to 60 mm petri dishes, and incubated under normal growth conditions for 2 hrs. Four ml of fresh RPMI 1640 medium were then added and the cells were grown overnight. The infected cells were then centrifuged, resuspended in selective medium containing 400 μg/ml G418 and transferred to 100 mm petri dishes. Every 3-4 day, half of the cell suspension was removed and replaced with fresh selective medium. By day 20, all of the uninfected cells had died and the remaining stably transformed cells were frozen and were used in the following experiments.
Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR analysis
Genomic DNA isolated (37) from the MT4 cells stably transformed with MoTN-t '" Gag" vector particles was used in PCR as follows. PCR reaction (100 μl) was performed in the presence of MgCl2 (1.5 mM), oligonucleotides (20 mM each; amplification buffer (1 x concentration; Promega), dNTPs (10 mM each), genomic DNA (1 μg), and Taq polymerase (2 units, Promega). The samples were overlaid with 100 μl mineral oil and amplified using Perkin-Elmer Cetus Instruments DNA Thermal Cycler by using three linked files as follows: File 1, STEP-CYCLE 1 min at 95 °C; File 2, STEP-CYCLE 1 min at 55 °C; File 3, STEP-CYCLE 1 min at 72°C; with a total of 45 cycles. PCR products (10 μl aliquots) were then analyzed by electrophoresis on a 3% agarose gel.
In order to assess the amount of φ~ Gag" RNA expressed, RT-PCR was performed using RNA isolated from the MT4 cells stably transformed with MoTN-^" Gag" vector particles. The cells were grown for 48 hrs and then the total RNA was extracted using the Guanidium thiocyanate-Phenol-Chloroform procedure (44) . Reverse transcription was performed as follows: total RNA (5 μg) was incubated with oligo dT (20 mM) for 10 min at 65°C in a total volume of 20 μl. The reaction mixture was chilled on ice for 2 min and RNA guard (75 units; Pharmacia), reverse transcription buffer (1 x concentration; BRL), DTT (5 mM), dNTPs (12.5 mM each), and
Superscript RTase (400 units; BRL) were added to make a total volume of 40 μl. The reaction mixture was incubated for 1 h at 37°C and then for 10 min at 65°C Five μl of this reaction mixture containing cDNA was then used in a PCR reaction which was performed as described above and the products were then analyzed by electrophoresis on a 3% agarose gel. HIV-1 infections
Actively dividing various MT4 transformants (1 x 10° cells/ml) were each infected with HTV-l as follows: a 2 ml cell culture was incubated with 20 μl HTV-l strain NL -3, NIH Cat #78, (1064 TdD50/ml) for 2 hrs at 37°C. The cells were then pelleted, washed 3 times with PBS, resuspended in 2 ml medium, transferred to 35 mm dishes and allowed to grow at 37 °C Every 3 days for up to day 30, a 1 ml sample containing cells and medium from each infected cell culture was removed and frozen at -70°C One ml of complete medium was added back to the culture each time. After day 30, the frozen samples were thawed and centrifuged at 250 x g for 10 min. The culture supernatants (200 μl each) were then diluted as required and tested for the presence of HTV-l p24 antigen using the HTVAG-1 Enzyme Immunoassay Kit (Abbott Laboratories) according to the manufacturer's instructions. A standard curve (OD492 as a function of p24 antigen concentration in ng/ml) was obtained using p24 antigen provided by the supplier. The OD492 values for the various samples collected at different time intervals following HTV-l infection were corrected for the dilution factor and were converted to ng p24 antigen released per ml cell culture supernatant. These experiments were repeated four times.
RESULTS Retroviral vectors allowing interfering RNA expression
MoMuLV-derived retroviral vector pUCMoTN was modified to express
HTV-l φ signal and Gag coding sequences in antisense orientation. This molecule was expressed as part of the 3' untranslated region of the neo mRNA in between the stop codon and the poly (A) site (Fig. 1). The pUCMoTN-^-" Gag" vector expressed a single copy of antisense RNA to both of the HTV-l φ signal and Gag coding region (Fig. 1). The expression of HIV-
1 φ signal and Gag coding region-containing RNAs in these vectors is under the control of the MoMuLV 5' LTR and HSV tk promoters.
Establishment of stable MT4 transformants expressing interfering RNA molecules
The aforementioned retroviral vectors were first used to transfect an ecotropic packaging cell line Psi-2; (41), and the vector particles released from this cell line were used to infect an amphotropic packaging cell line (PA317; 42). The resulting amphotropic pseudotyped retroviral vector particles capable of infecting human cells were then used to infect a human CD4+ lymphoid (MT4) cell line and the G418R stable transformants were selected. The presence of the anti-HTV-1 gene and the level of therapeutic RNA/protein produced in these cells were then monitored as described below.
Confirming CD* expression on stably transformed MT4 cells
The fact that various MT4 transformants expressed CD4 was confirmed by Fluorescence activated cell sorter (FACS) analysis using an anti-CD4 monoclonal antibody (T4-RD1/T11-FITC). Over 95% of cells examined were found to be CD4 positive.
Confirming the presence of interfering RNA Since various interfering RNA molecules are expressed as part of the neo gene, the fact that the cells are G418R confirms that the neo mRNA was expressed. Anti-HTV-1 resistance was obtained from MT4 cells stably transformed with the
MoTN-^" Gag" vector particle. The presence of HTV-l φ signal and Gag coding sequences within their genome was confirmed by PCR analysis. The presence of φ signal and Gag coding sequence-containing RNAs was confirmed by RT-PCR. As expected, a 315 bp PCR or RT-PCR product was visible in both cases.
Testing for the ability of interfering RNAs to confer HTV-l resistance
MT4 cells stably transformed with MoTN-^" Gag" vector particles expressing antisense RNA to HIV-1 φ signal and Gag coding sequences were challenged with HTV-l. MT4 cells transformed with the parental retroviral vectors lacking test DNA sequences served as control. Virus production was monitored by measuring the level of p24 antigen (HTV-l gag gene product) in the cell culture supernatant every 3 days for up to 30 days post-infection. The MT4 transformants expressing antisense RNA to the φ signal and Gag coding sequences delayed virus production for up to 30 days (Fig. 2).
HTV-l resistance of sense RNA-expressing cells was monitored as follows. MT4 cells stably transformed with MoTN-^+ Gag+ vector particles were subjected to challenge by HTV-l and virus production in the culture supernatant was measured every 3 days post-infection. MT4 cells expressing sense RNA to the HTV-l φ sequence and Gag coding sequences failed to prevent HTV-l multiplication (Fig. 2). In the studies of the present invention, retroviral vectors were, thus, engineered that expressed φ signal and Gag coding sequences in antisense orientation. The retroviral vector particles were used to infect the human CD4 + lymphocyte-derived MT4 cells and stable G418R transformants were selected. The pool of these transformants was then infected with HTV-l and virus production measured for up to 30 days post-infection.
Interference with genomic RNA packaging is believed to result in the production of replication-defective virions, thus limiting the spread of viral infection. In cells expressing HTV-l φ signal and Gag coding sequences in sense orientation, HTV- 1 production began even earlier than in the control cells (Fig. 2). If the HTV-l φ signal and Gag coding sequence-containing retroviral vector RNA was also packaged by HTV-
1 , the infectivity of these chimeric RNA-containing virus particles should have been reduced. The lack of resistance observed with the HTV-l φ signal and Gag coding sequences expressed in the sense orientation may be explained by the fact that the length of the HTV-l φ signal used in the present experiments was not sufficient to allow packaging of non-viral mRNA.
High level of resistance was observed in cells expressing HTV-l φ signal and Gag coding sequences in an antisense orientation as no virus could be detected in the culture supernatants from these cells for up to 30 days post-infection (Fig. 2). Note that for the antisense RNA approach to be effective, it is not required that the antisense RNA to the HTV-l φ signal be designed against the entire HTV-l φ signal. However, when MT4 transformants were challenged with a higher dose of HTV-l, some HTV-l production could be detected around day 20 but it remained quite low; by day 30, the amount of virus produced was 6-7 fold below the control. As expected, these cells were fully viable. The experiments described hereinabove were performed using a pool of transformed MT4 cells and therefore represent an average of the resistance conferred by each cloned MT4 transformant. This appears to be a more realistic view of what would happen should human gene therapy be performed using these retroviral vectors. While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as described and claimed.

Claims

I claim:
1. A method of inhibiting HTV-l multiplication in a mammal comprising treating said mammal with an effective amount of mammalian cells expressing RNA molecules containing HTV-l φ signal and/or Gag coding sequences in antisense orientation.
2. A method as claimed in Claim 1 wherein said mammal is a human and said cells are bone-marrow cells.
3. A method as claimed in Claim 1 wherein said cells are blood cells.
4. A method as claimed in Claim 1 wherein said cells are human CD4 containing lymphocytes.
5. A method as claimed in Claim 1 wherein said cells express RNA molecules containing HTV-l packaging signal coding sequence in antisense orientation.
6. A method as claimed in Claim 1 wherein said cells express RNA molecules containing HTV-l Gag coding sequence in antisense orientation.
7. Mammalian cells harboring DNA expressing RNA molecules containing HTV-l φ signal and Gag coding sequences in antisense orientation.
8. Mammalian cells as claimed in Claim 7 harboring proviral vector DNA expressing RNA molecules containing HTV-l φ signal and/or Gag coding sequences in antisense orientation.
9. Cells as claimed in Claim 7 being bone-marrow cells.
10. Cells as claimed in Claim 7 being blood cells.
11. Cells as claimed in Claim 7 being human CD4 containing lymphocyte cells.
12. A therapeutic composition comprising mammalian cells as defined in any one of Claims 7-11, harboring DNA expressing RNA molecules containing HTV-l φ signal and/or Gag coding sequences in antisense orientation; and a pharmaceutically acceptable diluent, adjuvant or carrier therefore.
13. A vector expressing RNA molecules containing HTV-l φ signal and/or gag coding sequences in antisense orientation.
14. A vector as claimed in Claim 13 expressing RNA molecules containing HTV-l φ signal coding sequence in antisense orientation.
15. A vector as calimed in Claim 13 expressing RNA molecules containing HTV-l gag coding sequence in antisense orientation.
16. A retroviral vector expressing RNA molecules containing HTV-l φ signal and/or Gag coding sequences in antisense orientation.
17. A retroviral vector as claimed in Claim 16 expressing RNA molecules containing HTV-l φ signal coding sequence in antisense orientation.
18. A retroviral vector as claimed in Claim 16 expressing RNA molecules containing Gag coding sequence in antisense orientation.
19. A retroviral vector as claimed in Claim 16 derived from Moloney murine leukemia virus (MoLuLV).
PCT/CA1995/000190 1994-04-06 1995-04-05 Inhibition of hiv-1 multiplication in mammalian cells WO1995027783A1 (en)

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