WO1996023876A1 - Expression system and method for inhibiting replication of human immunodeficiency virus (hiv) - Google Patents

Abstract

A method and expression system for inhibiting replication of the Human Immunodeficiency Virus (HIV) within cells. The expression system encodes RNA sequences that are complementary to strong stop DNA which is present during viral replication. It is found that such strong stop RNA has a protective effect that lasts for a substantial time. A method of treatment include isolation of haematopoietic stem cells of sufferers, introducing ex vivo the expression system of the strong stop RNA into the stem cells, and reintroducing the stem cells back to the sufferers to give progeny HIV resistant T cells, monocytes, macrophages and other cells, thus to restore the immune system of the patients.

Description

EXPRESSION SYSTEM AND METHOD FOR INHIBITING REPLICATION OF HUMAN IMMUNODEFICIENCY VIRUS (HTV)

FIELD OF THE INVENTION

This invention relates to an expression system and a method for use in treatment of individuals infected with Human Immunodeficiency Virus.

BACKGROUND OF THE INVENTION Human Immunodeficiency Virus HIV is a retrovirus that infects humans and can cause AIDS. Following infection there is generally a latent period where individuals test positive for HIV but do not have symptoms associated with the disease. The length of the latent period is quite variable amongst individuals and it is not clear whether all individuals that test HTV+ will progress to showing the symptoms of AIDS.

The primary symptom of AIDS is that the affected individual has progressive degeneration of d e immune systems, and is therefore progressively unable to mount an immune response to infection, leading to eventual immune failure concurrent with multiorganism opportunistic infection, as well as neurological symptoms. In certain cases, associated cancers such as Karposi's sarcoma and lymphomas are also seen. AIDS has no known effective treatment and has an apparent 100% mortality rate.

The destruction of the immune system is progressive and irreversible. Two of the primary cells whose function is affected are macrophages (which cells function as "scavenger" cells phagocytose or dispose of various foreign infectious material), and T cells, (which cells play a part in the immune responses in the recognition of intracellular infectious agents) with loss of CD4⁺ lymphocytes being most obvious.

The means by which HIV replicates is by infecting white blood cells primarily in CD4+ T cells, and replicating within such T cells. Replication of HIV when within a host cell has been studied in detail but is not totally understood, and several models of replication have been suggested, at the molecular level.

So far the only commercially available treatment is the administration of a nucleoside analogue compound known as AZT (azidothymidine) which acts to alleviate the symptoms of AIDS, and to some extent temporarily arrests the speed with which AIDS progresses, however the disease is not reversed. Furthermore there is as yet no means for reversing the HIV status of an individual, or of preventing replication of HTV within an infected individual, whether the individual shows the symptoms of AIDS or not

It is therefore desirable that some progress be made in the treatment of individuals who test positive for HTV, or for individuals who have contracted AIDS. It is desired to have an effective treatment to prevent at least a proportion of individual who test positive for HIV from developing AIDS, and preferably a treatment that results in a reversal of the HTV status of such individuals. It is also desirable that individuals who have contracted AIDS, may be treated so that the disease might be reversed, or at least stabilised.

One approach to these aims that has attracted some research has been by direct inhibition of HIV replication by gene therapy. Research on this approach has gathered considerable momentum in the past few years. A promising feature of this approach is the ability to design and deliver genes and gene products precisely to the selected target cells to achieve one of two goals: (i) abolishment or inhibition of HIV replication in virus infected cells, or (ii) provision of anti-HIV functions to extra corporeal CD_ι⁺ cells or bone marrow or blood stem cells which might then be returned to the body to reconstruct or repopulate the immune system. The rationale for directly attacking viral replication as an absolutely essential component of the total strategy in combating AIDS has been reinforced recently. Using quantitative competitive polymerase chain reaction (QC-PCR) high levels of HTV were detected in the patient blood during all stages of HIV infection (Piatak et al (1993) ). Massive latent and to a lesser extent productive, HlV-infection of CD_ι,⁺ T cells and macrophages throughout the lymphoid system, in patients from early to late stages of disease, has been reported. Simultaneous analysis of the virus burden in patient's blood and lymphoid tissues revealed that in early stages of the disease (asymptomatic patients) there were more HIV -infected cells in the lymph node than in the blood and that these infected cells in the lymph node were more likely to produce progeny virus than infected cells in the blood (Panteleo et α/ (1993); Embretson et _ι7 (1993)). These studies have affirmed the view that HIV infection is active and progressive during the clinically latent stage of disease and that HTV replication needs to be considered as a major target for therapy. Baltimore first proposed that gene transfer technology be used to dominantly interfere with intracellular HIV growth, and coined the words "intracellular immunization" (Baltimore (1988)). The general principle of the "intracellular immunization" strategy is that of using competitive or interfering gene products expressed in the target cells to specifically inhibit HTV replication. Expression of trans-dominant (negative) mutant HTV-Gag, -Rev and -Tat proteins (Trono et al

(1989); Malim etαi (1989) Cell 58, 205-214; Malim etα/ (1992) ; Bevec eta/

(1992); Bahner et al (1993)), or expression of anti-HTV immunologicals such as soluble CD4 or interferon α (Morgan etal (1990); Bednarik (1989)), or HTV- regulated expression of a simple bacterial toxin (Harrison et al (1992)), or expression of ami- tat and anti-rev antisense RNAs (Rhodes and James (1990); Sczakiel and Pawlita (1991); Joshi et al (1991); Sczakiel et al (1992); Lo et al (1992)), or expression of HTV-TAR or HIV-RRE decoy RNAs (Sullenger et al (1990a) ; Sullenger et al (1991)); all have been reported to inhibit expression or replication of HIV. An extended version of "intracellular immunization" with destructive approaches employed HIV-specific ribozymes to cleave HTV RNA (Lo et al (1992) ; Sarver et al (1990); Weerasighe (1991); Dropulic (1992); Yu et al (1993)), or HTV-regulated conditional-lethal 'suicide' gene constructs (Venkatesh etal (1990); Caruso and Klatzmann (1992); Brady etal (1994)). An active area in anti-HIV research (a hybrid of traditional antiviral drugs and gene therapy) is the use of anti-HTV antisense oligonucleotides and their thio- derivatives (Matsukura et al (1989) PNAS 86; Agawal et al (1989); Li et al (1993)) or the use of other novel approaches such as HIV-specific chemical nucleases (Jayasena and Johnson (1992)) and inhibitory triple helix formation in the poly-purine track of HIV proviral DNA (Giovannangeli et al (1993)). The various approaches have all been met with some success and one emerging trend in this research area is to combine different strategies to achieve more desirable anti-HIV effects (Lisziewicz et al (1993); Homann et al (1993)).

If we classify currently proposed effector molecules in anti-HIV gene therapy research into proteins and nucleic acids, it is evident that the effector proteins are diverse and targeted at different stages of the viral replication cycle. These include early/regulatory protein functions (transdominant negative Rev and Tat), late/structural protein functions (transdominant negative Gag), viral entry (transduced CD4) and viral release (transduced interferon α) etc. On the other hand, effector nucleic acids are exclusively RNA (antisense or ribozymal) and targeted almost exclusively at early viral RNAs (Tat/Rev mRNA and their protein products or 5' leader RNA). The rationale for attacking early regulatory RNAs is that these RNAs are made early in the HTV expression timetable, in relatively low abundance and their protein products are prerequisite for the expression of late viral structural mRNAs and proteins and thus represent a more sensitive "control button" in curbing intracellular HTV replication and growth. However, one can argue that when the virus starts making early viral RNAs, although it is earlier than the appearance of late viral RNAs and viral proteins, the unique and all important "true early" intracellular events in a retrovirus replication cycle i.e. reverse transcription and integration are all but finished. Gene transfer experiments designed to compete or interfere with these true early events have not been reported.

A description of aspects of HTV DNA replication is helpful for a better understanding of the present invention. A diagrammatic representation can be seen in FIG. 8. Reverse transcription (RT_n) of retroviruses such as HIV converts the incoming virion genomic RNA to yield a double stranded DNA. During this process two template switches are involved B-C and I. The first of these switches transfers the growing minus strand viral DNA (minus strand strong-stop DNA (shown in bold lines)) from the 5' end of one RNA molecule to the 3' end of either the same or a second RNA molecule. The second switch transfers the growing plus strand DNA (plus strand strong-stop DNA) from the 5' end to the 3' end of the same molecule of the newly synthesised minus strand viral DNA (Panganiban and Fiore (1988); Hu and Temin (1990); Jones et .2/(1994) ; Li et al (1993b)). Minus strand-, plus strand- and double-stranded strong stop DNAs have been shown to accumulated during RT_n in vitro and in vivo (Coffin and Haseltine (1977); Varmus and Shank (1976); Li et al (1993b)). The transient single stranded viral DNA sequences involved in the process of RT_n represent an unexplored target for antisense intervention. Their role and actual configuration is not yet clear.

There are several attractive aspects to the use of strong stop nucleic acid for antisense intervention. Firstly, all the reverse transcription models to date predict that only one DNA molecule is made from one virion genomic RNA which is degraded during RT_n (see Li et al (1993b) and refs therein). Thus the stoichiometry of the anti-strong stop DNA approach would be far superior to that of anti-Tat/Rev mRNAs that are continuously expressed in multicopy numbers from integrated proviral DNA (which is immune to antisense RNA and ever present as a potential breach in the antisense blockade). Secondly, a model study on the intracellular sense-antisense RNA hybrid duplex formation (which is envisaged as the molecular basis for antisense effects) suggested that to incorporate 50% of a sense RNA into sense-antisense duplex,

600- to 2800- fold molar excess of antisense RNA are needed depending on a hybrid index which is specific to each particular sense-antisense duplex (Wang and Dolnick (1993)). This might be one of the reasons underlining the need to over-express antisense RNA using strong promoters such as tRNA cassette driven by RNA polymerase HI (Sullenger et al (1990b)). Given this vastly inefficient sense-antisense RNA duplex formation, it might be anticipated that the present approach involving intracellular DNA-RNA interaction would be more efficient because of the lower tendency of the DNA component to form secondary structures.

Thirdly, targeting anti-strong stop DNA is different from the existing anti-HIV gene transfer designs and is aimed at the first major stage of HIV replication cycle, thus blocking virus replication earlier. Even if proven only partially successful clinically, it may still be used in combination with other later stage intracellular immunization regimens to summon a more effective attack on HIV.

HTV reverse transcription happens in the cytoplasm of infected cells in the context of viral replication complexes (Fennet and Haseltine (1990); Fennet and Haseltine (1991) ; Karageorgos et al (1993). It was not certain whether the sense and antisense RNAs expressed in the transformed T cells would have adequate access to the viral replication complexes.

It is also not certain that there will not be any adverse effect(s) on the growth of the transformed T cells in which over expression is occurring of the various sense and antisense RNAs from the integrated expression vector.

The term strong stop RNA will be used throughout this specification. The term is understood to mean RNA that is identical or complementary to that region in whole or in part of the HIV genome known as the U3, R U5, PBS and the Ψ regions whether they are sense or antisense. These region generally encompass nucleic acids between lb to 800 b on the sequence published for the HXB2 clone of HTV (Myers et al (1990)). The location of these elements on a representation of nucleic acid of HIV can be seen in FIG. 8. SUMMARY OF THE INVENTION

This invention results from a finding that HTV replication can be inhibited by the presence of strong stop RNAs and their derivatives within a cell infected with

HTV, and that such inhibition is much more effective than any earlier, published, attempts.

In a first form the invention could be said to reside in an expression system for producing strong stop RNAs and RNA derivatives, that interfere with corresponding sequences of HTV virus DNA during replication within a cell infected with HIV, which RNAs inhibit replication of HTV virus, so as to enhance the viability of the cell.

The produced strong stop RNAs may not be present in the normal HTV reverse transcription cycle in which case the produced strong stop RNA may act to bind the plus or minus sense DNA essential for replication, thereby inhibiting replication. Alternatively the produced strong stop RNA may be present in the normal HIV reverse transcription cycle but may be modified. It is also to be understood that in certain circumstances that it may be desired to modify produced strong stop RNA that normally is in the cell. In this case the produced strong stop RNA acts as a decoy, competing with normally present RNA, and binding to complementary RNA thereby producing a defective product The modification may by any one of several known in the art, and may include deletions, substitutions, insertions, base modifications or combination of two or more of these modifications. The choice of modification will depend upon the desired effect

The expression system may simply be nucleic acid which includes a nucleic acid promoter such as an RNA promoter, and a nucleic acid sequence allowing production of multiple copies of strong stop RNAs. The expression system may be present in several different forms, and for expression it may be desired to have the expression system integrated into endogenous DNA of the cell, alternatively it may be desired to have the expression system resident in a self-replicating vector.

Preferably the produced strong stop RNA is greater than 250 bases long, however it is possible that shorter pieces will also function and perhaps pieces as short as 50 bases will suffice. The produced strong stop RNA is identical or complementary to any one or more of the elements designated U5, PBS, U3, R, Ψ. Nucleotide number for these elements according to the HXB2 sequence (Ibid) are: U3 (1-453), R (454-551),

U5 (552-634); PBS (635-653), Ψ (653-790). It will be understood that DNA outside of these specific bases may also be present provided that binding with strong stop DNA can still occur.

The expression system may be carried on a vector, wherein the vector includes means to replicate in the target cells.

In one form the vector may be based upon a retroviral vector, which might, for example, be a Murine Leukemia Virus vector, which contains a 5' and a 3' LTR (long terminal repeat) sequence, a packaging signal, a selectable marker, and one or more promoters upstream of the site encoding the strong stop nucleotide sequence. This type of vector facilitates introduction of the expression system into the cell. It is to be understood that other suitable vectors may be employed.

The promoter(s) for producing strong stop RNAs may be one of several promoters, for example it may be the human tRNA _met or it may be the promoter located within the murine leukaemia or HTV LTRs. A separate promoter will generally act as a means for expressing the drug resistance gene. Thus Simian Virus 40 (SV40) may be employed to provide for expression of the drug resistance gene.

The selectable marker may be an antibiotic resistance gene for example neomycin resistance that is able to be selected both in eukaryotic and prokaryotic cells.

In a second form the invention could be said to reside in a method of inhibiting HIV replication in cells, comprising introduction of any one of the above expression systems into said cell, producing the said produced strong stop RNA so as to interfere with replication of HTV.

The invention in a third form encompasses cells containing an expression system for producing strong stop RNA that inhibits HIV replication.

In a fourth form the invention encompasses a method of treating HTV infection in a human, said method comprising harvesting potentially affected cells from said human, using the second form of the invention to introduce a strong stop expression system into said potentially affected cell, and then, re-introducing said potentially affected cells back appropriately into said human.

Preferably the potentially affected cells are derived from cells involved in the immune system. These cells may be T cells or monocytes/macrophages or bone cells.

The cells are also preferably mononuclear cells or stem cells and in particular haemopoietic stem cells, that are capable of giving rise to macrophages and or T cells.

EXAMPLES

For a better understanding the invention will now be described with reference to the examples.

BRIEF DESCRIPTION OF THE FIGURES.

Figure 1. Structure of anti-RTn RNA expression vectors (not drawn to scale). Abbreviations are: LTR (5'), Moloney murine sarcoma virus (MSV) 5' long terminal repeat: LTR (3"), Moloney murine leukemia virus (MLV) 3' long terminal repeat, which was modified in vector 1 to 5 and 7 as individually indicated; SV, Simian virus 40 early promoter; NEO, neomycin phosphotransf erase gene; HTV LTR, long terminal repeat of HIV (1-684); HTV RU5 (454-743); HIV mLTR, HIV LTR with a deletion (376-434) in the SPl sites and TATA box; a HTV LTR, antisense template of HTV LTR (684-1); aHIV LTR φ , antisense template of HTV LTR plus HTV RNA packaging signal (800-1; tRP, human tRNA^met promoter; TT, RNA polymerase in transcription termination signal. HTV sequence numbers were based on HTV NL43 (Genbank accession number M19921)

Figure 2 Is a bar graph that shows the uptake of (^H) Thymidine by each of 8 cell lines undergoing testing, as an indication of the growth rate, standard variations are shown by capped lines through the centre of each column,

Figure 3 Is a bar graph showing the level of CD4 expression in each of the cell lines used, the level of expression is shown in arbitrary units, and standard variations are shown by the capped lines through the centre of each bar, Figure 4a 4a is a photograph of an ethidium bromide stained gel showing the quantity of antisense RNA present in the total RNA pool produced by each of the cell lines being tested, compared with a standard β-actin RNA, 12 weeks after selection,

Figure 4b 4b is a photograph of an ethidium bromide stained gel showing the quantity of antisense RNA present in the cytoplasmic RNA pool produced by each of the cell lines being tested, compared with a standard β-actin RNA, 12 weeks after transduction,

Figure 5 is a graph of the level of HIV p24 production in the supernatant of all the cell lines of the test after challenge with live HIV (Strain NL43),

Figure 6 Inhibition of HIV transcription by anti-RTn RNA expression.

Total cellular RNA was isolated from die transduced cells 14 weeks after HTV-1 challenge. HIV-1 gag RNA (gag) was detected using RT-PCR. β-actin RNA (β- actin) was included as a control in RT-PCR using the same RNA samples to rule out the presence of possible RT or PCR inhibitors and die variation in RNA input between the samples.

Figure 7 is a photographic representation of an ethidium bromide stained gel showing the level of us HTV RNA, ms HTV RNA compared to control β-actin RNA, at 14 weeks post infection, giving an indication of the level of HIV RNA after challenge, compared to general cellular DNA production, (us is unspliced (about 9kb) HIV RNA; ms is multiply spliced (about 2kb) HTV RNA)

Figure 8a is a photographic representation of an ethidium bromide stained gel showing the level of HTV gag DNA compared to control β-globin DNA, at 12 weeks post infection giving an indication of the level of HIV DNA after challenge, compared to general cellular DNA production,

Figure 8b is a photographic representation of an ethidium bromide stained gel showing the level of HTV gag DNA compared to control β-globin DNA, at 15 weeks post infection giving an indication of the level of HTV DNA after challenge, compared to general cellular DNA production, Figure 9 is a flow diagram of the replication of HTV from initial stage A, through to the complete double stranded DNA from which RNA can be transcribed.

DETAILED DESCRIPTION OF ONE EXAMPLE OF THE INVENTION

EXAMPLE 1

Materials and Methods.

Construction of retroviral vectors

DNA used in the construction of retroviral vectors expressing sense and antisense strong stop RNAs were synthesised by polymerase chain reaction (PCR) amplification of HIV DNA using plasmids pNL43 (Adachi et al (1986), from ATDS Research and Reference Reagent Program, NIIAD, NIH, (ARP)) as template, using the strategy described before (Peng et al 1994). Human tRNA^met promoter was PCR amplified from Jurkat cellular DNA. All the PCR fragments were subcloned into pBluescriptllKS (-) (Strategene). Their sequences were subsequently verified by DNA sequencing. To construct 'double copy' retroviral vectors, the polylinker of plasmid pLXSN (Miller and Rosman, 1989) was deleted. A double stranded oligonucleotide was inserted into the Nhe I site of the plasmid. The resulting vector contains a polylinker and the RNA polymerase III transcription termination signal (RNA-P III TT) in the U3 region of the 3' LTR of HIV DNA. The tRNA promoter and the desired HTV DNA fragments were inserted into the polylinker upstream of RNA P in TT. Unless stated otherwise, DNA cloning was performed according to the established procedures (Sambrook etal, 1989).

Cell culture, transfection and transduction The amphi tropic packaging cell line PA317 (from ATCC) and HeLa cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS, from Commonwealth Serum Laboratories, Melbourne, Australia), 12 ng/ml penicillin, 160 ng/ml Gentamicin and 2mM L-glutamine. Jurkat cells (from ATCC) and H9 cells (from ARP) were grown in RPMM640 (from Gibco) with 10% FCS, 12ng/ml Penicillin, 160 ng/ml Gentamicin and 2mM L-glutamine. All the cells were free of mycoplasma contamination. The recombinant retroviral vectors were transfected into PA317 cells by using calcium phosphate precipitation procedure (Sambrook et al supra). Forty-eight hr after transfection, the conditioned medium containing the recombinant virus was collected by centrifuge at 4,000 rpm for 10 min and stored at -70°C.

To select the stably transfected cell line, the cells were divided 1:10 into selective medium (DMEM growtfi medium containing 400μg/ml active Geneticin, from

Gibco), every 3-4 days thereafter the medium was replaced. Resistant colonies grown up two or three weeks later, were pooled and stored in liquid nitrogen.

The human CD4+ lymphocyte-derived Jurkat cells were transduced by co- culturing with the conditioned medium containing recombinant defective retrovirus at the presence of 8μg/ml of polybrene (Sigma). Twenty-four hours after co-culture, the cells were transferred into RPMI selection medium (RPMI- 1640 growth medium with 400μg/ml active Geneticin). The medium was replaced twice a week thereafter. Resistant cells that had been selected by growth in selection medium for up to 4 weeks were pooled. The pooled resistant Jurkat cells transduced by retroviral vectors anti-RTn 1-5 were designated J anti RTn 1-5 respectively; the cells transduced by the control vectors Cl and C2, which are identical to the parental vector mentioned above but without anti-HIV sequences, were named JC1, JC2, respectively.

Cellular RNA and DNA analysis

Total RNA from parental or transduced Jurkat cells was isolated by acid guanidium thiocyanate (Chromczynski P and N Sacchi, 1987). The cytoplasmic RNA was prepared by NP-40 lysis and SDS-proteinase K digestion (Gilman M, 1992). To remove the possibly contaminating DNA, all the RNA samples were digested widi DNase in the following reaction: to 5 μg RNA, added 3U RNase- free DNase (Boehringer Mannheim Biochemicals), 12U RNasin (Promega), lOmM DTT, lOOmM NaAc, 5mM MgSO₄ in a lOOμl reaction, 37°C for 20 min. One unit of RNase free DNase had been shown to completely digest 1 μg of plasmid DNA in 15 min at 37°C.

The RNA (5μg) was reverse transcribed (RT) in the presence of the antisense primer to detect sense RNA, in the presence of sense primer to detect antisense RNA, using M-MLV reverse transcriptase (Gibco) at the conditions recommended by the manufacturer. One fourth (5μl) of the resulting DNA was amplified as follows. The reaction mixture composed of 50mM KC1 lOmM Tris- HC1, pH 8.3, 1.5mM MgCh, 200μM each dNTP, 1.25U Taq DNA polymerase (Perkin-Elmer Cetus), 50pmol each primers in 50mL was initially heated at 94°C for 3 min; then incubated at 94°C, and 64°C for 30 sees, and 2 min, respectively, the cycle was repeated 40 times in a DNA thermal cycler (Perkin-Elmer Cetus).

Ten-microliter portions of each PCR product were electrophoresed on 1.5% agarose gel. DNA was visualized by UV fluorescence after staining with ethidium bromide, blotted onto Hybond N+ nylon membrane (Amersham) using die protocol recommended by the manufacturer, and probed with specific oligoprobes labelled with ³²P r-dATP.

Extra chromosomal DNA and chromosomal DNA was purified by the Hirt method in the presence of 500μg/ml proteinase K (Hirt 1967; Li & Burrell 1992). Total cellular DNA was prepared by proteinase K digestion. Briefly the cells were pelleted by microfuging. To 10⁶ cells, added lOOμl of digestion buffer containing 50mM KC1, lOmM Tris HC1, pH8.3, 1.5mM MgCl2, 0.5% Triton x 100, 200μg/ml proteinase K. After incubation at 56°C for 1 hr and then heated at 95°C for 10 min and cooled to room temperature, 5μl of d e DNA preparation was directly used for PCR. The thermal cycle program was the same as that for RT-PCR. To prevent contamination, all the tubes containing the DNA samples were opened only inside the laminar flow hood dedicated to PCR work.

The standard RNAs were synthesised in vitro from a plasmid containing full length HTV gag using MEGAscript T7 in vitro Transcription Kit (Ambicon Inc.) with recombinant pBluescript IIKS(-) digested with appropriate restriction enzyme as templates. The in vitro transcribed RNA were digested with RNase free DNase, extracted with Phenolxhloroform twice, precipitated in ethanol and resuspended in DEPC-treated water. The concentration of RNA in mg/ml was converted into copy number. The diluted standard RNAs were supplemented wiu cellular RNA from parental Jurkat cells at concentration of lμg/μl.

Thvmidine uptake assay and quantitation of CD₄ on cell surface Cell proliferation was determined by the level of (³H) thymidine incorporated into the cells. Briefly parental Jurkat cells and transduced cells were seeded, in quadruplicate, into wells of 96- well microtitre plates at 2x10^s cells in 200μl per well. 20μl of (³H) thymidine (Amersham) diluted in RPMI growth medium at concentration of 50μCi/ml was added to each well. After 23 hr the cells were harvested on filter paper with a semi-automatic cell collector. After cell lysis and washing with distilled water and ethanol the radioactivity of the filter was measured in a scintillation counter. Quantitation of CD4 and CD3 on cell surface was performed by flow cytometry (Sattentau etal (1986))

HIV-1 infection and p24 assay

Virus stock HTV-1 NL43 was produced by transfecting HeLa cells using an infectious provirus clone pNL43 (ARP). The subconfluent HeLa cells were transfected by calcium phosphate precipitation as previously described. Forty- eight hours later, the conditioned medium containing the virus was collected and stored at -70°C. An aliquot of the virus was thawed and the TCID 50 was determined by the Endpoint Method (Dulbecco, R. 1988).

Parental and Transduced Jurkat cells (J anti RTn 1-5) and control cells (JC1 and JC2) were infected with HTV-1 NL43 at 0.01 MOI for 24 hr at 37°C. The infected cells were washed twice with serum free RPMI medium and resuspended in growth medium. The culture supernatant was collected for p24 assay twice a week from 1 to 4 weeks post infection (PI), once a week 5 to 10 weeks PI, once every two weeks 10-20 weeks PI.

HIV p24 concentration in the supernatant was determined by using a commercial HIV-1 p24 ELISA kit (Dupont). p24 expression on HTV-1 infected cells was determined by immunofluorescence using the protocol described before (Peng 1994 supra).

Virus rescue

Transduced Jurkat cells tiiat had been infected with HJV-1 and maintained for 20 weeks were mixed with parental Jurkat cells at a ratio of 1:2. After co-culture for 3 days, another aliquot of parental Jurkat cells were added at a ratio of 1 : 1. Every week after starting of the co-culture an aliquot of 10⁶ cells were taken for HTV DNA PCR.

Oligonucleotide primers and probes

All the oligonucleotides were synthesised by Bresatec (Adelaide, Soutfi

Australia). Their sequences are listed on table 1. Table 1. Sequences of oligonucleotide primers and probes

HIV-1 gag primers and probe

Gag 5 (+) 5' agt ace ctt cag gaa caa ata gga 3'

Gag 6 (-) 5' gta ccc ttc agg aac aaa tag gat 3'

Gag probe (-) 5' cat get gtc ate att tct tct agt gtc 3'

Human β-globlin primers β-glo 1 (+) 5' caa ctt cat cca cgt tea cc 3'

β-glo 2 (-) 5' gaa gag cca agg aca ggt ac 3'

Primers for anti-RTn RNA

R 1 (+) 5' ggg tct etc tgg tta gac cag ate 3'

LTR 2 (-) 5' aga tct cct ctg get tta ctt 13'

Human β actin primers β-act 1 (+) 5' etc ace atg gat gat gat ate gec gcg etc 3'

β-act 2(-) 5' cgc get egg tga gga tct tea tga ggt agt 3'

RESULTS

Construction of anti-RTn expression retrovirus vectors and transduction of T cells.

A series of retroviral vectors were constructed in which the expression of effector RNAs (anti RTns) was placed under the control of the human tRN™¹ promoter in the context of a double copy vector (Figure. 1). Anti-RTn 1 should express plus sense RNA containing the RU5 sequence. This transcript in theory, might hybridize with the minus strand strong stop DNA, the first intermediate of HTV reverse transcription, and thus inhibit HIV replication. However, there is no evidence for the existence of a free single strand minus strong stop DNA as target for hybridisation. Anti-RTn 1 dierefore may simply provide abundant initiating template to Trick start' minus strand strong-stop DNA synthesis and thus enhance reverse transcription of die main body of viral RNA from the 3' end (Li et al (1993b)). Anti-RTn 2 was designed to express plus sense RNA containing the full U3RU5 sequence of HTV. Anti-RTn 3 was a similar construct apart from the fact that the SPl sites and TATA box were deleted from the U3 region. This should result in the expression of plus sense RNA containing a U3RU5 sequence with a defect in HTV promoter sequence. Anti-RTn 2 and anti-RTn 3 may compete witii the free, transient single-stranded plus strong stop DNA and thus interfere with the second template switch and inhibit HTV replication.

Anti-RTn 4 should express minus sense RNA complementary to the plus strand strong stop DNA. Anti-RTn 5 had a similar structure to anti-RT 4 except that the minus sense RNA should also contain sequence complementary to the packaging sequence of HTV. Anti-RTn 4 and anti-RTn 5 RNAs may therefore anneal to the free plus- strand strong stop DNA and interfere with the second template transfer thus inhibit virus replication. The control vectors had exacdy the same structure as other vectors but HTV sequences.

The recombinant retrovirus DNA constructs were converted into defective virions by transfecting the DNA into the amphotropic packaging cell line PA 317 (Johnson and Byington, 1990). Two days after transfection, each type of conditioned medium containing different defective retrovirus particles was harvested and used to transduce Jurkat cells. Each group of Jurkat transductants was separately selected in medium supplemented with Geneticin for 55 days to 61 days until the number of cells reached similar levels in each group. Pooled cells corresponding to each construct were then used for further experiments without cell cloning. The Jurkat transductants are named after the corresponding vectors, such as J Anti-RTn 1 for Jurkat cells transduced with anti-RTn 1, etc..

Anti-RTn RNA expression in Jurkat transductants

Intracellular expression of the anti-RTn RNA transcripts in Jurkat transductants was assessed by RT-PCR. Total cellular RNA was isolated from Jurkat transductants ten weeks after selection in Geneticin-containing medium as described. Sense primer (R 1) was used to syndiesise cDNA from any antisense RNA present, while antisense primer (LTR 2) was used to syntiiesized cDNA from the sense RNA (Table 1). The cDNA was then amplified by PCR after addition of the second primer. Jurkat cells transduced by all the vectors expressed the expected sense or antisense RNAs (Figure 2a). The expression level of sense RNAs (anti-RTn 1, 2, 3) was lower compared to diat of antisense RNA (anti-RTn 4, 5). The strand specificity of the RT-PCR procedure was confirmed by experiments using syndietic oligonucleotides complementary to the sense (or anti-sense) RNA as primers; these experiments demonstrated that RT- PCR with sense primers amplified only antisense target and vice versa (data not shown).

To determine whether die anti-RTn RNA transcripts could be transported to die cytoplasm, we isolated cytoplasmic RNA from transduced cells and used RT- PCR to detect the anti-RTn RNAs (Figure 2b). After allowing for a reduced yield of RNA in the cytoplasmic preparations, d e expression pattern of anti-RTn RNA in die cytoplasm was similar to d at in total cellular RNA. This demonstrates diat die anti-RTn RNAs could be transported to cytoplasm.

Anti-RTn RNA expression did not alter the rate of cell proliferation and surface CD4 expression.

To determine die effect of anti-RTn RNA expression on die proliferation of T cells, [³H] diymidine uptake assays were performed. Figure 3a shows t_hat die rates of [³H] thymidine incorporation in J anti-RTn 1 to J anti-RTn 5 cultures were similar to each other and to that of control cells and parental Jurkat cells. Cell proliferation rates as indicated by cell counts over a period of 4 weeks also did not show significant difference between the various groups of anti-RTn RNA expressing cells and the control cells (data not shown). These results demonstrated that die expression of anti-RTn RNA did not have substantial effects on die proliferation of T cells.

The effect of anti-RTn RNA expression on cell surface CD4 expression was evaluated using flow cytometry. The overall levels of CD4 expression of the parental cells and each of die six groups of transduced cells were similar although individual measurements of die cells in die same group varied, as indicated by die extent of standard deviation in each group (Figure 3b). These results indicate diat expression of anti-RTn RNA did not significandy alter die cell surface CD4 level.

Long term inhibition of HTV replication in T cells transduced by anti-RTn expression vectors.

One of die possible approaches to gene dierapy against HIV infection involves ex vivo transduction of autologous haemopoietic stem cells which are then engrafted back into HTV infected patients. The transduced stem cells and their progeny cells, such as CD4⁺ T cells, monocytes and macrophages may express anti-HTV RNA and therefore may resist HTV infection. It is clinically impractical and undesirable to clone die transduced stem cells before engrafting back into patients. Furthermore, individual clones derived from cells transduced with one particular construct may demonstrate variable degrees of resistance to HTV infection (Lee et al, 1994). We dierefore used the pooled transduced Jurkat cells derived from each construct ratiier than homogenous cloned cell lines, to test the ability to resist HTV infection. Results derived from analysing a whole population of transduced cells may minimize the bias associated with analysing clonal cells.

Cultures of Jurkat cells transduced witii each of five constructs or control constructs were challenged at M.O.I, of 0.01 witii HIV-1 virus stock prepared from die pNL43 infectious clone. NL43 was chosen for its ability to cause marked cytopathic effects and cell killing in parental Jurkat cells. To monitor the progress of infection, culture supematants and cells were collected at designated time points after challenge for HTV p24 assay, HTV RNA RT-PCR and HTV DNA PCR.

After HIV-1 challenge, die two separate control cultures diat had been transduced by control vectors (without anti-RTn sequence) showed syncytium formation within one week which reached a peak by four weeks. Most of the cells were killed by that time. Marked syncytium formation also appeared in Jurkat cells transduced by anti-RTn 1(J anti-RTn 1) within one week, which progressed more rapidly tiian in control cells, and reached a peak on day seventeen. No cytopatiύc effects were observed in J anti-RTn 2, 3, 4 and 5 during 20 weeks of follow up.

HTV replication as indicated by die appearance of p24 in die culture supematants correlated well witii die cytopatiiic effects (Figure 4). P24 appeared in die supernatant of the two control cultures 10 days after infection, and reached a peak at 4 weeks, while in Jurkat cells expressing sense RU5 RNA (J antiRTn 1), p24 appeared earlier and reached a peak in a shorter time than in the control cells. In contrast, J anti-RTn 2, 3, 4 and 5 showed no detectable p24 secretion throughout the 20 week experiment. This suggested that no detectable amount of vims was released by J anti-RTn 2, 3, 4, 5 following HTV challenge. To test whedier die production of viral protein was also inhibited, we performed immunofluorescence assay at 9 weeks after infection to detect p24 inside or on die surface of die cells.

The results (data not shown) were in good agreement with mat of culture supernatant p24 assay: HTV p24 was found only in J antiRTn 1 and die control cells. No p24 immunofluorescence was detected in J anti-RTn 2, 3, 4 and 5 cells.

The level of HIV proviral DNA produced following HTV challenge of Jurkat cells transduced witii different anti-RTn vectors or control vectors were also monitored using semi-quantitative PCR. To obtain more accurate quantitation and avoid cross contamination we used a simple and highly reproducible protocol to prepare cellular DNA samples from infected cells (see Materials and Mediods). One million cells were harvested, lysed whh Triton x-100 and digested witii proteinase K. After inactivating proteinase K, an aliquot of die digested mixture was used direcdy for PCR. The whole process of DNA sample preparation did not involve Phenol-chloroform extraction nor edianol precipitaton. The loss of DNA during preparation was tiierefore avoided. This simplified procedure could be conveniendy carried out inside a dedicated laminar flow hood, so that d e possibility of cross contamination was also minimised. Representative results of DNA PCR are shown in Figure 5A and B. After viral challenge control cells and J anti-RTn 1 contained high levels of HTV proviral DNA at all d e time points tested (>10³ copy in 5x10* cells). In contrast, J antiRTn 2, 3, 4, and 5 showed no detectable HIV DNA at most of the time points, and only low level of HTV DNA at some time points tested.

Three possible explanations may account for die absence or very low level of HTV DNA in these cultures. (1) The cells were infected by HTV, but in die presence of anti-RTn sequences, die vims was unable to complete reverse transcription and any incomplete viral DNA produced was subsequently lost from die culture; (2) proviral DNA was produced but was rendered defective by some of the anti-RTn RNA sequences during the second template transfer. Further viral replication was inhibited; (3) die vims may have established infection in a minority of cells diat had inadequate anti-RTn levels (as transduced cells were not cloned) but further viral replication and spread was continuously inhibited by d e anti-RTn RNA expressed in d e majority of transduced cells. To test these possibilities, we performed a vims rescue experiment The rationale behind tiiis experiment was diat if die culture contained replication competent virus diat was continuously suppressed by die anti-RTn RNA, tiien following co-culture witii permissive cells, such as parental Jurkat cells, tiiis should undergo a burst of replication.

After coculture witii parental Jurkat cells for one week, high level (>10³ copies per 5 10⁴ cells) of HIV DNA could be detected from J anti-RTn 2, 3, 4, 5 (Figure 5C). These results suggest diat during d e 20 weeks following initial HTV challenge, viable vims remained in the system, which was fully replication competent More importandy, it appeared diat the expression of anti-RTn 2, 3, 4 and 5 continuously suppressed vims replication in die face of continuing HTV challenge.

To examine die effects of anti-RTn RNA expression on the transcription of HTV RNA after HTV challenge, we performed RT-PCR to detect HIV RNA. Total cellular RNA was isolated from Jurkat transductants 14 weeks after HTV infection. After digestion widi RNase fixe DNase to remove contaminating DNA, the RNA was subjected to RT-PCR to detect HTV gag RNA. As shown in Figure 6, J anti-RTn 1 and the control cells contained HIV RNA transcripts, while J anti¬ RTn 2, 3, 4 and 5 did not have detectable HIV gag RNA. These results demonstrated diat die production of HTV RNA was inhibited in Jurkat cells expressing anti-RTn 2, 3, 4 and 5.

DISCUSSION

Our experiments demonstrated diat intracellular expression of a number of different RNAs targeted at HIV plus strand strong stop DNA could confer long term inhibition of HTV replication in otherwise fully permissive human T cells. In contrast, die expression of sense RNA complementary to die minus strand stong stop DNA actually enhanced HTV replication. It seems diat die RU5 sequences expressed in the form of RNA were unable to anneal witii die minus strong stop DNA in d e process of first template transfer. Instead, die RU5 RNA molecules may have served as initiating templates to enhance the synthesis of die minus strand strong-stop DNA which facilitated die first template transfer and subsequent synthesis of the major body minus strand DNA. This scenario would predict diat U5 region of minus-strand strong stop DNA be synthesized using die vector-expressed RU5 RNA as template (see Li et al, 1993b). The mechanism involved in die enhanced HTV replication in J anti-RTn 1 Cells is currently under investigation.

A high level of inhibition of HTV replication was observed in cells transduced by anti- RTn 4, which expressed minus sense RNA complementary to die plus strand strong stop DNA sequence. This minus sense RNA may inhibit die second template switch (transfer of the plus-strand strong stop DNA) by anneaUng with plus strand strong stop DNA. It could also bind to all the HTV mRNA diat contained R and U5 sequence and tiierefore inhibit die translation of HTV proteins. A slighdy less efficient inhibition of HTV production (as indicated by transient presence of low level HIV DNA, (data not shown) was observed in cells transduced by anti-RTn 5; which has a similar structure to anti- RTn 4 except diat die minus sense RNA would include a sequence complementary to part of d e packaging sequence of HTV RNA. That part of the packaging sequence is not present in the plus- strand strong stop DNA, but is present in die unspliced HTV RNA transcripts. The less potent inhibition by anti-RTn 5 therefore may suggest diat anti-RTn 4 or 5 may have exerted tiieir effects on HTV replication mainly by binding to die strong stop DNA intermediates, although inhibition by anneaUng to HIV transcripts could not be ruled out.

Expression of anti-RTn 2 (sense RNA representing HTV U3RU5 sequence) and anti-RTn3 (as above but widi deletions in die Spl sites and TATA box) may inhibit HIV repUcation by competing widi die transfer of functional plus strand strong stop DNA to newly synthesised minus-strand DNA. The sense U3RU5 RNA (anti-RTn2) may also anneal to die minus strand viral DNA and subsequendy serve as a primer for plus strand viral DNA extension, resulting in creation of a disabled double stranded viral DNA widi a DNA-RNA hybrid in die critical 5* LTR region. If the deleted U3RU5 RNA (anti-RTn 3) annealed to the RU5 PCS sequence of minus strand HTV DNA, die subsequent copying of die deleted U3 part into viral DNA wdl also generate a viral DNA without functional promoter. Such a structure has been proved replication deficient even in die presence of cellular activator (Parrot et al , 1990). Of die three sense constructs die enhancing (anti-RTn 1) or inhibitory efforts (anti-RTn2 and 3) on HTV repUcation could be best interpreted if we assume that the second template switch involves a free single stranded (plus) strong stop DNA while die first template switch does not (Li et al , 1993b). Our results indicate diat strong stop DNA as reverse transcription intermediates which are involved in a different and earlier stage in d e HIV repUcation cycle tiian die "traditional" antisense targets such as tat or rev mRNA, can be efficiently targeted by intraceUular immunication. A combination strategy targeting the early stage of HTV repUcation (anti-RTn) as weU as die HIV expression stage (such as anti-tat or anti-rev) may enhance die efficiency of protection against HIV infection and may be useful for gene dierapy against HTV infection and AIDS.

FURTHER EXAMPLES

Multipotent haemopoietic stem and progenitor ceUs can be harvested from patients' bone marrow or peripheral blood and tiiese primitive ceUs can be transduced by the antiRTn retroviral vectors using established protocols (Nolta et al 1992; Hughes et al 1992, Bregni et al 1992) These in vitro transduced cells can tiien be transferred back to patients from whom those haemopoietic stem and progenitor cells were obtained using approved protocols (MUler 1992; Morgan and Anderson 1993). These transduced stem cells and their progeny cells including monocytes-macrophages and T cells would then reconstruct part of the patients immune ceUs diat are refractory to HJV infection. The antiRTn constructs can also be linked to conditional letiial genes, such as the Herpes Simplex Virus (HSV) tK gene, tiien used in the human gene dierapy protocols. This would aUow eUmination of the transduced cells, in die case of HSV-tK constructs by administration of antiviral dmg gangcyclovir, should unacceptable side effects occur (Venkatesh et al 1990, Morgan and Anderson 1993)

REFERENCES

Adachi, A. et al (1986) J. Virol. 59, 284-291.

Adeniyi- Jones, S., et al (1984). Nucleic Acids Res. 12, 1101-1115

Agawal et al (1989) Proc. Natl. Acad. Sci. USA 86, 7790-7794

Banner, I., et al (1993). /. Virol. 67, 3199-3207.

Baltimore (1988) Nature 335, 395 Bednarik (1989) Proc. Natl. Acad. Sci. USA 86, 4958-4962)

Bevec, D., et al (1992). Proc. Natl. Acad. Sci. USA 89, 9870-9874.

Brady et al (1994) Proc. Natl. Acad. Sci. USA 91, 365-369.

Bregni et al 1992 Blood 80: 1418-1422

Caruso and Klatzmann (1992) Proc. Natl. Acad. Sci. USA 89, 182-186 Chatterjee, S., et al (1992). Science 258, 1485-1488.

Chen, S.-Y. et al (1994). Proc. Natl. Acad Sci. USA 91, 5932-5936.

Chirmule, N., et al (1995). J Virol. 69 492-498

Chromczynski, P., and Sacchi, N. (1987). Anal. Biochem. 162 156-159

Coffin and Haseltine (1977) Proc. Natl. Acad. Sci. USA 74, 190-1917 Crisell, P., et al (1993). Nucleic Acids Res 21, 5251-5155.

Cullen, B.R. et al. (1984) Nature 307, 241-245

Cupp, C etal (1993). Oncogene 8, 2231-2236

Dayton, A.I., et al (1986). Cell 44, 941-947.

Dropulic, B., et al. (1992)./ Virol. 66, 1432-1441. Embretson et al (1993) Nature 362, 359-362.

Ensoli, B et al (1990). Nature 345, 84-86

Ensoli, B et al (1994). Nature 371, 674

Feinberg, M.B. et al (1986) Cell 46, 806-817

Felber, B.K. et al (1989) Proc Natl Acad Sci USA 86, 1495-1499 Fennet and Haseltine (1990) Proc. Natl. Acad. Sci. USA 89, 4164-416,

Fennet and Haseltine (1991) J Virol 65, 1910-1915

Fisher, A.G. et al (1986). Nature 320, 367-371.

Freed, E.O. et al (1992). Proc Natl Acad Sci USA 89, 70-74

Furtura. R.A. et al (1995). J Virol 69, 1591-1599 Gilman, M. (1992). Preparation of cytoplasmic RNA from tissue culture ceUs. In:

"Current Protocols in Molecular Biology" (Ausubel, F.M., et al ed) pp 4.1.2 -

4.1.6, John Wiley & Sons Inc., New York.

Giovannangeli et al (1993) Pro. Natl. Acad. Sci. USA 90, 10013-10017 Graham, F. L., and Van Der Eb, A. J. (1973). Virology 52, 456.

Green, M., et al (1989). Cell 58, 215-223.

Hantzopoulos, P., et al (1989). Proc. Natl. Acad. Sci. USA 86, 3519-3523.

Harrison et al (1992) AIDS Res Human Retroviruses 8, 39-45) Hill, A., and Ploegh, H. (1995). Proc. Natl. Acad. Sci. USA 92, 341-343.

Hirt, B. (1967) J. Mol. Biol. 26, 365-369.

Ho, D. D., et al (1995). Nature 373, 123-126.

Ho, W.-Z., et al (1990). J. Gen. Virol. 71, 97-103.

Homann, M., et al (1993). Nucleic. Acids. Res. 21, 2809-2814. Hu and Temin (1990) Science 250, 1127- 1233.

Hughes et al 1992, J Clin Invest 89: 1817-1824

Karageorgos et al (1993) AIDS Res. Human Retroviruses 9, 819-823

Jayasena and Johnson (1992) Pro. Natl. Acad. Sci. USA 89, 3526-3530

Johnson, V. A., and Byington, R. E. (1990). Quantitative assays for vims infectivity. In "Techniques in HTV Research" (A. Aldovini, B. D. Walker, Ed), pp. 71-76, M Stockton Press, New York.

Jones etfl/(1994) 7 Virol 68 207-216

Joshi, S., et al (1991). J. Virol. 65, 5524-5530.

Lee, S. W., et al (1994). J. Virol. 68, 8254-8264. Li et al (1993a) J Virol 67, 6882-6888

Li et al (1993b) Virology 194, 82-88)

Li, C. J., et al (1995). Science 268, 429-431.

Li, P., and Burrell, C.J. (1992). AIDS. Res. Human Retroviruses 8, 253-259.

Lisziewicz, J., et al (1993). Pro. Natl. Acad Sci. USA 90, 8000-8004. Lo, K. M. S.,et al (1992). Virology 190,176-183.

Lu, Y., et al (1989). J. Virol. 63, 4115-4119.

Maciejewski, J. P., et al (1995). Nature Med. 1, 667-673.

Malim, M. H., et al (1989). Cell 58, 205-214.

MaUm, M. H., et al (1989). Nature 338, 254-257. Malim et al (1992) J Exp med 176, 1197-1201

Matsukura et al (1989) Proc. Natl. Acad Sci. USA 86, 4244-4248

Mhashilkar, A. M., etal (1995).The EMBO J. 14, 1542-1551.

MiUer, A. D., and Buttimore, C. (1986). Mol. Cell. Biol 6, 2895-2902.

Miller, A.D. and Rosman, G.J. ( 1989) BioTechniques 7, 980-990. MiUer 1992 Nature 357; 455-460

Morgan etal (1990) AIDS RES Human Retroviruses 6, 183-191.

Morgan and Anderson 1993 Annu Rev Biochem 62: 191-217 Myers et al (1990) Human Retrovimses and AIDS, Los Alamos National

Laboratory, Los Alamos New Mexico

Nolta et al 1992 Expt Hemeto. 20: 1065-1422

Ojwang, J. O., et al (1992). Proc. Natl. Acad. Sci. USA 89, 10802-10806. Panganiban and Fiore (1988) Science 241, 1064-1069

Panteleo et al (1993) Nature 362, 355-358.

Parrot et al (1990) J. Virol 65, 1414-1419

Pearson, L., et al (1990). Proc. Natl. Acad Sci. USA 87, 5079-5083.

Peng, H., et al (1995). Virology 206, 16-27. Piatak et al (1993) Science 259, 1749-1754

Proudfoot, N. J. (1986). Nature 322, 562-565.

Rhodes, A., and James, W. (1990). J. Gen. Virol. 71, 1965-1974.

Rhodes, A., and James, W. (1991). AIDS 5, 145-151.

Rice et al (1993) Proc. Natl. Acad Sci. USA 90, 9721-9724 Rosen, C.A., Sodroski, J.G., and Haseltine, W. (1985). Cell 41, 813-823.

Sakaguchi (1993) Proc. Natl. Acad. Sci. USA 90, 5219-5223

Sambrook, J., et al. (1989). In: "Molecular Cloning: A Laboratory Manual" 2nd

Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY.

Santos, T., and Zasloff, M. A. (1981). Cell 23, 699-709. Sarver, N., et al (1990). Science 247, 1222-1225.

Sattentau, Q.J., et α/ (1986). Science 234, 1120-1123.

Sczakiel, G., and Pawlita, M. (1991). J. Virol. 65, 468-472.

Sczakiel, G., et al (1992)./ Virol. 66, 5576-5581.

St Johnston, D. (1995). Ce// 81, 161-170. Sullenger, B. A., et al (1990a). Cell 63, 601-608.

Sullenger et al (1990b) Mol. CelLBiol 10, 6512-6523

Sullenger, B. A., et al (1991). J. Virol. 65, 6811-6816.

Sun, L.-Q., et al (1994). Proc. Natl. Acad Sci. USA 91, 9715-9719.

Trono, D., et al (1989). Cell 59, 113-120. Varmus and Shank (1976) J Virol 18, 567-573

Venkatesh et al (1990) Proc. Natl. Acad Sci. USA 87, 8746-8750,

Viscidi, R. P., et al (1989). Science 246, 1606-1608.

Wang and Dolnick (1993) Nucleic Acid Res 21, 4383-4391)

Weerasinghe, M., et al (1991). J. Virol. 65, 5531-5534. Wei, X., et al (1995). Nature 373, 117-195.

Yamada, O., et al (1994). Gene Therapy 1, 38-45.

Yu, M., et al (1995). Proc. Natl. Acad Sci. USA 92, 699-703.

Yu, M., et al. (1993). Proc. Natl. Acad Sci. USA 90, 6340-6344. SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: MEDVET SCIENCE PTY LTD

(U) TITLE OF INVENTION: "Expression System And Method Of Inhibiting RepUcation Of Human Immunodeficiency Vims (HTV)"

(iii) NUMBER OF SEQUENCES: 9

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: A.P.T. Patent and Trade Mark Attorneys

(B) STREET: 1 King WilUam Street

(C) CITY: Adelaide (D) STATE: South AustraUa

(E) COUNTRY: AustraUa

(F) ZIP: 5000

(v) COMPUTER READABLE FORM: (A) MEDTUM TYPE: 5.25" floppy disc

(B) COMPUTER: Macintosh

(C) OPERATING SYSTEM: Macintosh System 7.1

(D) SOFTWARE: Microsoft Word 5.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) ATTORNEY/AGENT INFORM ATION

(A) NAME: Paul J Wyk

(B) REGISTRATION NUMBER:

(C) REFERENCE DOCKET NUMBER: 406PCT:PJW

(vii) TELECOMMUNTCAΉON INFORM ATION

(A) TELEPHONE: 61 84105040

(B) TELEFAX: 61 84105042 (2) INFORMATION FOR SEQ ID NO. 1;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1

AGTACCCTTC AGGAACAAAT AGGA

(3) INFORMATION FOR SEQ ID NO. 2;

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 GTACCCTTCA GGAACAAATA GGAT

(4) INFORMATION FOR SEQ ID NO. 3; (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CATGCTGTCA TCATTTCTTC TAG TGT C

(5) INFORMATION FOR SEQ ID NO. 4;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 nucleic acid residues

(B) TYPE: nucleic acid (C ) STRANDEDNESS : single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4

CAACTTCATC CACGTTCACC

(6) INFORMATION FOR SEQ ID NO. 5;

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 5 GAAGAGCCAA GGACAGGTAC

(7) INFORMATION FOR SEQ ID NO. 6; (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6 GGGTCTCTCT GGTTAGACCA GAT C

(8) INFORMATION FOR SEQ ID NO. 7;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7 AGATCTCCTC TGGCTTTACT TT

(9) INFORMATION FOR SEQ ID NO. 8;

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CTCACCATGG ATGATGATAT CGCCGCGCTC

(10) INFORMATION FOR SEQ ID NO. 9; (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9 CGCGCTCGGT GAGGATCTTC ATGAGGTAGT

(4) INFORMATION FOR SEQ ID NO. 3 ;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CATGCTGTCA TCATTTCTTC TAG TGT C

(4) INFORMATION FOR SEQ ID NO. 3 ;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CATGCTGTCA TCATTTCTTC TAG TGT C

(4) INFORMATION FOR SEQ ID NO. 3; (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CATGCTGTCA TCATTTCTTC TAG TGT C

(4) INFORMATION FOR SEQ ID NO. 3 ;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: Linear

(ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1 CATGCTGTCA TCATTTCTTC TAG TGT C

(4) INFORMATION FOR SEQ ID NO. 3;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleic acid residues

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (iv) ANTISENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO. CATGCTGTCA TCATTTCTTC TAG TGT C

Claims

CLAIMS:

1. An expression system for producing strong stop RNA or derivatives dierof , diat interfere widi corresponding target sequences of HTV virus DNA during viral repUcation within a ceU, in which strong stop RNA inhibits replication of HTV vims, so as to enhance the viability of the ceU.

2. An expression system as in claim 1 wherein the strong stop RNAs are not present in the normal HTV replication cycle.

3. An expression system as in claim 1 wherein the produced strong stop RNA are die same as nucleic acid sequences present in the normal HTV but wid inactivating modifications.

4. An expression system as in claim 3 wherein the modification is selected from the group comprising deletions, substitutions, insertions, base modifications or combination of two or more of these deletions.

5. An expression system comprising solely nucleic acid which includes an RNA promoter, and a nucleic acid sequence aUowing production of multiple copies of the strong stop RNA.

6. An expression system as in claim 5 wherein the nucleic acid is to be integrated into endogenous nucleic acid of die target cell.

7. An expression system as in claim 5 wherein die nucleic acid is part of a replicating vector system.

8. An expression system as in claim 1 wherein die strong stop RNA is greater tiian 250 bases long.

9. An expression system as in claim 1 wherin die strong stop RNA is greater than 50 bases long.

10. An expression system as in claim 1 wherein die strong stop RNA is identical or complementary to any one or more of the elements designated U5, PBS, U₃, R, Ψ.

11. An expression system as in claim 1 wherein die strong stop RNA is antisense.

12. An expression system as in claim 1 wherein die strong stop RNA is sense but expressed with one or more modifications.

13. An expression system as in claim 1 wherein the strong stop RNA produced is complementary to any one or more of the sequences number of die HIV genome: U₃ (1-453), R (454-551), U₅ (552-634); PBS (635-653), Ψ (653- 790)

14. The expression system as in claim 7 wherein the vector comprising a Murine Leukemia Vims vector, which contains a 5' and a 3' LTR (long terminal repeat) sequence, a packaging signal, a selectable marker encoding sequence, and one or more promoters upstream of die site encoding the strong stop nucleotide sequence.

15. The expression system as in claim 14 wherein the promoter(s) for producing strong stop RNAs is human tRNA _me_t promoter.

16. The expression system as in claim 14 wherein the promoter for producing strong stop RNAs is the promoter located within die murine leukemia or HIV LTRs.

17. The expression system as in claim 14 wherein a separate promoter act as a means for expressing die selectable marker encoding sequence.

18. The expression system as in claim 17 wherein the Simian Vims 40 (S V40) acts to provide for expression of the selectable marker encoding sequence.

19. The expression system as in claim 14 wherein the selectable marker encoding sequence is an antibiotic resistance gene diat is able to be selected in eukaryotic cells.

20. The expression system as in claim 14 wherein die selectable marker encodes neomycin phosphotransferase.

21. The expression system as in claim 1 wherein die strong stop RNA is not solely unmodified R U5 sense nucleic acid.

22. A method of inhibiting HTV repUcation in ceUs, comprising introducing die expression system of any one of the above claims into a ceU, and allowing the production of die said produced strong stop RNA so as to interfere widi repUcation of HIV.

23. The ceUs produced by die metiiod of claim 22.

24. A metiiod of treating HIV infection in a human, said metiiod comprising harvesting potentially affected ceUs from said human, introducing d e expression system of any one of claims 1 to 21 using the metiiod of claim 22, and tiien, re¬ introducing said potentiaUy affected ceUs back appropriately into said human.

25. The method of claim 24 wherein die potentially affected cells are derived from cells involved in the immune system or/and haematopoietics.

26. The metiiod of claim 25 wherein die potentially affected cells are derived from the group comprising T ceUs, monocytes/macrophages or bone marrow cells.

27. The method of claim 25 wherein die potentiaUy affected cells are mononuclear cells or bone marrow stem cells or blood stem ceUs.

28. The metiiod of claim 25 wherein die potentiaUy affected ceUs are haemopoietic stem cells, diat are capable of giving rise to macrophages and/or T cells.