WO2005095607A2

WO2005095607A2 - ANTI-SENSE OLIGONUCLEOTIDES DIRECTED TOWARDS THE HIV ψ DOMAIN

Info

Publication number: WO2005095607A2
Application number: PCT/GB2005/001273
Authority: WO
Inventors: Michael Gait; Andrew Lever; Douglas Brown
Original assignee: Cambridge University Technical Services Limited; Medical Research Council
Priority date: 2004-03-31
Filing date: 2005-03-31
Publication date: 2005-10-13
Also published as: GB0407382D0; WO2005095607A3

Abstract

Anti-sense oligonucleotides directed at the ψ domain of the HIV genome, in particular, the SL3 loop/stem region of the HIV ψ domain, are shown herein to disrupt the interaction of the ψ domain with Gag NC, thereby inhibiting genome encapsidation and reducing the production of infectious HIV particles. These anti-sense oligonucleotides are useful as anti-retroviral therapeutics.

Description

Anti-sense oligonucleotides directed towards the HIV PSI Domain

This invention relates to anti-sense oligonucleotides wb-ich are useful in the treatment of HIV infection.

The Human Immunodeficiency Virus (HIV) is the aetiological agent that causes Acquired Immunodeficiency Syndrome (AIDS). Since the first isolation and identification of the virus in the early 1980' s, its seroprevalence amongst adults has increased dramatically. It is now estimated that 40 million people worldwide are either infected with HIV or suffer from AIDS. Even though global educational and awareness programs are active, infection is still spreading. Several therapeutics have been developed which target two main areas of the virus life cycle, the reverse transcription step and protease cleavage step. At present, there are three classes of drug and a combination of these must be administered at any one time to reduce the emergence of drug resistant viruses. If drug resistance occurs, that particular set of drugs is no longer able to reduce viral load and another set is prescribed. This process continues until all available drugs currently on the market are exhausted. Despite the success of current drugs in prolonging the life span of HIV infected individuals, they do not clear the infection and resistant forms of the virus emerge which eventually lead to the onset of AIDS.

There is therefore a need for improved anti-retroviral agents for the treatment of HIV/AIDS.

The ψ domain of the HIV genome is involved in the encapsidation of viral RNA through the binding of Gag NC protein. The present inventors have discovered that anti-sense oligonucleotides directed at a particular region of the ψ domain can disrupt the interaction of the ψ domain with Gag NC and thereby inhibit genome encapsidation. This inhibition reduces the production of infectious HIV particles . Anti-sense oligonucleotides as described herein may therefore be useful as anti-retrovixal therapeutics. One aspect of the invention provides an oligonucleotide comprising or consisting of a nucleic acid sequence which is complementary to SL3 loop/stem region of the HIV ψ domain.

An oligonucleotide is an oligomer of RNA, DNA or a mimetic or analogue thereof. An oligonucleotide may thus be composed of naturally-occurring nucleobases, sugars and covalent internucleoside linkages, or non-naturally-occurring components which have similar function.

A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base, such as a purine or pyrimidine. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3 ' to 5 ' phosphodiester linkage.

Preferably, an oligonucleotide of the invention reduces or inhibits the binding of the gag NC polypeptide (SEQ ID NO: 5) to the domain sequence. An oligonucleotide of the invention may thus be useful in inhibiting encapsidation. Through the inhibition of encapsidation of the viral genome, an oligonucleotide may reduce viral spread and thereby reduce HIV pathogenicity.

In addition to reducing pathogenicity, inhibition of encapsidation may lead to the production of viral particles that lack RNA. The production of such non-virulent particles may have a beneficial immunostimulatory effect. Preferably, the HIV ψ domain is an HIV-1-.

domain. The core region of the HIV-1 ψ domain, including the SL3 -loop/stem region, is shown in Figure 1 (SEQ ID NOr 1) .

An oligonucleotide of the invention may comprise all or part of a nucleic acid sequence which is complementary to the SL3 loop/stem region, for example an oligonmcleotide may comprise all or part of the nucleic sequence; UCC UUC UAG CCU CCG CUA GUC AAA A (SEQ ID NO: 2)

An oligonucleotide described herein may comprise or consist of at least 12, 13, 14, 15 or 16 contiguous nucleotides from the nucleic acid sequence of SEQ ID NO: 2.

An oligonucleotide described herein may comprise or consist of 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, or 18 or less nucleotides,

In some preferred embodiments, the oligonucleotide may comprise or consist of the nucleic acid sequence

UCUAGCCUCCGCUAGU (SEQ ID NO: 3) or UAGCCUCCGCUAGU (SEQ ID NO: 4) .

In preferred embodiments, an oligonucleotide of the invention may comprise one or more modifications (i.e. it may comprise one or more modified bases or base analogues) .

For example, an oligonucleotide may comprise or consist of one or more 2' -0-methyl substituted bases, ffor example 2, 3, 4, 5, 6 or more 2 '-O-methyl substituted bases, or one or more locked nucleic acid (LNA) bases, for example 2, 3, 4, 5, 6 or more locked nucleic acid (LNA) bases .

2 '-O-methyl and 2 ' -O-methoxy-ethyl RNA substitutions are described, for example, in Kurreck, J. et al (2002) Nucleic Acids Res. 30,19 11-1918. Locked nucleic acids (LNA) are ribomucleotides which contain a methylene bridge that connects the 2 ' -oxygen of the ribose with the 4' -carbon. LNAs are described, for example, in 0rum,H. & Wengel,J. (2001) Curr. OpLnion Mol . Ther. 3, 2 39- 24. and are commercially available -from Proligo (Paris, France and Boulder, CO,USA) .

Other suitable modifications may incrlude, for example, phosphorothioate (PS) linkages (Eckstein, F. (2000) Antisense Nucleic Acids Drug Dev. 10,11 7-121) , peptide nucleic acids (PNAs) (Braasch,D .A.& Corey, D. R. ( 2002) Biochemistry 41,45 03-4509), N3'-P5' phosphoroamidates (NPs) (Gryaznov,S. et al (1994) J. Am. Chem. Soc. 116,3143 -3144), 2 ' -deoxy-2' -fluoro- (S-D-arabino nucleic acids (FANA) (Da.τnha,M. et al (1998) J. Am. Chem. Soc. 120,1 2976-12977), morpho lino nucleotides (MF) (Gene Tools LLC, Corvallis, OR ,USA) , cyclohexene nucleic acids (CeNA) (Wang, J. et al (2000) J. Am. Chem. Soc. 122,8 595-8602) and tricyclo-DNA, (tcDNA) ( -Renneberg, D. & Leumann, C.J. (2002) J. Am. Chem. Soc. 124,5993-6002).

In some preferred embodiments, an ol igonucleotide of the invention may be a chimeric oligonucleotide i.e. an oligonucleotide which contains two o_r more chemically distinct regions, each made up of at least one monomer unit. Each chemically distinct region may comprise for example, a particular modification, for example a modification described above. The chemically distinct regio-ns may be in any order or arrangement within the oligonucleotide .

For example, an oligonucleotide may comprise one or more regions of 2 '-O-methyl substituted biases and one or more regions of LNA bases, more preferably 2, 3, 4, 5 or 6 regions consisting of one or more 2' -O-methy-1 substituted bases and 2, 3, 4, 5 or 6 regions consisting of o-cie or more LNA bases. In some embodiments, an oligonucleotide may consist of LNA and 2' -O-methyl substituted bases. An oligonucleotide as described herein may have any order, combination or arrangement of LNA and 2 ' -O-methxyl substituted bases. A skilled person is readily able to des-Lgn suitable combinations of modifications within the oligonucleotide sequence .

In some preferred embodiments, the oligonucleotide may comprise or consist of alternate or substantial-ly alternate LNA and 2 'OMe substituted bases. For example, an oligonucleotide may comprise or consist of the sequence; ϋCUAGCCUCCGCUAGU or UAGCCUCCGCUAGU, where bold denotes a 2 -OMe substituted base and underlining denotes an LNA- base .

An olignucleotide may be attached or linked by a covalent or non-covalent bond to one or more functional groups which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Suitable groups include peptides, proteins, cholesterols, lipids, phospholipids , biotin, phenazine, folate, phenanthridine, anthraquinon-e, acridine, fluoresceins, rhodamines, coumarins, and dyes. The use of such groups to improve the properties of anti-sense molecules is well known in the art.

An oligonucleotide as described herein or a salt, solvate, chemically protected form or prodrug thereof, may be used in the treatment of the human or animal body, in particular, for use in the treatment of an HIV infection, such as an HIV-1 infection, or a condition associated with HIV infection.

An antisense oligonucleotide may include any pharmaceutically acceptable salts, esters, or salts of such estexs, or any other compound, such as a prodrug, that, upon a ministration to an animal including a human, is capable of p-roviding the biologically active antisense oligonucleotide.

A prodrug is a therapeutic agent that is prepared in an inactive form that is converted to an active fo-rm (i.e. the active antisense oligonucleotide) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides may be prepared as SATE [ (S-acetyl-2- thioethyl) phosphate] derivatives using techniques well-known in the art (see for example WO 93/24510 or WO 94/26764) .

Suitable pharmaceutically acceptable oligonucleotide salts include (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or nitric acid; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, or polygalacturonic acid; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

An oligonucleotide as described herein, a salt, solvate, chemically protected form, or prodrug thereof, may be used in the manufacture of a medicament for use in the treatment of a HIV infection or a condition associated with HIV infection.

A method of treatment of an HIV infection or a condition associated with HIV infection may comprise administering an oligonucleotide of the invention or a salt, solvate, chemically protected form, or prodrug thereof, to an individual in need thereof .

An oligonucleotide may be administered in the form of a pharmaceutical composition. A pharmaceutical composition may comprise an oligonucleotide of the invention, or a salt, solvate, chemically protected form or prodrug thereof, and a pharmaceutically acceptable excipient.

A pharmaceutical composition may be produced by a method comprising; synthesising or producing a oligonucleotide of the invention or a salt, solvate, chemically protected form or prodrug thereof, and admixing the oligonucleotide, salt, solvate, chemically protected form or prodrug, with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials.

The precise nature of the excipient, vehicle or carrier wi-11 depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, intramuscular or intravenous, or intranasally.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with, a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing ini_.o association the oligonucleotide, salt, solvate, chemically protected form or prodrug with the carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the oligonucleotide with liquid carriers or finely divided solid carriers or both, and then, if necess-ary, shaping the product . Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols .

Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of oligonucleotide; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients . Compressed tablets may be prepared by compressing in a suitable machine the oligonucleotide in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose) ; fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) ; surface-active or dispersing or wetting agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid) . Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered oligonucleotide moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile . Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Liquid pharmaceutical compositions for oral administration generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs . Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the oligonucleotide in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml . The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs .

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular peptide, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient . The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. In general, a suitable dose of oligonucleotide may be in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day.

All documents mentioned in this specification are hereby incorporated herein by reference .

It will be understand that the invention encompasses each and every combination and sub-combination of the features of the invention described above .

Aspects of the present invention will now be illustrated with reference to the accompanying figures described above and experimental exemplification, by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art.

Figure 1 shows the HIV-1 Ψ sequence and secondary structure, including the SL3 region.

Figure 2 shows the SL3 region of the ψ domain with the target oligonucleotide sequences (16mer3 shown in grey and 14mer3 shown in black) .

Figure 3 shows the reduction of Gag binding in the presence of chimeric oligonucleotide.

Figure 4 shows a fusion index graph of the syncitia produced during HIV-1 infection with and without chimeric oligonucleotide present.

Figure 5 shows the reduction of Gag binding in the presence of 2 "OMe Phos 16mer3 oligonucleotide. Ficjure 6 shows a fusion index graph of the syncitia produced during HIV-1 infection with and without 2 "OMe Phos 16mer3 oligonucleotide present .

Figure 7 shows inhibition of HIV-1 release from Jurkat T cells by 2"0me/LNA oligonucleotide (L16m3) , scrambled oligonucleotide controls (L16mScr) and mock transfections (no oligo) . P.T. = post transfection of oligonucleotide; P.I. = post infection of HIV-1; RT = reverse transcriptase .

Figure 8 shows inhibition of HIV-1 release from Jurkat T cells by 2 "OMe Phos 16mer3 oligonucleotide (pl6m3) , scrambled oligonucleotide controls (pl6mScr) and mock transfections (no oligo) . P.T. = post transfection of oligonucleotide; P.I. = post infection of HIV-1; RT = reverse transcriptase.

Table 1 shows examples of chimeric antisense oligonucleotides where bold denotes a 2 "OMe substituted base and underlining denotes an LNA base.

SEQ ID NO: 1 is the nucleotide sequence of the HIV-1 ψ domain.

SEQ ID NO: 2 is the nucleotide sequence which is complementary to the SL3 region of HIV-1 ψ domain.

SEQ ID NO: 3 is the nucleotide sequence of the 2"OMe/LNA 16mer3 oligonucleotide.

SEQ ID NO: 4 is the nucleotide sequence of the 2"OMe/LNA 14mer3 oligonucleotide.

SEQ ID NO: 5 is the amino acid sequence of the Gag NC polypeptide.

SEQ ID NO: 6 is the complement of the core sequence of the SL3 region. Experiments Ψ target and antisense oligonucleotide sequence Antisense oligonucleotides were designed targeted to the SL3 region (figs 1 and 2) and tested for binding affinity in binding assays.

The oligonucleotides used were 2'-0-Methyl oligoribonucleotide (2 OMe) locked nucleic acid (LNA) chimeras (Table 1). A third oligonucleotide (2 "OMe Phos 16mer3) had the same sequence as the 16mer 2"OMe/LNA oligonucleotide but consisted of 2 "OMe substituted bases containing phosphorothioate linkages and did not contain LNA bases. Equal concentrations of radioactively labelled RNA were mixed • with binding buffer (50mM Tris-Cl, 50mM KC1, 50mM MgCl₂) , 4μg tRNA and varying concentrations of oligonucleotide (7.81nM to 500nM) . A scrambled sequenced 2"0Me/LNA 16mer3 oligonucleotide which has no homology for target was used as a control. The reaction mix was heated at 95°C for 3 minutes and snap cooled on ice before incubation at room temperature for 20 minutes. Samples were then loaded on 10% polyacrylamide gel and run at 20mA at 4°C. Gels were then dried and exposed to x-ray film. The intensity of oligonucleotide bound and unbound RNA species was then measured.

A graph of oligonucleotide concentration vs percentage of RNA in complex with the oligonucleotide was plotted and an affinity value calculated by observing the concentration of oligonucleotide required to cause 50% binding of the RNA. The affinities of the 16mer and 14mer 2"OMe/LNA oligonucleotides are 70nM and 80nM respectively. These affinity values reflect the ability of the oligonucleotide to bind to the target sequence with high affinity. The affinity of the 2 "OMe Phos 16mer3 oligonucleotide was found to be 290nM, which indicates that it binds to target RNA more weakly compared with the 2"OMe/LNA chimeras.

Oligonucleotide inhibi tion of Gag binding to SL3

The Gag protein was added into the binding assays in order to assess the ability of the oligonucleotide to inhibit Gag binding to SL3.

Binding assays were carried out as described above except oligonucleotide concentration remained constant (to give 100% binding of target RNA) and Gag protein was added in varying concentrations (300nM-3000nM) to the reaction mix. The Gag was added after the 20 min incubation of RNA and oligonucleotide and the reaction incubated for a further 20 minutes before being loaded on to a polyacrylamide gel .

Gag binding to the target RNA was observed to -be reduced in the presence of oligonucleotide (fig 3) . This effect was seen with both the 14mer and 16mer oligonucleotides. The reduction in Gag binding was quantified by measuring the concentration of Gag protein required to cause a 50% shift of RNA, in the presence and absence of oligonucleotide. When oligonucleotide was present in the reaction, a reduction of Gag binding of up to 10 times was observed (i.e. Gag binding to RNA was reduced to 10% of the level observed in the absence of oligonucleotide) .

The 2 "OMe Phos 16mer3 oligonucleotide was found to reduce protein binding to SL3 to about 60% of the level of wild type

Gag binding (fig. 5) . This represents an inhibitory effect although the reduction in binding was less than seen with the chimeras .

Oligonucleotide inhibi tion of cell to cell spread of HIV-1

The cell line HeLa T4 LTR LacZ contains the receptor to allow HIV entry and a stably integrated LTR-LacZ construct . The LTR promoter is activated by the virally produced Tat protein and drives expression of LacZ, allowing identification of individually infected cells

HeLa T4 LTR LacZ cells were plated onto a 96 well plate at 10% confluency and. incubated overnight. Oligonucleotide was then transfected into the cells using lipofectamine 2000 and incubated for 3 hours. Cells were then washed and infected with wild type HIV-1 and incubated for 3 days. After incubation, cells were stained for LacZ production.

In the wild-type infected samples (without pre-treatment by oligonucleotide) blue cells were observed, as well as many multi-nuclei foci. This is due to infected cells producing infectious vi-trus, which is transported to the cell membrane and transmitted to an adjacent cell. The presence of the viral proteins on trie cell surface of the infected cells promotes fusion of the adjacent cells forming multi-nuclei syncitia. However, when the cells were pre-treated with oligonucleotide then the abundance of these multi-nuclei syncitia was greatly reduced, as well as the number of nuclei per syncitium, providing indication that viral replication has been inhibited

The syncitia were scored for number of nuclei per syncitia and number of syncitia observed and a fusion index value was calculated. This equation was:

Fusion Index = (N - S) T Where N = number nuclei in syncitia S = number of syncitia T = total number of nuclei counted

The fusion index values were calculated for each concentration of oligonucleotide used and plotted relative to the fusion index of wild type infection (fig. 4) . Figure 4 shows that the chimeric oligonucleotides reduce the formation of syncitia in a dose dependent manner and inhibit cell-to-cell transmission of the HIV virus.

HIV challenge experiments were also carried out as described above for the 2 "OMe Phos 16mer3 oligonucleotide. Syncitia production was then scored and fusion index values plotted relative to wild type infection (fig. 6) . No significant reduction of syncitia production was observed when the cells are pre-treated with the 2 "OMe phos 16mer3 oligonucleotide.

In summary, antisense LNA/2' -0'methyl chimeric oligonucleotides have been identified that bind with high affinity to the SL3 region of the HIV-1 packaging signal. The oligonucleotides were observed to reduce the ability of the Gag protein to bind to SL3 in vitro and to reduce, in a cellular assay, the formation of multi-nuclei syncitia which is seen in wild type infection. The reduction of these syncitia is due to the inability of the viral genome to be packaged and transported to the cell surface . The presence of the oligonucleotide thus prevents spread of infectious virus from an infected cell to adjacent cells by inhibiting encapsidation of the genomic RNA.

Oligonucleoti de inhibi tion of HIV-1 release from Jurkat T cells lx 10⁷ Jurkat T cells were transfected with oligonucleotide using Effectin-12 (Cambio, Cambridge, UK) and incubated for 3 hours to allow uptake. Cells were then washed, to remove residual oligonucleotide-lipid complexes, and then infected with wild type HIV-1, in a final volume of 10ml. Cells were incubated for 19 days and virus release into the supernatant was measured at regular intervals using the reverse transcriptase (RT) assay.

2"0me/LNA 16m3 and 2"0me Phos 16m3 were both analysed at lOOOnM and 500nM concentrations and compared with scrambled oligonucleotide controls and mock transfections. In the case of both chemistries of oligonucleotide the release of virus from cells into the supernatant was reduced in a specific manner (fig. 7 and. fig. 8) .

In conclusion, viral challenge of oligonucleotide transfected cells resulted in a reduction in the level of virus released from the cells into the supernatant.

Table 1

SEQUENCES

1. (SEQ ID NO: 1) CUCUCUCGAC GCAGGACUCG GCUUGCUGAA GCGCGCACGG CAAGAGGCGA GGGGCGGCGA CUGGUGAGUA CGCCAAAAAU UUUGACUAGC GGAGGCUAGA AGGAGAGAGA UGGGUGCGAG AGCGU

2. (SEQ ID NO: 2) UCCUUCUAGC CUCCGCUAGU CAAAA

3. (SEQ ID NO: 3) UCUAGCCUCCGCUAGU

4. (SEQ ID NO: 4) UAGCCUCCGCUAGU

5. (SEQ ID NO: 5) IQKGNFRNQR KTVKCFNCGK EGHIAKNCRA PRKKGCWKCG KEGHQMKCDT ERQANFLGKP IWSHKGRPGN FL

6. (SEQ ID NO: 6) CCUCCG

Claims

Claims :

1. An antisense oligonucleotide comprising a nucleotide sequence which is complementary to all or part of the SL3 loop/stem region of the HIV ψ domain.

2. An antisense oligonucleotide according to claim 1 comprising at least eight contiguous nucleotides from the nucleic acid sequence of SEQ ID NO: 2.

3. An antisense oligonucleotide according to claim 1 or claim 2 wherein the oligonucleotide consists of 25 or less nucleotides .

4. An antisense oligonucleotide according to claim 3 wherein the oligonucleotide consists of 12 to 16 nucleotides.

5. An antisense oligonucleotide according to any one of the preceding claims wherein the oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 6.

6. An antisense oligonucleotide according to any one of the preceding claims consisting of the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

7. An antisense oligonucleotide according to any one of the preceding claims comprising one or more modifications .

8. An antisense oligonucleotide according to claim 7 wherein the oligonucleotide comprises one or more 2' -O-methyl substituted (2 "OMe) bases or locked nucleic acid bases.

9. An antisense oligonucleotide according to claim 7 comprising two or more different modifications.

10. An antisense oligonucleotide according to claim 9 comprising one or more 2' -O-methyl substituted (2"OMe) bases and one or more locked nucleic acid bases .

11. An antisense oligonucleotide according to claim 10 wherein each base of said oligonucleotide is a 2 '-O-methyl substituted (2 "OMe) base or a locked nucleic acid base

12. An antisense oligonucleotide according to claim 11 comprising alternating 2' -O-methyl substituted (2"0Me) base and locked nucleic acid base.

13. An antisense oligonucleotide according to claim 11 or claim 12 consisting of the sequence UCUAGCCUCCGCUAGU or UAGCCUCCGCUAGU, where bold denotes a 2 "OMe base and underlining denotes an LNA base .

1 . An antisense oligonucleotide according to any one of the preceding claims which is bound to a conjugate group.

15. An antisense oligonucleotide according to any one of the preceding claims which, on binding to target RNA within a cell, does not induce cleavage of the target RNA.

16. An antisense oligonucleotide according to any one of the preceding claims for use in the treatment of the human or animal body.

17. An antisense oligonucleotide according to any one of the preceding claims for use in the treatment of an HIV infection.

18. Use of an oligonucleotide according to any one of claims 1 to 15 in the manufacture of a medicament for use in the treatment of an HIV infection.

19. A method of inhibiting the binding of the HIV gag protein to an RNA ψ sequence comprising contacting said sequence with an oligonucleotide according to any one of claims 1 to 15.

20. A method of treatment of an HIV infection comprising administering an antisense oligonucleotide according to any one of claims 1 to 15 to an individual in need thereof.

21. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

22. A method of making a pharmaceutical composition comprising; admixing an oligonucleotide according to any one of claims with a pharmaceutically acceptable excipient .