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• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
  • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

• the present invention relates to genetic elements that suppress the activities of the human immunodeficiency virus (HIV) .
• the invention relates to polynucleotides isolated from the HIV-1 genome, methods for isolating, identifying and designing such polynucleotides, and methods for using them for the protection of human cells against HIV infection and/or replication.
• HIV acquired immunodeficiency syndrome
• HIV is a member of the lentivirus family of retroviruses (Teich et al . , 1984, RNA Tumor Viruses, Weiss et al . , eds . , CSH-Press, pp. 949-956) .
• Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally- encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-1439).
• HIV-1 Barre-Sinoussi et al .
• CD4 + T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al . , 1984, Nature 312:763-767; Klatzmann et al . , 1984, Nature 312:767-768; Maddon et al . , 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gpl20 binding to the cellular CD4 receptor molecule (McDougal et al . , 1986, Science 231:382- 385; Maddon et al . , 1986, Cell 47:333-348), and a chemokine co-receptor such as CXCR4 or CCR5 (Moore, 1997, Science 276:51-52) .
• a chemokine co-receptor such as CXCR4 or CCR5
• HIV infection is pandemic, and HIV-associated diseases have become a world-wide health problem.
• anti-HIV modalities there is, thus far, no successful prophylactic or therapeutic regimen against AIDS.
• several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al . , 1991, FASEB J. 5:2369-2381).
• virally-encoded reverse transcriptase has been a major focus of drug development.
• reverse-transcriptase- targeted drugs including 2', 3'-dideoxynucleotide analogs such as AZT, ddl, ddC, and ddT have been shown to be active against HIV (Mitsuya et al . , 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al . , 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
• 2', 3'-dideoxynucleotide analogs such as AZT, ddl, ddC, and ddT have been shown to be active against HIV (Mitsuya et al . , 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of
• Late stage processing is dependent on the activity of a virally-encoded protease, and drugs including nelfinavir, saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al . ,
• tk herpes simplex virus type 1 thymidine kinase
• Diphtheria toxin gene has also been used, and the gene was placed under the control of cis-acting HIV regulatory sequences (U.S. Patent 5,306,631, issued April 26, 1994). Others have utilized replication incompetent mutants of HIV which have the potential to express an inhibitory gene product in the presence of HIV tat (WO 94/16060, July 21, 1994) .
• Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Efforts to identify appropriate protective genes have, in large part, been based on an understanding of the molecular biology of HIV replication. A few examples of this approach are as follows.
• the HIV-1 Rev gene encodes a protein that is necessary for the expression of full length HIV-1 transcripts in infected cells and the production of HIV-1 virions .
• Transfection with one Rev mutant known as RevMlO has been shown to protect the cells against HIV infection (Malim et al., 1992, J.Exp. Med. 176:1197; Bevec et al . , 1992, Proc. Natl. Acad. Sci. USA 89:9870-74).
• the transfectants are resistant to HIV-1 infection for about 2 weeks from the time of inoculation before resistant variants appear (Woffendin et al . , 1994, Proc. Natl. Acad. Sci. USA 91: 11581-85) .
• Rev function can be interfered with by producing an excess of the binding site of the Rev protein, termed Rev Response Element (RRE) , which prevents the binding of Rev to RRE of viral transcripts.
• RRE Rev Response Element
• a "decoy” which consisted of a chimeric RNA composed of an RRE and a tRNA prevented infection of cultured cells for a period of greater than about 40 days (Lee et al . , 1994, J. Virology 68:8254-64).
• fusion proteins capable of binding to viral env proteins have been made to prevent the production of HIV-1 virions.
• examples include a fusion protein composed of CD4 and a lysosomal targeting protein procathepsin D, and an anti-env Fv which is secreted into the endoplasmic reticulum (Lin et al . , WO 93/06216; Marasco et al . , 1993, Proc. Natl. Acad. Sci. USA 90:7889-93).
• Antisense polynucleotides have also been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al . , WO 94/10302; Kretschmer et al . , EP 594,881; and Chatterjee et al . , 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The ribozyme approach to forming an HIV-1 resistant hematopoietic cell line has been reported (Ojwang et al . , 1992, Proc. Natl. Acad. Sci.
• Roninson et al described a method for isolating genetic fragments from the HIV-1 genome capable of protecting a cell from HIV-1 infection (U.S. Patent No. 5,217,889 and WO 92/07071) .
• the method involves the preparation of an expression library known as a Random Fragment Expression (RFE) library that contains random sequence fragments of the HIV-1 genome.
• RFE Random Fragment Expression
• Gene fragments referred to as HIV-1 Genetic Suppressor Element (HIV-1 GSE) are then selected from the RFE library following an extensive selection procedure.
• the selection step involves transfection of the RFE library into a cell line to which HIV-1 infection is normally cytotoxic .
• the low sensitivity of this selection step greatly limits the practical use of the procedure.
• no specific GSE sequences were reported using this method that were capable of suppressing HIV-1 infection.
• the present invention relates to specific HIV-derived polynucleotides herein referred to as GSEs that suppress HIV infection and/or replication in human cells, methods for isolating and identifying such polynucleotides, methods for designing such polynucleotides, and methods for using them in the prevention or treatment of HIV infection.
• the invention is based, in part, on the Applicants' discovery that nucleotide fragments can be isolated from the HIV-1 genome, based on their ability to suppress the activation of latent HIV-1 in a CD4 + cell line or on their ability to prevent cells from productive infection by HIV-1.
• any cellular or viral marker associated with HIV replication can be used to monitor the activation of latent HIV or productive infection of cells.
• a number of novel HIV-1 GSE polynucleotides are selected on the basis of their ability to sustain CD4 expression by the induced cells, and several of such sequences are further shown to protect uninfected T cells from productive infection by HIV-1.
• the GSEs may function in the form of an RNA product or protein product, both of which are within the scope of the invention.
• Another exemplary marker is the intracellular p24. Replication of HIV in susceptible cells is associated with accumulation of intracellular p24, concomitant with down modulation of surface CD4.
• the expression of GSEs capable of interfering with productive infection should result in enrichment of protected cells displaying the CD4 + , p24 ⁇ phenotype . Such cells can be separated by FACS from infected population.
• This invention is also based upon the discovery that isolated GSEs cluster around narrowly defined regions of the HIV-1 genome, particularly, the surprising discovery that inhibitory or protective clusters of GSEs were also isolated from previously untargeted regions ⁇ RT and nef) .
• Preferred embodiments of the invention provide methods for designing GSEs based upon the consensus sequences of such GSE clusters.
• GSE may be transferred into T cells or hematopoietic progenitor cells in vi tro followed by their engraftment in an autologous, histocompatible or even histoincompatibile recipient.
• any cells susceptible to HIV infection may be directly transduced or transfected with GSE in vivo .
• TNF- induction TNF-induced cells, - ⁇ -; uninduced cells, - ⁇ -.
• TNF- ⁇ induction TNF-induced cells, - ⁇ -; uninduced cells, - ⁇ -.
• FIG. 3 Location of isolated HIV GSE on the HIV-1 BRU genome. Arrows indicate antisense orientation elements, while shaded boxes indicate sense orientation elements. Scale of the HIV-1 genome is in kilobases.
• Figure 5A&B Time course of viral infection of CEM-ss cells containing GSEs. Results are presented as the percentage of p24 positive cells at specified days post infection. All results are of a representative experiment.
• Figure 5A CEM-ss cells containing IGX-117 (vpr/tat sense, SEQ ID NO:9), IGX-201 (RRE antisense, SEQ ID N0:15) and controls were infected with a TCID 50 of 500 of HIV-1 SF2 .
• Figure 5B CEM-ss cells containing IGX-104 (RT sense, SEQ ID NO: 2) and controls were infected with a TCID 50 of 3000 of HIV-1 IIIB .
• the present invention relates to specific HIV-derived polynucleotides identified by an improvement of the method disclosed by Roninson et al . in United States Patent No. 5,217,889. More specifically, the improvement of the method includes the use of a cell line containing a latent and inducible HIV-1 provirus such as OM10.1. In addition, the improvement also encompasses the use of a marker associated with HIV infection such as CD4 to select for polynucleotides from an HIV-1 RFE library that effectively suppress HIV-1 infection. The GSEs selected by this procedure are also capable to protect uninfected cells from HIV infection.
• GSEs are isolated based upon their ability to prevent cells from productive infection by HIV-1.
• the method of the invention includes the steps of: 1) fragmenting the HIV-1 genome into 100-700 base pair (bp) fragments; 2) inserting the fragments into expression vectors such that the fragments are transcribed and translated to form an expression library; 3) transferring the expression library into a population of cells containing an inducible latent HIV-1 provirus or cells susceptible to HIV infection; 4) selecting a subpopulation of cells which contain a subset of the expression library enriched for HIV-1 GSEs by monitoring the expression of a cellular or viral marker associated with HIV infection; and 5) recovering the GSEs from the selected cell population.
• the method further includes repetition of the aforementioned steps with a secondary or tertiary library so that many rounds of successive selection can be performed.
• the selection of GSEs can be performed by monitoring the continued expression of a cellular marker such as CD4 or the decreased expression of a viral marker such as p24 or gpl20 using an antibody.
• An HIV RFE library can be constructed from the DNA of a plasmid or multiple plasmids that contain an HIV provirus insert. HIV proviral DNA is first treated with enzymes to produce randomly cleaved fragments. This can be conveniently performed by DNase I cleavage in the presence of Mn ++ (Roninson et al . , Patent No. 5,217,889, column 5, lines 5-20). Thereafter, the randomly cleaved genomic DNA are size fractionated by gel electrophoresis . Fragments of between 100 and 700 bp are the preferred lengths for constructing RFE libraries. Single strand breaks of the size-selected fragments are repaired, e.g., by Klenow or T4 polymerase, and ligated with 5 ' and 3 ' adaptors .
• the 5' and 3' adaptors are selected to have non-cohesive restriction sites so that each fragment can be inserted into an expression vector in an oriented fashion. Further, the 5' adaptor contains at least one start (ATG) codon to allow the translation of the fragments which contain an open reading frame in the correct phase .
• ATG start
• the fragments are inserted into appropriate expression vectors.
• Any expression vector that results in efficient expression of the fragments in host cells can be used.
• viral based vectors such as the retroviral vectors LXSN and a modified LSXN vector, LXSNgfr, are exemplified (Miller and Ros an, 1989, BioTechniques 7:980).
• adeno- associated virus vectors may also be used for this purpose.
• the ligated vectors are first transfected into a packaging cell line to produce viral particles.
• a packaging cell line for retroviral vectors, any amphotropic packaging line such as PA317 (Miller and Buttimore, 1986, Mol. Cell. Biol. 6:2895-2902; ATCC CRL #9078) or BING may be used to efficiently produce virus.
• the viral vector also contains a selectable gene, such as the neo r gene or a truncated nerve growth factor receptor (NGFR) gene, which allow isolation of the cells that contain the vector.
• NGFR nerve growth factor receptor
• the number of independent clones present in each GSE expression library may vary. In a preferred embodiment, libraries of about 5 x 10 4 to 10 6 independent clones may be used.
• OM10.1 cells are used to select for GSEs, and they are maintained in conventional tissue culture as described in Butera (U.S. Patent No. 5,256,534) .
• the purpose of using OM10.1 cells for the selection of HIV-1 GSEs is that they contain a latent HIV- 1 provirus which is inducible by TNF- ⁇ .
• Other cell lines may be similarly engineered with an inducible HIV provirus. Examples of cell lines that are infected with latent HIV include, but are not limited to, Ul , U33, 8E5, ACH-2, LL58, THP/HIV and UHC4 (Bednarik and Folks, 1992, AIDS 6:3-16).
• agents have been shown to be capable of inducing latent HIV-infected cells, and these include TNF- ⁇ , TNF- ⁇ , interleukins-1 , -2, -3, -4 and -6, granulocyte-macrophage colony stimulating factors, macrophage-colony stimulating factors, interferon- ⁇ , transforming growth factor- ⁇ , PMA, retinoic acid and vitamin D3 (Poli and Fauci, 1992, AIDS Res. Human Retroviruses 9:191-197) .
• the HIV-infected cells may be transduced with the HIV-1 RFE library by any technique well known in the art that is appropriate to the vector system employed.
• the viral vector also contains a selectable marker in addition to a random fragment of the HIV-1 genome.
• a suitable marker is the neo r gene, which permits selection by the drug G-418.
• the multiplicity of infection of the virions of the library is adjusted so that pre-selection for cells that are transduced by the vector is not needed.
• the transduced population is treated with 10 U/ml TNF- ⁇ for a period of 24-72 hours and preferably about 24 hours according to the method of Butera.
• the activation of the latent HIV-1 provirus in OMlO.l can be detected by the suppression of the cell surface CD4. It is believed that viral protein gpl20 binds to CD4 in the cytoplasm, which prevents subsequent expression of CD4 on the continue to express cell surface CD4.
• Such clones can be selected by cell sorting using any conventional antibody staining technique for CD4 and a fluorescence activated cell sorter (FACS) .
• FACS fluorescence activated cell sorter
• the OMlO.l cells harboring putative GSE and sorted after TNF- ⁇ induction are used to purify genomic DNA and the inserts amplified by the polymerase chain reaction (PCR) .
• the selected OMlO.l cells can be re-cultured under the selection conditions for the marker gene, e.g., in G-418, to ensure that the cells have retained the GSE derived from the HIV-1 RFE library.
• the fraction of CD4 + cells that have been transduced with an HIV-1 RFE library can be compared with cells transduced with an expression library consisting of the vector only.
• An increased relative difference between the HIV-1 RFE library and the control library can be found with each additional round of TNF- ⁇ induction.
• GSE sequences can be recovered from cells that continue to express CD4 following induction of the latent HIV provirus by TNF- ⁇ .
• the HIV-1-associated GSEs in this population are recovered by amplification in PCR using the primers according to the sequence of the linkers.
• the recovered GSEs can be introduced into an expression vector as discussed in Section 5.1, supra .
• 1 GSE expression library is known as a secondary library.
• the secondary library may utilize the same or a different vector from that used for the construction of the primary library.
• the secondary library may be transduced into another cell population and the resultant population selected, recultured and processed as described herein.
• each individually recovered element can be inserted into cloning vectors for determining its specific nucleotide sequence, its orientation and the portion of HIV genome from which it is derived.
• the isolated GSEs can be analyzed to determine their minimal core sequences and tested for their ability to protect previously uninfected cells from HIV infection.
• nucleotide sequences capable of hybridizing to these sequences or their complementary sequences under highly or moderately stringent hybridization conditions are well within the scope of the invention.
• Highly stringent hybridization conditions may be defined as hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS) , ImM EDTA at 65°C, followed by washing in 0.1 x SSC/0.1% SDS at 68°C (Ausubel F.M. et al . , eds , 1989, Current Protocols in Molecular Biology, Vol.
• the present invention also includes methods for determining the core sequence of each GSE. This may be done by comparing overlapping sequences of independently derived GSEs.
• Figure 3 depicts a number of clusters of independently derived GSEs.
• a surprising discovery of the invention is that isolated GSEs are clustered around narrowly defined regions of the HIV-1 genome.
• clusters of GSEs from previously untargeted regions i.e., reverse transcriptase and nef
• a number of GSEs selected on the basis of their ability to prevent productive infection are clustered in the region (RT) with coordinates 2426-2511 of the HIV-1 isolate BRU (GenBank accession number K02013) .
• GSEs also showed inhibitory effects upon in virally-infected cells (Figure 4) .
• sequences selected from this region are expected to be active in preventing productive infection and in inhibiting induction of latently infected cells.
• a number of GSEs are clustered around the region 8195-8599 of the HIV-1 genome in both sense and antisense orientation. It would be apparent to those of skill in the art that nucleotides containing sequences selected from this region are likely to be active in preventing productive infection and/or inhibiting virus induction in latently infected cells.
• the length of GSE sequences may affect their ability of inhibiting HIV-1 infection.
• the sequences selected from the regions of the viral genome depicted in Figure 3 are preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length.
• GSEs may be altered by additions, substitutions or deletions and assayed for retention of HIV- suppressive function.
• Alterations in the GSE sequences may be generated using a variety of chemical and enzymatic methods which are well known to those skilled in the art.
• oligonucleotide-directed mutagenesis may be employed to alter the GSE sequence in a defined way and/or to introduce restriction sites in specific regions within the sequence.
• deletion mutants may be generated using DNA nucleases such as Bal 31 or Exo III and SI nuclease. Progressively larger deletions in the GSE sequences may be generated by incubating the DNA with nucleases for increased periods of time (See Ausubel, et al . , 1989 Current Protocols for Molecular Biology, for a review of mutagenesis techniques) .
• altered sequences may be evaluated for their ability to suppress expression of HIV proteins such as p24 in appropriate host cells. It is well within the scope of the present invention that any altered GSE sequences that retain their ability to suppress HIV infection may be incorporated into recombinant expression vectors for further use.
• the GSEs may be transferred into HIV susceptible host cells, followed by HIV infection. Protection experiments can be performed in any cell type that takes up the potential HIV-1 GSEs and which is otherwise susceptible to HIV infection.
• the CEM-ss cell line is used (Foley et al., 1965, Cancer 18:522-29). The use of CEM-ss cells as targets for quantitative infectivity of HIV-1 has been described by Nara & Fischinger (1988, Nature 322:469-70).
• Other cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLa CD4 + and C8166.
• the test of the potential HIV-1 GSEs can be performed using the same expression vector system as that employed in the RFE library transduction of cells during initial selection steps.
• the vector system can be modified to achieve higher levels of expression, e.g., linkers can be employed to introduce a leader sequence that increases the translational efficiency of the message.
• linkers can be employed to introduce a leader sequence that increases the translational efficiency of the message.
• One such sequence is disclosed by Kozak, 1994, Biochemie 76:815-821.
• Another way of testing the effectiveness of a GSE against HIV is to determine how rapidly HIV-1 variants develop that can negate the effects of the potential HIV-1 GSE.
• Such a test includes infection of a culture of susceptible cells such as CEM-ss cells at a low multiplicity of infection and repeatedly assaying the culture to determine whether and how quickly HIV-1 infection becomes widespread.
• the range of useful multiplicities of infection is between about 100 to 1000 tissue culture infectious units (TCID 50 ) per 10 6 CEM-ss cells.
• the TCID 50 is determined by an endpoint method and is important for determining the input multiplicity of infection (moi) .
• a parameter that correlates with the development in the test culture of HIV-1 strains that are resistant to the effects of the potential HIV-1 GSE is the fraction of cells that are infected in the culture. This fraction can be determined by any means. Immunofluorescent staining with an antibody specific for the HIV-1 p24 antigen of fixed permeabilized cells is a convenient method for determining the fraction of cells that is infected. Commercially available reagents are suitable for performing such tests (Lee et al . , 1994, J. Virol. 68:8254-8264).
• GSEs were tested for their ability to protect CEM-ss cells from infection with HIV-1 strains SF 2 and IIIB. Uninfected cells were transduced with a LXSN construct containing either an irrelevant DNA or a GSE sequence. Non-transduced cells were eliminated by exposure to the selection agent, G-418. The percentage of p24 + cells was determined at specific time points post infection. The results demonstrate that GSEs tested are able to protect a productive HIV-1 infection in susceptible host cells.
• GSE operably linked to a regulatory sequence such as a promoter that controls its expression may be transferred in vi tro into any HIV- susceptible host cells such as CD4 + T cells or hematopoietic progenitor cells such as CD34 + cells obtained from bone marrow or mobilized peripheral blood, by any DNA transfer techniques well known in the art such as electroporation, transfection or transduction, followed by transplantation of the cells into a recipient.
• any HIV- susceptible host cells such as CD4 + T cells or hematopoietic progenitor cells such as CD34 + cells obtained from bone marrow or mobilized peripheral blood
• DNA transfer techniques well known in the art such as electroporation, transfection or transduction
• GSEs may be directly administered in vivo using a gene therapy expression vector.
• anti- HIV GSEs can be delivered or transferred into CD4 + T cells in both HIV-infected or uninfected individuals to protect against development of HIV infection.
• GSEs can also be transferred into stromal cells, including macrophages .
• Expression vectors derived from viruses such as retroviruses, adeno-associated virus, herpes viruses, or bovine papilloma virus may be used for delivery of a recombinant GSE into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing a GSE sequence operably linked to a promoter that controls its expression (Sambrook et al . , 1989, Molecular Cloning A
• GSE sequences were inserted into a retroviral vector.
• a GSE sequence may be ligated to an adenovirus transcription-translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vi tro or in vivo recombination.
• Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing GSE in infected hosts (Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).
• recombinant GSE nucleic acid molecules can be reconstituted into liposomes for delivery to target cells.
• Liposomes are spherical lipid bilayers with aqueous interiors. All molecules that are present in an aqueous solution at the time of liposome formation (in this case, oligonucleotides) are incorporated into this aqueous interior.
• the liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm, obviating the need to neutralize the polynucleotides' negative charge.
• initiation signals may also be required for efficient translation of inserted GSE sequences.
• Exogenous transcriptional control signals including the ATG initiation codon, must be provided.
• the initiation codon must be in phase with the reading frame of the GSE sequence to ensure translation of the entire insert.
• exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al . , 1987, Methods in Enzymol. 153: 516-544).
• the isolated GSE sequences suppress HIV activity by either encoding protein or RNA products.
• the present invention encompasses any such protein product, including fusion proteins, leader peptides and localization signals.
• anti-sense RNA, DNA molecules and ribozymes that function to inhibit HIV infection are also within the scope of the invention.
• Anti- sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
• GSEs may be represented by structural RNAs which act as decoys .
• GSEs may also form triplexes.
• Oligodeoxyribonucleotides can form sequence-specific triple helices by hydrogen bonding to specific complementary sequences in duplexed DNA. Formation of specific triple helices may selectively inhibit the replication and/or gene expression of targeted genes by prohibiting the specific binding of functional trans-acting factors.
• Polynucleotides to be used in triplex helix formation should be single stranded and composed of deoxynucleotides .
• the base composition of these polynucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
• Polynucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.
• the pyrimidine-rich polynucleotides provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
• polynucleotides may be chosen that are purine-rich, for example, containing a stretch of G residues.
• polynucleotides will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
• Switchback oligonucleotides are synthesized in an alternating 5' -3', 3'- 5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
• the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of HIV RNA sequences.
• GSE represented by antisense RNA showing high affinity binding to target sequences can also be used as ribozymes by addition of enzymatically active sequences known to those skilled in the art.
• RNA molecules may be generated by in vi tro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
• DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters .
• antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into host cells.
• nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5 ' and/or 3 ' ends of the molecule or the use of phosphorothioate or 2 ' O- methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone .
• Methods for introducing polynucleotides into cells or tissues include the insertion of naked polynucleotide, i.e., by injection into tissue, the introduction of a GSE in a cell ex vivo, i . e . , for use in autologous cell therapy, the use of a vector such as a virus, retrovirus, phage or plasmid, etc. or techniques such as electroporation which may be used in vivo ox ex vivo .
• the GSEs may be formulated and administered through a variety of means, including systemic, localized, or topical administration. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition. The mode of administration may be selected to maximize delivery to a desired target site in the body.
• route of injection includes, intramuscular, intravenous, intraperitoneal, and subcutaneous.
• the polynucleotides of interest are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks ' s solution, Ringer's solution, or physiological saline buffer.
• physiologically compatible buffers such as Hanks ' s solution, Ringer's solution, or physiological saline buffer.
• the polynucleotides may be formulated in solid or lyophilized form, then redissolved or suspended immediately prior to use.
• HIV-1 genomic DNA included pBENN7 (Cat. No. 342) derived from 5' half of HIV-1 BRU (AIDS Research and Reference Reagent Program, Rockville, MD) (ARRRP), 9B/6R derived from 3' half of HIV-1 SF2 and HIV-1 HXB2 genomic DNA.
• RFE library was constructed using HIV-1 BRU and HIV-1 ⁇ F2 -derived genomes as the DNA source.
• a second library was made from HIV-1 HXB2 genomic DNA. The plasmids were partially digested with DNAsel in the presence of manganese (Sambrook et al . , 1989, Molecular
• OMlO.l cells are available from the American Type Culture Collection, Rockville, MD as CRL 10850 (Butera, United States Patent No. 5,256,534). OMlO.l cells were a chronically infected promyelocytic clone of HL-60 which contained a single copy of the HIV-1 BRU isolate.
• the CEM-ss cells are available from the ARRRP as Cat. No. 776. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C and 5% C0 2 .
• the amphotropic packaging cell line BING was maintained in DMEM supplemented with 10% FBS at 37°C and 5% C0 2 .
• Persistently HIV-infected cells, HUT78/HIV-1 SF2 and H9/HIV-1 IIIB were obtained from ARRRP and used to prepare HIV- 1 SF2 and HIV-1 IIIB viral stocks.
• anti-CD4 antibodies Q4120PE (Sigma, St. Louis, MO) and L120 (Becton Dickinson Immunocytometry Systems, San Jose, CA)
• anti-p24 antibodies KC-57 FITC, Coulter, Hialeah, FL
• TNF- ⁇ was obtained from Boehringer Mannheim.
• G418 was purchased from Gibco/BRL as neomycin (Gaithersburg, MD) .
• the plasmid DNA prepared according to the method of Section 6.1.1. supra was transfected into the packaging cell line, BING, using a standard calcium phosphate method.
• the packaging cells were co- cultivated with OMlO.l cells or CEM-SS cells for 2-3 hours. Three cocultivations were used at 24, 48 and 72 hours after transfection. Cells were then grown under neomycin selection for two weeks. The surviving cells were purified using "FICOLL" and grown in neomycin-free media before any further manipulations. A different method was used for LXSNgfr based libraries and GSEs.
• Filtered retroviral supernatants from BING cells (24 and 48 hour virus) were used to infect the target cells by centrifugation at 1200xg for 90 minutes.
• cells were stained with a NGFR monoclonal antibody and the transduced cells represented by NGFR positive population were sorted using a FACS Vantage (Becton Dickinson) equipped with a 488 nm argon laser. Cells were cultured in nedia and checked for NGFR prior to use.
• CD4 + or NGFR T cells 10 7 cells were washed twice with Assay Buffer (500 ml PBS, 1 ml of 0.5 mM of EDTA at pH 8, 0.5 ml of 10% sodium azide and 10 ml of fetal bovine serum) , and resuspended in 500 ⁇ l PBS to which 50 ⁇ l of an anti-CD4 antibody, Q4120PE or L120, or PE-conjugated anti- NGFR antibody 20.4 (ATCC) was added. After incubation at 4°C for 30 min . , 5 ml of Assay Buffer was added and the cells centrifuged at 1200 rpm for 4 min. The cells were washed twice with Assay Buffer before sorting by FACS. The aforementioned procedure was performed under sterile conditions .
• Assay Buffer 500 ml PBS, 1 ml of 0.5 mM of EDTA at pH 8, 0.5 ml of 10% sodium azide and 10 ml of feta
• the cells were first washed twice with Assay Buffer. About 10 6 cells were suspended in 100 ⁇ l Assay Buffer, mixed with 2 ml of Ortho PermeaFix Solution (Ortho Diagnostics, Raritan, NJ) , and incubated for 40 min. at room temperature. After centrifugation at 1200 rpm for 4 min. at room temperature, the cells were resuspended in 2 ml Wash Buffer (500 ml PBS, 25 ml fetal bovine serum, 1.5% bovine serum albumin and 0.0055% EDTA) for 10 min. at room temperature.
• Assay Buffer 500 ml PBS, 25 ml fetal bovine serum, 1.5% bovine serum albumin and 0.0055% EDTA
• the cells were resuspended in 50 ⁇ l Wash Buffer and mixed with 1:500 dilution of an IgG 2a antibody for 20 min. at room temperature, followed by incubation with 5-10 ⁇ l of anti-p24 antibody (KC57-FITC, Coulter, Hialeah, FL) for 30 min. at room temperature. The cells were then washed twice with Wash Buffer and analyzed by flow cytometry.
• the transduced populations of the OMlO.l cells were washed once with PBS, then induced with 0.1 ng/ml of TNF- ⁇ (Boehringer Mannheim, Indianopolis, IN) in RPMI supplemented with 10% FBS at a density 5xl0 5 cells/ml. After 24 hours, cells were stained with a PE-conjugated anti-CD4 monoclonal antibody. Propidium iodide was added to a final concentration of 10 ⁇ g/ml immediately prior to sorting. The propidium iodide negative, CD4 positive population was sorted by flow cytometry. The cells were lysed and the genomic DNA was purified.
• Inserts were amplified by PCR using vector-derived primers, the mixture was digested with BamHI and EcoRI, column purified, ligated to BamHl/EcoRI digested vector, and transformed into E. coli . Purified DNA from transformants were either used as a pool for subsequent rounds of selection and/or individually isolated and sequenced using the ALF DNA Sequencer (Pharmacia LKB, Piscataway, NJ) .
• Transduced CEM-ss cells (GSE library or insert free vector) were infected with HIV-1 IIIB at a tissue culture infectious doses (TCID 50 ) of 3000 per 10 6 cells in the presence of polybrene .
• TID 50 tissue culture infectious doses
• 10 ' cells were stained for CD4 and p24.
• the p24 negative, CD4 positive population was sorted. Genomic DNA purification, insert amplification and subcloning were performed as described above.
• Genomic DNA was isolated from the selected population of OMlO.l cells or CEM cells harboring putative GSE by resuspending the cell pellet in 0.1% Triton X-100, 20 ⁇ g/ml proteinase K in lx PCR buffer, incubating at 55°C for 1 hour, and boiling for 10 minutes. Genomic DNA was used for PCR amplification using vector-derived primers, cloned into the LXSN vector, and transformed into E. coli using techniques well known in the art. Individual plasmids were purified from E. coli clones using QIAGEN plasmid kits. Inserts were sequenced by the dideoxy procedure (AutoRead Sequencing Kit, Pharmacia Biotech) and run on a Pharmacia LKB A.L.F. DNA sequencer. Sequences were analyzed using the DNASTAR program. 6.2. RESULTS
• the HIV-1 life cycle consists of two distinct stages: productive or acute infection and latency. An ideal HIV inhibitor should be effective against both stages. Accordingly, two selection approaches of HIV-1 GSEs were developed. The first approach, designed to inhibit virus induction in latently infected cells, was based on the unique properties of OMlO.l cells. For selection of GSEs in OMlO.l cells, an HIV-1 RFE library was constructed from plasmids containing the genome of the virus. Following transfection of the entire library into a packaging cell line, virus was transferred into OMlO.l cells by co-cultivation. The virally- transduced cells were selected in culture medium containing G- 418 to ensure the retention of the viral vector.
• the second selection scheme was designed to select GSEs capable of inhibiting productive HIV infection. Replication of HIV in susceptible cells is associated with accumulation of intracellular p24, concomitant with down-modulation of surface CD4.
• an HIV-1 RFE library was constructed from plasmids containing the genome of the virus. The entire library was transfected into CEM-ss cells. The cells were infected with HIV-1 IIIB and sorted by flow cytometry for CD4 positive, p24 negative cells.
• RFE libraries from various HIV-1 isolates were cloned into retroviral vectors.
• a library from isolates HIV-1 BRU and HIV-1 SF2 in LXSN vector was used in OMlO.l selection.
• a library from HIV-1 HXB2 in LXSN vector was used in productive infection selection.
• a third library from HIV-1 HXB2 in LXSNgfr vector was used in two independent experiments in OMlO.l cells.
• Each RFE library was transferred into the target cells (OMlO.l or CEM-ss) and two rounds of selection were performed. The reproducibility of the system was demonstrated by independent transfers and selections of the same RFE library.
• nef sense cluster the rev/tat sense cluster
• vpr/tat antisense cluster three clusters (the nef sense cluster, the rev/tat sense cluster, and the vpr/tat antisense cluster) were identified- in all libraries and selections.
• a cluster of sense-oriented elements from the reverse transcriptase (RT) gene was found only in the productive infection selection, presumably because reverse transcription preceded integration.
• SEQ ID NOS : 1-25 present nucleotide sequences of the selected polyncleotides which corresponded to GSEs in either sense or antisense orientation. More specifically, GSE IGX-149 in SEQ ID NO:l is in the gag
• IGX 104 SEQ ID NO:2
• IGX-109 SEQ ID NO:3
• IGX-102 SEQ ID NO : 4
• IGX-111 SEQ ID NO:5
• IGX- 013 SEQ ID NO:6
• IGX-018 SEQ ID NO:7
• IGX-117 SEQ ID NO:8
• IGX-217 SEQ ID NO:9
• IGX-050 SEQ ID NO:10
• IGX- 103 SEQ ID NO:12
• IGX-031 SEQ ID NO:13
• IGX-201 SEQ ID NO: 14
• IGX-171 SEQ ID NO: 16
• IGX-171 SEQ ID NO: 16
• IGX-222 SEQ ID NO:17
• IGX-003 SEQ ID NO:18
• IGX-219 SEQ ID NO:19
• IGX-210 SEQ ID NO:20
• IGX-221 SEQ ID NO:21
• IGX-151 SEQ ID NO:22
• IGX-078 SEQ ID NO:23
• IGX-121 SEQ ID NO:24
• IGX-140 SEQ ID NO:25
• GSEs with overlapping sequences from narrowly defined regions of the HIV-1 genome were isolated in different selection systems using different HIV-1 isolates indicates that these GSEs are from functionally important and conserved regions of the viral genome.
• clusters of GSEs from previously untargeted regions were also isolated.
• GSEs from the first OMlO.l selection were further tested for their ability to prevent HIV-1 induction by TNF- ⁇ .
• negative controls OMlO.l cells and OMlO.l cells with the LXSN vector
• Figure 4 Two additional negative controls containing anonymous DNA inserts from plasmid vectors (#34 and #220) were 8-11% CD4 positive.
• individual GSE showed a constant and reproducible inhibitory effect by allowing 18- TNF- ⁇ ( Figure 4) .
• OMlO.l cells containing the transdominant mutant, RevMlO (Malin et al . , 1989, Cell 58:205), were not effective at inhibiting induction of virus from latency.
• GSEs isolated by OMlO.l selection were tested for their ability to protect uninfected human T cells from a productive HIV-1 infection. Plasmids containing each of these sequences were transferred into CEM-ss cells followed by G418 selection. The Rev transdominant mutant, RevMlO and a LXSN vector containing an irrelevant piece of plasmid DNA (#34) were used as controls. The G418 resistant cells were 99% CD4 + , and were then infected with low titers (TCID 50 of 500-1000) of HIV-l st , or high titers (TCID 50 of 3000) of HIV-1 IIIB . The cells were removed at various days after infection, and stained with a fluorescinated-anti-p24 antibody as an indicator of HIV infection.
• GSEs have been isolated from the HIV-1 genome based on their ability to maintain CD4 expression in OMlO.l cells after activation of latent HIV by induction with TNF- ⁇ or based on their ability to inhibit productive HIV infection in CEM-ss cells.
• the isolated GSEs contain nucleotide sequences in both sense and anti-sense orientations, and are mapped to different regions of the HIV-1 genome.
• Several elements corresponding to portions of the in tegrase, Nef and Rev/Ta t genes are able to suppress HIV-1 infection of T cells by reducing p24 levels in infected cells.
• Such polynucleotides are useful in protecting the infection by and/or suppressing the replication of HIV-1 in human host cells .

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