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  • 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
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3517—Marker; Tag

Definitions

• the present invention is directed to processes for the inhibition of induction of viruses in latently or chronically infected cells into the actively replicating form.
• Retroviruses were so named because in their life cycle the normal flow of genetic information (DNA to mRNA to protein) is reversed.
• DNA DNA
• mRNA messenger RNA
• the genes of a retrovirus are encoded in RNA; the genes must be converted into DNA before they can be expressed. Only then are the viral genes transcribed into mRNA and trans- lated into proteins in the usual sequence.
• a retrovirus infects a cell by binding to the outside of the cell and injecting its core.
• the core contains the RNA encoding the viral genome as well as structural proteins (gag gene), viral enzymes and regulatory proteins (tat, rev, nef).
• One enzyme is responsible for converting the viral genetic information into DNA.
• This DNA polymerase (pol gene) first makes a single-stranded DNA copy of the viral RNA.
• An associated enzyme, ribonuclease destroys the original RNA, and the polymerase makes a second DNA strand, using the first one as a template. (The polymerase and ribonuclease together are often called "reverse transcriptase").
• the viral genome now in the form of double-stranded DNA (the same form in which the cell carries its own genes), migrates to the cell nucleus.
• a third viral enzyme called an integrase, may then splice the viral genome — its full complement of genetic information - into the host cell's DNA.
• the viral DNA (the "provirus") will be duplicated together with the cell's own genes every time the cell divides.
• the infection is a permanent part of the cell. During this latent or chronic infection phase, new virus particles are not generally produced.
• RNA long terminal repeat
• RNA strands serve as mRNAs that guide cellular machinery in producing the structural proteins and enzymes of the new virus.
• induction or rescue The conversion of the virus from the latent proviral stage to the active replicating stage is called induction or rescue.
• viruses besides retroviruses, such as Herpes viruses and the Hepatitis B virus have similar cycles with latent or chronic infection phases and active replicating phases.
• the provirus With many viruses, especially retroviruses, after initially infecting a cell the viral genome is integrated into the DNA in the cell's nucleus, in what is called the provirus form.
• the provirus may remain integrated in the cell's DNA during a long period of chronic or latent infection.
• the latent or chronic infection period ends when the provirus (virus) is induced and enters an active replicating phase.
• Mammalian viruses which exhibit such a life cycle often having long periods of latency (or chronic infection) as well as active replication phases include retroviruses such as the AIDS virus (human immunodeficiency virus or HIV), the various Herpes viruses and Hepatitis B.
• the AIDS virus, HIV is an example of a virus that would be advantageous to maintain in the provirus form.
• the virus After initial infection with HIV, the virus often replicates abundantly, and free virus may appear in the brain and spinal cord and in the bloodstream. Fevers, rashes, flu-like symptoms and sometimes neurological complaints can accompany this first wave of HIV replication. Then, within a few weeks, the amount of virus in the circulation and the cerebrospinal fluid drops precipitously and the initial symptoms disappear. However, the virus is still present (primarily in the provirus form). It can be found not only in the T4 lymphocytes, the subset of immune system cells originally thought to be its only target, but also in other classes of immune system cells, in cells of the nervous system and the intestine and probably in some bone marrow cells. From about two to ten years after the start of this asymptomatic period, replication of the virus flares again and the infection enters an active replicating phase, generally the final stage.
• HIV Underlying this variable course of infection are complex interactions between HIV and its host cells.
• the virus behaves differently depending on the kind of host cell and the cell's own level of activity.
• HIV can lie dormant indefinitely, inextricable from the cell but hidden from the victim's immune system.
• the cells When the cells are stimulated (induced), however, it can destroy them in a burst of replication.
• macrophages and their precursors, called monocytes the virus may be latent or grow continuously, but slowly, sparing the cell but probably altering its function.
• tat a regulatory gene responsible for the burst of viral replication seen, for example, in T4 cells that have been stimulated by an encounter with an antigen.
• the tat gene is unusual in both its structure and its effects. It is made up of two widely separated sequences of nucleotides; after it is transcribed into mRNA the intervening genetic material must be spliced out before the transcript can be made into protein.
• the tat gene has sites termed splice/- acceptor ("S/A”) and splice/donor ("S/D”) which allow the splicing out of non-transcribed material.
• S/A splice/- acceptor
• S/D splice/donor
• Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected foreign nucleic acid sequence can selectively inhibit the expression or function or expression of that particular nucleic acid without disturbing the function or expression of other nucleic acids present in the cell, by binding to or interfering with that nucleic acid.
• U.S. Patent No. 4,469,863 and 4,511,713 The use of complementary nuclease-resistant nonionic oligonucleoside methylphos- phonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including Herpes simplex virus-1 is disclosed in U.S. Patent No. 4,757,055.
• anti-sense oligonucleotides or phosphoro- thioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral DNA into protein has been proposed.
• the anti-sense constructs can bind to viral RNA and were thought to obstruct the cells ribosomes from moving along the RNA and thereby halting the translation of mRNA into protein, a process called "translation arrest” or "ribosomal-hybridization arrest.” (See, Yarchoan et al., "AIDS Therapies,” Scientific American, pp. 110-119 (October 1988)).
• oligonucleotide complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or expression is disclosed in U.S. Patent No. 4,806,463.
• the oligonucleotides were found to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins pl5 and p24 (gene expression).
• the present invention is directed to methods of inhibiting or controlling of the induction of a viral infection from a latent or chronic infection to an active replicating infection.
• the present invention is also directed to novel methylphosphonate nucleoside oligomers which are useful for inhibiting the induction of such viruses.
• the present invention is directed to a method of treating virally infected cells to inhibit induction of a latent or chronic viral infection into an active replicating infection which comprises treating said cells or their growth environment with an Oligomer which is complimentary to an Enhancer Site as defined herein- below.
• Suitable nucleoside oligomers include oligonucleotides, nonionic oligonucleoside alkyl- and aryl- phosphonate analogs, phosphorothioate analogs, neutral phosphate ester analogs of oligonucleotides, phosphoro- amidate analogs or other oligonucleotide analogs and modified oligonucleotides.
• the present invention is also directed to the treatment of isolated cells, or individuals or animals whose cells or fluids have such latent or chronic viral infections or contain viruses capable of entering a latent or chronic infection state.
• the method of the present invention is especially suited to treating infections which typically have periods of latent or chronic infection followed by especially virulent active replicating phases.
• viruses include retroviruses such as HIV, HTLV-I, HTLV-II and the like; the Herpes viruses; Hepatitis B virus; and the like.
• Oligomers having various internucleo- side phosphorous linkages may be used according to the present invention, due to their increased resistance to enzymatic metabolism, it is particularly preferred to use oligonucleoside alkyl-and aryl-phosphonate analogs in to the methods of the present invention.
• the present invention also provides certain novel oligonucleoside methyl phosphonate analogs ("MP- Oligomers”) which are particularly active in inhibiting induction of virus replication in latent and/or chronic virus infections.
• MP- Oligomers novel oligonucleoside methyl phosphonate analogs
• the present invention also provides Oligomers capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA.
• the present invention also provides hybridization probes for a virus which maintains or is capable of maintaining a chronic or latent infection which comprises an Oligomer of at least 8 nucleosides wherein said Oligomer is substantially complementary to an Enhancer Site of said virus.
• diagnostic methods including methods for detecting the presence of in a test sample of a virus which maintains or is capable of maintaining a chronic or latent infection.
• viral infection refers to a viral infection wherein the provirus is integrated into the genetic nuclear structure (DNA) of the host cell and wherein there is no unintegrated viral DNA, no viral RNA and no viral proteins.
• chronic infection refers to a viral infection having the provirus or virus material in the nucleus or cytoplasm of the host cell and which, until induced, has little or no detectable viral RNA or protein.
• End Region refers to all sequences of U3 of a retrovirus on the viral DNA or to the corresponding sequences of the viral RNA which affect viral replication and to similar sites in Herpes or Hepatitis B viruses which have equivalent activity.
• Oligonucleotides refers to oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothionate analogs of oligonucleotides, phosphoamidate analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, and other oligonucleotide analogs and modified oligonucleotides.
• methylphosphonate Oligomer refers to nucleotide oligomers (or oligonucleotide analogs) having internucleoside phosphorus group linkages wherein at least one methylphosphonate internucleoside linkage replaces a phosphodiester internucleoside linkage.
• nucleoside includes a nucleosidyl unit and is used interchangeably therewith.
• p in, e.g., as in ApA represents a phosphodiester linkage
• p in, e.g., as in CpG represents a methylphosphonate linkage
• FIG. 1 depicts a generalized genetic map of a retro- virus whose induction may be inhibited according to the present invention.
• FIG. 2 depicts a genetic map of HIV.
• FIG. 3 shows the position in the HIV genome of the sequences complimentary to some of the oligomers tested.
• the present invention is directed to methods of inhibiting the induction of a viral infection using Oligomers which are complimentary to and which bind to an Enhancer Site on the viral DNA or a corresponding sequence of the viral RNA.
• anti-sense Oligomers are constructed to be complimentary to a specific region of the viral nucleic acid. Accordingly, such anti-sense oligomers when complimentary to the first splice acceptor region (S/A-1) of the tat gene may be termed “anti-tat S/A-l” or anti- S/A-1, when complimentary to the secord splice acceptor region (S/A-2)of the tat gene may be termed anti-tat-s/A- 2 or anti-S/A-2, when complimentary to the target region of tat (TAR) may be termed "anti-TAR" or when complimentary to the initiator region of env may be termed "anti- env", and so forth.
• Oligomers having at least 8 nucleosides which is usually a sufficient number to allow for specific binding to a desired nucleic acid sequence. More preferred are Oligomers having from about 8 to about 40 nucleotides; especially preferred are Oligomers having from about 10 to about 25 nucleosides. Due to a combination of ease of synthesis, with specificity for a selected sequence, coupled with minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, it is believed that Oligomers having from about 12 to about 15 nucleosides comprise a particularly preferred group. Preferred Oligomers
• Oligomers may comprise either oligoribonucleo- sides or oligodeoxyribonucleosides; however, oligodeoxy- ribonucleosides are preferred.
• nucleotide oligomers i.e., having the phosphodiester internucleoside linkages present in natural nucleotide oligomers, as well as other oligonucleotide analogs
• preferred Oligomers comprise oligonucleoside alkyl and aryl-phosphonate analogs, phosphorothioate oligonucleoside analogs, phosphoroamidate analogs and neutral phosphate ester oligonucleotide analogs.
• oligonucleoside alkyl- and aryl-analogs which contain phosphonate linkages replacing the phosphodiester linkages which connect two nucleosides.
• MP-Oligomers Preferred synthetic methods for methylphosphate Oligomers are described in Lee, B.L. et al, Biochemistry 27:3197-3203 (1988) and Miller, P.S., et al., Biochemistry 2555092-5097 (1986), the disclosures of which are incorporated herein by reference.
• oligonucleoside alkyl- and aryl- phosphonate analogs wherein at least one of the phospho- diester internucleoside linkages is replaced by a 3' - 5' linked internucleoside methylphosphonyl (MP) group (or "methylphosphonate”).
• MP internucleoside methylphosphonyl
• the methylphosphonate linkage is isosteric with respect to the phosphate groups of oligonucleotides.
• MP-oligomers should present minimal steric restrictions to interaction with complimentary polynucleotides or single-stranded regions of nucleic acid molecules.
• MP-oligomers should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules. It has been found that certain MP-oligomers are more resistant to nuclease hydrolysis, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent No, 4,469,863).
• labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o-phenanthroline-copper or EDTA- iron may be incorporated in the MP-Oligomers.
• MP-oligomers having at least about 8 nucleosides which is usually sufficient to allow for specific binding to the desired nucleic acid sequence. More preferred are MP-oligomers having from about 8 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides. Due to a combination of ease of preparation, with specificity for a selected sequence and minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, particularly preferred are MP-oligomers of from about 12 to 15 nucleosides.
• MP-oligomers where the 5'- internucleoside linkage is a phosphodiester linkage and the remainder of the internucleoside linkages are methyl- phosphonyl linkages. Having a phosphodiester linkage on the 5' - end of the MP-oligomer permits kinase labelling and electrophoresis of the oligomer and also improves its solubility.
• MP-oligomers complimentary to the sense strands of those regions were prepared by the methods disclosed in the above noted patents.
• MP-oligomers complimentary to an Enhancer Site of U3 of the viral DNA or the corresponding sequence in viral RNA are especially preferred.
• One preferred group of MP-oligomers may include a sequence complimentary to all or a portion of a tract immediately 5' to U3 having the sequence:
• MP-oligomers which are complimentary to the portion of an Enhancer Site of U3 region which binds the NF-/cB protein in the viral DNA or the corresponding sequence in the viral RNA to be especially effective in inhibiting the induction of HIV in chronically infected cells, even when the cells are treated with a strong inducer such as the protein kinase C activator phorbol myristate acetate (PMA).
• PMA protein kinase C activator phorbol myristate acetate
• MP-oligomers were effective in inhibiting viral induction without significantly inhibiting cell proliferation or other normal cellular functions. This is in contrast to MP-oligomers complimentary to some other regions of the HIV genome wherein activity in decreasing infection by HIV appeared to correspond to activity in inhibiting cell proliferation and other normal cell functions.
• the present invention is directed to novel MP-oligomers which are complimentary to an Enhancer Site in HIV and which are capable of inhibiting induction of chronically infected cells without significantly inhibiting cell proliferation.
• examples of such preferred MP-oligomers of the present invention include:
• the present invention is directed to a method of inhibiting the induction of HIV virus in cells which have a chronic or latent HIV infection.
• Oligomers complimentary to viral regulatory genes were directed to two possible targets: (a) the initiation codon AUG of a particular gene and the sequence that followed, or (b) the sequences around the site known as the splice/acceptor site, abbreviated S/A, which would give the added advantage of possibly being able to block expression of two different messages simultaneously. It has been shown that sequences complimentary to the particular sites can bind and inhibit expression of the virus. However, it has been found that oligomers which bind to those sites and inhibit the virus inhibit normal cellular functions, including cell division.
• RNA The genetic material of HIV and other retroviruses is RNA. Once the virus infects a cell, it directs synthesis of a double-stranded DNA version of its genome which is incorporated into the cell's genome as the provirus.
• the U3 region is the farthest down- stream part of the viral RNA, since oligomers complimentary to the DNA Enhancer Site represented once in RNA would directly bind to the viral message and block reverse transcription, and if the viral DNA synthesis had begun, the oligomers would bind to the DNA copy (when one of these viruses infects the cell, DNA synthesis commences within the first two to three hours after infection).
• the U3 region is duplicated and copies form on either end of the viral gene sequence.
• the region including U3 is called the long terminal repeat or LTR, and includes an important enhancer site for the virus.
• these Oligomers are complimentary to and capable of binding to the binding region for enhancer proteins rather than just bind to the RNA to block transcription.
• these oligomers complimentary to the enhancing region are capable of inhibiting viral replication by two means. First they can block transcription by targeting the sequence that is represented in the RNA, and, second they can inhibit induction and, thus, replication by binding to the viral DNA in the enhancer site of U3 in order to block the binding of a regulatory protein to that region and thus inhibiting induction.
• the present invention is directed to the use of these Oligomers in determining the presence or absence of a virus which maintains or is capable of maintaining a chronic or latent infection state or the presence of such a chronic or latent viral infection in samples including isolated cells, tissue samples or bodily fluids.
• these Oligomers may be used in determining the presence or absence of HIV or infection by HIV. (See, e.g. US Patent No. 4,806,463).
• the present invention is directed to hybridization assay probes comprising these Oligomers and to detection assays using these Oligomers. These probes may also be used in diagnostic kits.
• Oligomers may be labelled by any of several well known methods.
• Useful labels include radioisotopes as well as non-radioactive reporting groups.
• Isotopic labels include 3 H, 35 S, 32 P, 125 I, Cobalt and 14 C.
• Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription.
• hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling.
• Non-isotopic materials can also be used for labelling, and may be introduced by the incorporation of modi fied nucleosides or nucleoside analogs through the use of enzymes or by chemical modification of the Oligomer, for example, by the use of non-nucleotide linker groups.
• Non- isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.
• One preferred labelling method is acridinium esters.
• Such labelled Oligonucleotides are particularly suited as hybridization assay probes and for use in hybridization assays.
• Oligonucleotide methylphosphonate analogs complimentary to the initiator codon regions of the gag-pol polypeptide and of tat were tested for their effect as specific inhibitors of HIV replication.
• An Oligomer specific to B-globulin mRNA and a d-AT polymer were used as negative controls.
• Varying concentrations of the Methylphosphonate (“MP”) oligomers were incubated with phytohemagglutin (“PHA”) stimulated human peripheral blood mononuclear cells (PBMC) for two hours of 37oC and then exposed to 100 ID-50 of Stock HIV from HTLV-IIIB-infected H9 cells. Eighteen hours later, fresh medium and additional oligomer were added.
• MP Methylphosphonate
• PHA phytohemagglutin
• PBMC peripheral blood mononuclear cells
• RT reverse transcriptase
• oligomers specific to the initiator codon region of gag-pol and tat were ineffective at inhibiting HIV replication, although they were non-cytotoxic to activated PBMC at concentrations up to 200 ⁇ M.
• Methylphosphonate oligomers complimentary to the tat initiation and splice/acceptor regions were tested for their ability to inhibit HIV-associated transactivation as measured by the ability of tat to enhance expression of the chloramphenicol acetyl transfer gene (CAT) upon trans- fection of a human T4+ lymphoblastoid T cell line with either a CAT plasmid alone or together with a tat plasmid (See: Laurence, J., et al., J. Clin. Invest. 80:1631-1639 (December 1987)).
• CAT chloramphenicol acetyl transfer gene
• the CAT plasmid pC15CAT and the control tat plasmid pCV-1, which lacked the splice/acceptor sites, were provided by Dr. Wong-Staal of the NCI.
• the experimental tat plasmid containing the two tat-associated exons, pBR322/pIIIextat lll was provided by Dr. Haseltine of Harvard Medical School.
• CAT activity was assessed by TLC analysis of acetylated forms of [ 14 C]-chloramphenical by standard techniques. (See, Laurence, J., et al., J. Clin. Invest. 80:1631-1639 (1987)).
• Anti-tat MP-oligomers were tested for their ability to block HIV infection of target cells (in order to see if that activity paralleled activity against trans- activation).
• Oligomer identification MP oligomer Sequence anti-tat S/A-1 dCpApCpCpApApTpTpCpTpG anti-tat S/A-1A dApApApTpGpGpApTpApApA anti-tat S/A-2 dTpGpGpGpApGpTpT
• MP-oligomers were assayed for their effect on phorbol-ester (13-phorbol-12-myristate acetate or "PMA") mediated induction of HIV.
• NF- ⁇ B a factor that regulates transcription and binds to the HIV enhancer, also mediates, phorbol-ester and mitogen or antigen activation.
• MP-oligomers complimentary to the tat-linked target element TAR and to the enhancing region in U3 which binds NF-kB were tested for their ability to inhibit PMA- induction of HIV replication-related effects.
• a stock solution of 100 ⁇ g/ml PMA (Sigma) was prepared in absolute ethanol diluted in RPMI-1640 and used in final concentrations of 5 to 500 ng/ml.
• U1.l cells were obtained from T.M. Folks of the NIH; they were subcloned from U1, a clone of the human monocytic cell line U937 which had been infected with the lymphadenopathy- associated virus (LAV) strain of HIV.
• LAV lymphadenopathy- associated virus
• H9 a human CD3+, CD4+ lymphoblastoid cell line permissive for the replication of HIV annd partially resistant to its cytotoxic effects was obtained from R.C. Gallo of the NIH.
• Stock Samples of these cells were cultured in RPMI-1640 (Flow Laboratories, McLean, VA); plus 10% fetal bovine serum (FBS), at a concentration of 5 x 10 5 cells/ml.
• FBS fetal bovine serum
• Varying concentrations of Oligomer (2 to 100 ⁇ M) were incubated with U1.1 cells (a chronically infected macro- phage cell line) at 37o C for one hour prior to exposure of the cells to PMA (5 ng/ml). Cultures were evaluated for HIV activity after 48 hours.
• HIV antigens were quantitated in supernatants by an ELISA-based assay for viral p24 core protein.
• Human immunoglobulin directed against p24 epitopes (Abbott Labs, Chicago, IL) contained in polystryene beads was added to supernatants and maintained overnight at room temperature. Plates were washed with citrate phosphate buffer; and rabbit anti-HIV IgG, followed by addition of horseradish peroxidase-labeled goat anti-rabbit antibody (Abbott). Color was developed with O-phenylenediamine as a substrate, followed by IN H 2 SO 4 to stop the reaction. Absorb- ance was read at 492 nm; data were expressed as pg/10 4 cells. The sensitivity of this assay was ⁇ 66 pg/ml.
• DNA synthetic response was assayed according to the following procedure. Cells were collected, washed three times with PBS, and viability assessed by trypan blue dye exclusion. 1 x 10 4 viable cells were resuspended in 0.2 ml of medicine in polystryene flat-bottom microwell plates. Selected cultures were treated with inducing agent. All groups were assayed in triplicate. Cells were incubated for 48 hours; eighteen hours before culture termination, they were pulsed with 0.1 mCi of [ 3 H- methyl]-thymidine (1.9 Ci/ ⁇ M sp. act.. New England Nuclear). The contents of each well were harvested and incorporation of radioactivity was measued by liquid scintillation counting.
• the cells were also assayed for PMA-induced enhancement of HIV-LTR driven CAT activity.
• the ability of the tat transcription unit of HIV to enhance the expression of the chloramphenical acetyl transferase (CAT) gene when CAT is linked to the LTR of HIV was measured as described in Laurence, J. et al., J. Clin. Invest. 80:1631-1639 (1987) with the following modifications. 2 X 10 6 U1.1 cells per condition were washed with serum-free RPMI-1640 and resuspended in 1ml of 5mM Tris (pH7.3) containing 250 ⁇ g/ml DEAE-dextran (Sigma) and 2 or 4 ⁇ g of total plasmid DNA.
• Plasmids Two plasmids (See Arya, S.K., et al., Science 229:69 (1985); plasmids were obtained from the sources noted in Example 2) were used either singly (HIV-LTR-CAT alone) or together (co-transfeetion of CAT and tat containing vectors).
• the tat plasmid pCV-1 contains a 1.8 kb fragment of HIV-1 cDNA encompassing the tat gene.
• the CAT plasmid pC15CAT contains SV-40 regulatory sequences, and the LTR and a portion of nef (3'-orf) of HIV-1.
• CAT activity was determined by incubating 50 ⁇ l aliquots of cell extracts with [ 14 C]-chloramphenicol (New England Nuclear) and 2.5M acetyl coenzyme A (P- L Biochemicals, Inc., Piscataway, NJ) at 37oC for 2 hours and extracting with ethyl acetate.
• the acetylated forms of chloramphenicol were separated from the unacetylated form by ascending thin layer chromatography using a chromatogram sheet (Eastman Kodak, Rochester, NY) in a chamber containing chloroform and methanol (19:1, v/v). The chromatogram was then autoradiographed. Areas of radioactivity were marked, cut from the sheet, and counted in scintillation fluid.

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Citations (4)

• 1990
  • 1990-04-18 ZA ZA902912A patent/ZA902912B/xx unknown
  • 1990-04-19 AU AU55511/90A patent/AU5551190A/en not_active Abandoned
  • 1990-04-19 EP EP19900907966 patent/EP0469080A4/en not_active Withdrawn
  • 1990-04-19 JP JP2506795A patent/JPH04505615A/ja active Pending
  • 1990-04-19 WO PCT/US1990/002011 patent/WO1990012578A1/en not_active Application Discontinuation
  • 1990-04-19 KR KR1019910701402A patent/KR920700654A/ko not_active Application Discontinuation
  • 1990-04-20 CA CA002014990A patent/CA2014990A1/en not_active Abandoned
• 1994
  • 1994-06-09 AU AU64651/94A patent/AU6465194A/en not_active Abandoned

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