WO1999033992A1 - Murine intracisternal a particle constitutive transport elements and uses thereof - Google Patents

Murine intracisternal a particle constitutive transport elements and uses thereof Download PDF

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Publication number
WO1999033992A1
WO1999033992A1 PCT/US1998/027792 US9827792W WO9933992A1 WO 1999033992 A1 WO1999033992 A1 WO 1999033992A1 US 9827792 W US9827792 W US 9827792W WO 9933992 A1 WO9933992 A1 WO 9933992A1
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cte
loop
hiv
nucleic acid
ncte
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PCT/US1998/027792
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French (fr)
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Barbara K. Felber
Carlos Tabernero
Andrei S. Zolotukhin
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Priority to AU20981/99A priority Critical patent/AU2098199A/en
Publication of WO1999033992A1 publication Critical patent/WO1999033992A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention pertains to the field of virology and vaccine development.
  • this invention pertains to the discovery that a novel post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) from a novel murine intracisternal-A particle (IAP) can be used to construct an attenuated retrovirus.
  • NCTE post-transcriptional RNA nucleo-cytoplasmic transport element
  • IAP novel murine intracisternal-A particle
  • IAPs Intracistemal A-type particles
  • IAP retroviral genomes share extensive sequence homologies with D-type retroviruses. IAP genes in variable copy number, up to 2000 in a genome, exist in all rodent species studied and are dispersed throughout the genome. Because of this high copy number, IAP elements have been used as genetic markers. Kaushik (1994) Mamm. Genome 5:688-695.
  • Murine IAPs are constitutively expressed in high levels in many mouse tumors and at basal levels in most normal adult mouse tissues. Kuff (1990) Cancer Cells 2:398-400. While they are not infectious retroviruses, IAPs can be transcriptionally active. Expression of some murine IAPs results in translation products corresponding to retroviral gag and Pol polyproteins. Fehrmann (1997) Virology 235:352-359. Under certain circumstances, IAPs can transpose to different positions in the genome, in some cases acting as insertional mutagens. When these transpositions affect the expression of a gene that can either positively or negatively regulate cell growth, a malignant or transformed cell or tumor may arise. IAP transpositions have been frequently observed in hematopoietic cells, often affecting cytokine gene expression. Wang (1997) Cytokines Cell. Mol. Ther. 3:3-19.
  • CD4 + T-cell deficiency who was negative for known human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV) infection.
  • HIV human immunodeficiency virus
  • HTLV human T-lymphotropic virus
  • the human IAP particle was distinct from HIV and HTLV.
  • Supernatants of activated mononuclear cells showed significant reverse transcriptase activity. While the patient's serum was negative for antibodies to known HIVs and HTLVs, his serum was positive for antibodies against the human IAP. Similar IAP particles were detected in a lymphoblastoid cell line exposed to mononuclear cells from the patient's daughter, who also showed CD4 + T-cell dysfunction.
  • retroviruses including HIV type 1 (HIV-1), simian retrovirus type 1 (SRV-1), SRV-2, and Mason-Pfizer monkey virus (MPMV) (the later three are type D simian retroviruses), regulate their expression by controlling the nuclear transport of unspliced mRNA encoding structural proteins. They do this using cts-acting post- transcriptional RNA nucleo-cytoplasmic transport elements ("NCTEs," designated “CTEs” for some type D retroviruses). NCTEs fold into highly structured secondary structures, typically stem-loop structures. Trans-ac ⁇ ng proteins bind to the NCTE loop structures to regulate RNA expression at several levels.
  • HIV-1 HIV type 1
  • SRV-1 simian retrovirus type 1
  • MPMV Mason-Pfizer monkey virus
  • NCTEs cts-acting post- transcriptional RNA nucleo-cytoplasmic transport elements
  • CTEs cts-acting post- transcriptional RNA nucleo-cytoplasmic transport elements
  • Retroviruses such as Simian type D retroviruses, including SRV-1, do not encode their own tr ⁇ s-acting, NCTE-binding proteins and instead utilize cellular NCTE binding proteins.
  • retroviruses such as HIV-1, utilize a retrovirally-encoded NCTE RNA binding protein, called "Rev.” HIV-1 regulates the expression of its structural proteins encoded by the g g/pol- and ewv-encoding transcript using this NCTE system.
  • HIV-l's NCTE binding protein "Rev” interacts with a specific NCTE sequence, designated the “Rev-responsive element,” or “RRE,” contained in its gag/pol and env encoding transcript. HIV-l's RRE does not bind cellular NCTE-binding proteins. Rev interacts directly with RRE as part of the RNA export machinery which transports REE-containing transcripts to the cytoplasm from the nucleus. As a result, Rev and RRE are needed to produce infectious virus.
  • Simian retroviruses contain an NCTE, or "CTE,” element located between their env gene and 3' long terminal repeat (LTR). HIV-1 lacking a functional Rev RRE control system is uninfectious, but can be reconstituted with simian retroviral CTE ⁇ e.g., CTE from SRV-1, SRV-2, MPMV) to produce transcripts and infectious virions (Bray (1994) Proc. Natl. Acad. Sci. USA 91 :1256-1260; Tabernero (1996) J. Virol. 70:5998-
  • cis- and trans-acting post-transcriptional nucleo-cytoplasmic transport regulatory system in retroviruses produces an attenuated virus, decreasing their replicative ability and pathogenicity in vivo. Such attenuated infection, results in an asymptomatic state in the infected individual.
  • cis- and transacting post- transcriptional RNA regulatory elements capable of attenuating a retrovirus, such as HIV- 1.
  • the present invention fulfills this and other needs.
  • the isolated CTE 1AP nucleic acid is defined as having the following properties: (i) the CTE IAP , when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a NCTE in place of wild-type NCTE in the hybrid HIV-1, and when the CTE IAP -containing hybrid HIV-1 virus infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24 gag is between about 50 fold and about
  • the secondary structure of the CTE IAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid comprising the loop structure as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
  • the invention provides for an isolated nucleic acid CTE 1AP comprising a ribonucleic acid comprising a motif of contiguous base pairs consisting of X A 10.500 - GUC AAUGAC GGGU AAGA - X B 16 . 24 - ACCU AAGACA GG - X c 6 . 100 - CAA U GUU - X D 6 . 100 - CC GAGGAC AGGU - X E 16 .
  • the isolated nucleic acid CTE IAP comprises the sequence as set forth in SEQ ID NO:l; and, in the isolated nucleic acid CTE IAP the distance between the loop A domain and the loop B domain is between about 21 to about 26 base pairs in length.
  • the invention also provides for an isolated transcription product of a CTE IAP nucleic acid, the CTE IAP nucleic acid as defined above.
  • the invention also provides for an expression vector comprising a nucleic acid encoding a CTE IAP and a non-naturally occurring nucleic acid sequence, the nucleic acid as defined above, and, a transfected cell comprising a polynucleotide encoding a CTELJJ. and a non-naturally occurring nucleic acid sequence, the CTE IAP nucleic acid as defined above.
  • the invention provides for a recombinant retrovirus either lacking or having a non-functional endogenous NCTE and further comprising a CTE 1AP operatively inserted into the retrovirus and capable of acting as a functional exogenous NCTE to reconstitute the lacking or non-functional endogeous NCTE and to reconstitute the infectivity of the retrovirus in a mammalian cell, the CTE jAP as defined above.
  • the recombinant virus can be HIV-1
  • the NCTE can be RRE
  • the HIV-1 can further lack a functional Nef.
  • the invention further provides for a vaccine for the prophylaxis or amelioration of a viral infection in a mammal comprising an attenuated retrovirus, where the retrovirus, when administered as a vaccine in sufficient amounts, is capable of eliciting an immune response to the retrovirus in a mammal with a functional immune system; and the attenuated retrovirus lacks an endogenous functional NCTE and/or the ability to express an endogenous functional NCTE binding protein, and the attenuated retrovirus further comprises a CTE IAP nucleic acid as defined above.
  • the attenuated retrovirus can HIV- 1
  • the attenuated HIV- 1 can also lack a functional Nef
  • NCTE can be RRE, and the NCTE binding protein can be Rev.
  • kits for the prophylaxis or amelioration of a virus infection in a mammal comprising the above described vaccine and a pharmacologically acceptable carrier.
  • the kit can further comprise an instructional material teaching the use of the vaccine, where the instructional material indicates that the vaccine is used for the prophylaxis or amelioration of HIV- 1 infection in a mammal; that the vaccine is to be administered to a mammal in a therapeutically effective amount sufficient to express a viral protein; wherein the vaccine will not cause clinically significant CD4 + cell depletion; and, the expression of the viral protein elicits an immune response to the attenuated HIV-1 virus.
  • the invention also provides for use of a CTE ⁇ in the manufacture of a medicament for the prophylaxis or amelioration of a viral infection, such as an HIV-1 infection, where the CTE IAP is as described above.
  • the invention provides for a method for eliciting an immune response to a virus in a mammal, comprising administering to a mammal a therapeutically effective amount of an attenuated recombinant virus, wherein the virus comprises the CTE IAP as defined above.
  • the invention provides for a method for screening for a NCTE binding protein comprising the following steps: a) providing a composition comprising a loop A and/or a loop B of a CTE IA as defined above; b) contacting the composition with a test compound; and, c) measuring the ability of the test compound to bind the NCTE.
  • Figure 1 illustrates two RNA sequences: the novel CTEL AJ . of the invention (SEQ ID NO:l, Figure 1A) and SRV-1 CTE (SEQ ID NO:2, Figure IB).
  • SEQ ID NO:l novel CTEL AJ . of the invention
  • Figure IB SRV-1 CTE
  • Figures 1 A and IB the two sequences are shown folded in a secondary structure whose configuration is based on intramolecular base-pairing.
  • This secondary structure includes two loops, A and B, a four nucleotide AAGA "bulge" adjacent to loop A, and a hairpin turn. Loops A
  • Figure 2 illustrates how the novel IAP, including the CTEL ⁇ of the invention, is inserted into the transcribed osteocalcin related gene (ORG) between its promoter (designated "P") and the ORG coding region, thus displacing ORG's functional promoter by 3.4 kilobases.
  • ORG transcribed osteocalcin related gene
  • the Figure shows that the murine osteocalcin gene consists of osteocalcin 1 ("mOGl"), osteocalcin 2 ("mOG2”), and ORG. Exons are designated as Roman numerals; exons lb and Ic are non-coding exons.
  • the sequence of the ORG promoter is also shown, with the identified start of transcription 47 nucleotides downstream of the ORG TATA box in the 5' LTR (long terminal repeat) of the ORG ⁇ .
  • CTE ⁇ of the invention within the ORG gene, shown as "CTE," and splice sites are indicated (see Figure 4, where the DNA subsequence encoding CTE 1AP is between nucleotides 3029-3204 of SEQ ID NO:3).
  • Figure 3 illustrates data demonstrating that the novel CTE 1AP is able to replace Rev-mediated activation of HIV- 1 in both Jurkat and PBMCs, as shown in Figure 3A and 3B, respectively; and explained in detail in below.
  • Figure 4 illustrates the nucleic sequence of the novel intracistemal A particle of the invention, ORG ⁇ , which is 3,402 base pairs in length, and flanking imperfect direct repeats (SEQ ID NO:3).
  • the DNA subsequence encoding the CTE ⁇ of the invention is located between nucleotides 3029-3204, as numerated in this figure.
  • the present invention provides for a novel post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE), designated “CrE ⁇ ,” derived from a novel intracistemal A particle (IAP).
  • NCTE post-transcriptional RNA nucleo-cytoplasmic transport element
  • IAP intracistemal A particle
  • the novel CTE ⁇ , of the invention can be used functionally replace the NCTE of HIV- 1, or "RRE.” It was discovered that when CTE ⁇ is used in place of RRE to construct an HIV-1 hybrid clone, a slower growing, attenuated virus results. Significantly, while capable of producing infectious virions in vivo, this HIV-1 hybrid (a CTE j ⁇ -containing RRE-negative recombinant virus) has lower replicative activity than wild-type vims, resulting in an attenuated HIV-1 strain. The level of attenuation can be quantitated using in vitro or in vivo assays, such as determining the amount of HIV- 1 p24 ⁇ g synthesized by hybrid viruses.
  • the CTE IAP of the invention when inserted in an RRE/Rev deficient HIV-1 , is capable of functionally reconstituting this post-transcriptional RNA nucleo-cytoplasmic transport system.
  • the CTE ⁇ -containing hybrid HIV-1 vims infects activated huPBMCs
  • the level of expression of HIV- 1 p24 g ⁇ is between about 50 fold and about 200 fold less than levels of p24 gag expression when HIV-1 wild type vims, utilizing wild-type NCTE (i.e., RRE), infects activated huPBMCs.
  • CTE SRV-1 CTE
  • RRE wild-type HIV-1 NCTE
  • CTE(+)/ RRE(-) HIV-1 clones were used to infect Thy/Liv implants (implanted human thymus and liver, see Kollmann (1995) J. Immunol. 154:907-921) in SCID-hu mice.
  • these viruses propagated slower than both wild-type and Nef-negative HIV-1 clones, demonstrating that they have lower replicative capacity in human lymphocytes.
  • CTE(+) RRE(-) attenuated HIV-1 clones were not lymphocytopathic, no depletion of CD4 + -bearing cells was observed.
  • CTE ⁇ -attenuated HIV-1 of the invention infects activated human lymphocytes in vivo, it will also produce low levels of infectious virions without any lymphocytotoxic effects, i.e., levels of CD4 + T cells will not decline.
  • the invention's CTE IAP -containing HIV-1 is significantly more attenuated than SRV-1 CTE-attenuated virus, as indicated by that fact that the level of expression of p24 g ⁇ g by CTEL ⁇ .
  • -containing HIV-1 is between about 50 fold less than wild type vims, while SRV-1 NCTE produces a hybrid vims only about 10 fold less than wild type.
  • this CTE ⁇ -attenuated vims will elicit an immune response in the infected, yet asymptomatic, individual.
  • an IAP element can be utilized in the attenuation of a retrovirus whose productive infection does not lead to loss of CD4 + cells is especially unexpected in view of past findings that a human IAP has been found to be associated with CD4 + T-cell immunodeficiency and dysfunction.
  • the presence of IAP sequence has also been associated with the occurrence of carcinogenesis, as discussed above.
  • This invention also provides for a vaccine in the form of a pharmacological compositions and a kit.
  • the pharmacological compositions can comprise a pharmaceutically acceptable carrier and the attenuated vims of the invention.
  • the kit can comprise a container containing a vaccine formulation.
  • CTE IAP refers to a family, or genus of nucleic acid sequences defined as having the following stmctural and functional properties: the CTE IAP , when inserted in an HIV-1 is capable of functioning as an NCTE in place of wild-type HIV-1 NCTE (i.e., RRE), and when the CTE IAP -containing hybrid HIV-1 vims infects activated huPBMCs, the level of expression of HIV- 1 p24 g ⁇ g is between about 50 fold to about 200 fold less than levels of p24 g ⁇ g expression when HIV-1 wild type vims, utilizing wild-type NCTE, infects activated huPBMCs; and, the secondary stmcture of the CTE IAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid comprising the loop s
  • IAP intracistemal A particle
  • Murine IAPs can have reverse transcriptase activity and can accumulate in the endoplasmic reticulum. IAPs are further described by, e.g., Wilson (1974) supra; Leuders (1977) supra; Kaushik (1994) supra; Kuff (1990) supra; Fehrmann (1997) supra; and, Wang (1997) supra.
  • IAP 0RG refers to the novel intracistemal A particle (IAP) of the invention, including the TE ⁇ of the invention, which was found inserted in a murine osteocalcin-related gene (ORG) between its promoter and coding region (thus termed
  • RNA nucleocytoplasmic transport element refers to cw-acting, post-transcriptional RNA nucleo- cytoplasmic transport elements. Simian retrovims NCTE is also referred to as "CTE.”
  • RNA sequences typically have a high degree of secondary stmcture in the form of stem-loop structures, which can interact with tn s-acting NCTE-binding proteins, hairpin rums, and the like.
  • Some retrovimses including HIV and simian retrovimses, regulate their growth through expression of their RNA using NCTEs and corresponding NCTE- binding proteins.
  • NCTE binding with viral or cellular tram-acting NCTE-binding proteins stabilizes unspliced viral transcripts and allows interaction with cellular machinery to transport the message from the nucleus to the cytoplasm.
  • HIV-l's NCTE is designated the "Rev-responsive element," or "RRE,” contained in its gag/pol and env encoding transcript.
  • NCTE-binding protein refers to a tr ⁇ ns-acting polypeptide which binds to RNA NCTE sequences, typically interacting with specific secondary stmctures. This RNA binding protein functions with other cellular proteins in the cellular nuclear export machinery to constitutively transport message nucleic acid (mRNA) from the nucleus to the cytoplasm.
  • An NCTE-binding protein can be encoded by endogenous cellular (eukaryotic) gene or a viral gene. HIV-1 encodes for its own specific, tr ⁇ r ⁇ -acting NCTE-binding polypeptide, termed "Rev.” Rev is further described, e.g., Hua (1996) supra; Iversen (1995) supra.
  • HIV refers to a lentivims usually called “human immunodeficiency vims” which is believed to the causal agent of acquired immune deficiency syndrome, or AIDS.
  • human immunodeficiency vims There are several known subtypes of HIV, including HIV-
  • HIV and AIDS are well described in the literature and, e.g., are further described by Gottfredsson (1997) Front Biosci. 2: D619-D634; Burton (1997) Proc. Natl. Acad. Sci. USA 94:10018-10023; Barnadas (1997) J. Cutan. Pathol. 24:507-510; Doms (1997) Virology 235:179-190; Cossarizza (1997) AIDS 11:1075-1088; Carpenter (1997) JAMA 277:1962-1969; Klein (1995) Trends Microbiol. 3:386-391.
  • P 24 g ⁇ g antigen or "p24 ⁇ ” refers to the 24 kd HIV-1 polypeptide found associated with the vims' RNA genome in the core of the virion, as described, e.g., by Jones (1996) Nat. Struct. Biol. 3:818-820 (1996); Doe (1996) AIDS 10:793-794; Klenerman (1996) AIDS 10:348-350; Klenerman (1994) Nature 369:403-407.
  • Nef refers to a 27 - 34 kD myristoylated protein unique to primate lentivimses. A functional Nef gene is important for development of high viremia and AIDS.
  • NCT ⁇ post-transcriptional RNA nucleo- cytoplasmic transport element
  • NCTE is still considered “capable of functioning as an NCTE in place of wild-type NCTE in a hybrid HIV-1 " even if it is less efficient, less accurate, or less capable, of splicing and/or transferring mRNA (of which it is a part) from the nucleus to the cytoplasm.
  • the CTE IAP of the invention when inserted in HIV-1, is considered capable of functioning as an NCTE in place of wild-type NCTE (RRE) in the hybrid HIV-1 even though it is less efficient than RRE; i.e., when the CTE IAP -containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24 ⁇ is between about 50 fold and about 200 fold less than levels of p24 g ⁇ g expression when HIV-1 wild type vims, utilizing wild-type NCTE (RRE), infects activated huPBMCs.
  • RRE wild-type NCTE
  • activated refers to a non-dormant cellular state, for example, as when a lymphocyte has been activated by an antigen, cytokine(s) or other mitogen.
  • wild-type refers to any form (e.g., tertiary stmcture), stmcture (e.g., secondary stmcture) or sequence (e.g., primary stmcture) of a composition, as a nucleic acid or polypeptide, as found in nature, versus stmctures or sequences which have been manipulated by the hand of man, i.e., recombinant nucleic acids or polypeptides.
  • stmcture e.g., secondary stmcture
  • sequence e.g., primary stmcture
  • second stmcture refers to the stmcture formed by the intramolecular folding of a polypeptide or a nucleic acid, such as the stem and loop stmctures formed by the CTE L ⁇ of the invention.
  • peripheral blood mononuclear cell refers to any peripheral mononuclear white blood cell.
  • expression vector refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • the term includes linear or circular expression systems.
  • the term includes expression systems that remain episomal or integrate into the host cell genome.
  • the expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell.
  • the term includes recombinant expression "cassettes” which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • isolated when referring to a molecule or composition, such as, for example, a polypeptide or nucleic acid, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g.,
  • RNAs or other contaminants with which it is associated in vivo or in its naturally occurring state.
  • a polypeptide or nucleic acid is considered isolated when it has been isolated from any other component with which it is naturally associated, e.g., cell membrane, as in a cell extract.
  • An isolated composition can, however, also be substantially pure.
  • An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).
  • SDS-PAGE polyacrylamide gel electrophoresis
  • HPLC high performance liquid chromatography
  • nucleic acid molecule refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleo tides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid.
  • the term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs, which contain non-ionic backbones, such as N-(2- aminoethyl) glycine units); see Oligonucleotides and Analogues, A Practical Approach, edited by F.
  • nucleic acid is used interchangeably with gene, DNA, cDNA, RNA, mRNA, oligonucleotide primer, probe and amplification product.
  • exogenous nucleic acid refers to a molecule (e.g., nucleic acid or polypeptide) that has been isolated, synthesized, and/or cloned, in a manner that is not found in nature, and/or introduced into and/or expressed in a cell or cellular environment other than or at levels or forms different than the cell or cellular environment in which said nucleic acid or protein can be found in nature.
  • the term encompasses both nucleic acids originally obtained from a different organism or cell type than the cell type in which it is expressed, and also nucleic acids that are obtained from the same organism, cell, or cell line as the cell or organism in which it is expressed.
  • endogenous refers to a molecule, e.g., a nucleic acid or polypeptide, in a form, stmcture and/or sequence found in nature.
  • Sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the nucleotides (or residues) in the two sequences which are the same when aligned for maximum correspondence over a specified "comparison window.” Sequence identity analysis is used to determine whether a nucleic acid is within scope of the invention. For example, to identify a specie of the CTE ⁇ family of the invention, a nucleic acid must have a secondary stmcture with at least a two loop A domain and/or a loop B domain, where the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid as set forth in Figure 1 , SEQ ID NO: 1.
  • Sequence identity can be analyzed by optimal alignment of sequences for comparison using any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed "PILEUP”; by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms, e.g.
  • BLAST, GAP, BESTFIT, FASTA, and TFASTA in, e.g., the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI; or, by inspection. See also Morrison (1997) Mol. Biol. Evol. 14:428-441, as an example of the use of PileUp, ClustalW, TreeAlign, MALIGN, and SAM sequence alignment computer programs.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J.
  • recombinant when used with reference to, e.g., a cell, nucleic acid, polypeptide, expression cassette or vector, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell (i.e., "exogenous nucleic acids”) or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all.
  • recombinant means refers to techniques where, e.g., a recombinant nucleic acid such as a cDNA encoding a protein or an antisense sequence, is inserted into an expression cassette, such as an expression vector, the resultant constmct is introduced into a cell, and the cell expresses the nucleic acid, and the protein, if appropriate.
  • “Recombinant means” also encompass the ligation of nucleic acids to coding or promoter sequences from different sources into one expression cassette or vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein.
  • test compound refers to any synthetic or natural compound or composition.
  • the term includes all organic and inorganic compounds; including, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids and the like.
  • motif or domain refers to a nucleic acid or amino acid sequence pattern, or stmcture, which is shared between related molecules.
  • Ameliorating refers to any indicia of success in the treatment of a ' pathology or condition, including any objective or subjective parameter such as abatement, remission or diminishing of symptoms or an improvement in a patient's physical or mental well-being. Amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination and/or a psychiatric evaluation.
  • prophylaxis refers to any form of prevention, delay or abatement a pathology or condition or symptom thereof, including any objective or subjective parameter.
  • infectious refers to a state wherein an infectious agent, i.e., a pathogen, such as a microbial or viral agent, has a phenotype manifested by a lessened ability to grow, proliferate, or cause pathogenesis, in a host, i.e., the non-wild type, attenuated phenotype is less vimlent.
  • a CTE L ⁇ -attenuated HIN-1 vims is capable of replication, infection and production of infectious virions without causing clinically significant pathology in its host.
  • immuno response in a host refers to both cellular and humoral (antibody) mediated responses to an immunogen, i.e., a compound or composition capable of eliciting an immune response.
  • an immunogen i.e., a compound or composition capable of eliciting an immune response.
  • the immune response can be elicited by a foreign substance or a pathogen, and the immunogen can be a carbohydrate, a nucleic acid, a polypeptide, a lipid, or a combination of these elements.
  • vaccine is used in its ordinary sense, meaning an agent which is capable of eliciting a humoral and/or cell-mediated immunoprotective immune response when administered to an individual with an at least partially functioning immune system.
  • This invention has for the first time provided for the characterization, cloning and expression of a novel NCTE, CTEJ A P, derived from a novel intracistemal A particle, IAP ORG .
  • the invention also provides for novel means of expressing the IAP ORG and CTE ⁇ AP of the invention in vitro and in vivo. In a further embodiment, this expression provides for a means to screen for novel NCTEs.
  • nucleic acids of this invention may be isolated from a variety of sources or may be synthesized in vitro.
  • Nucleic acids of the invention can be expressed in transgenic animals, transformed cells, in a transformed cell lysate, or in a partially purified or a substantially pure form. Sequencing methods typically use dideoxy sequencing (Sequenase, U.S. Biochemical), however, other kits and methods are available and well known to those of skill in the art.
  • Nucleic acids and proteins are detected and quantified in accordance with the teachings and methods of the invention described herein by any of a number of general means well known to those of skill in the art.
  • immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
  • Dot-blot analysis Dot-blot analysis, gel electrophoresis, RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography, to name only a few.
  • the invention provides for a novel IAP and CTE ⁇ and methods to identify alleles, isoforms and polymorphisms.
  • DNA can be isolated from a genomic or cDNA library using labeled oligonucleotide probes having sequences complementary to the sequences or subsequences disclosed herein, such as CTEL ⁇ and IAP 0RG , SEQ ID NOS: 1 and SEQ ID NO:3, respectively (the DNA subsequence encoding the CTEL ⁇ , of the invention is located between nucleotides 3029-3204 of SEQ ID NO:3).
  • probes can be used directly in hybridization assays to isolate DNA encoding IAP ORG or CTE ⁇ isoforms and polymorphisms.
  • probes can be designed for use in amplification techniques, such as, e.g., PCR, and nucleic acid encoding IAP 0RG or CLE ⁇ can be isolated by using methods (see infra).
  • mRNA is isolated, reverse transcribed from the mRNA according to procedures well known in the art and inserted into vectors.
  • the vectors are transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known. See, e.g., Gubler (1983) Gene 25:263-269, Sambrook, Ausubel.
  • DNA is extracted and purified by well- known methods (see, e.g., Sambrook).
  • DNA of appropriate size is produced by known methods, such as mechanical shearing or enzymatic digestion, to yield DNA fragments, e.g., of about 12 to 20 kb.
  • the fragments are then separated, as for example, by gradient centrifugation, or gel electrophoresis, from undesired sizes.
  • Selected fragments can be inserted in bacteriophage or other vectors. These vectors and phage can be packaged in vitro, as described, e.g., in Sambrook.
  • Recombinant phage can be analyzed by plaque hybridization described, e.g., in Benton (1977) Science 196:180; Chen (1997) Methods Mol Biol 62:199-206. Colony hybridization can be carried out as generally described in, e.g., Grunstein (1975) Proc. Natl. Acad. Sci. USA 72:3961-3965; Yoshioka (1997) J. Immunol Methods-20 ⁇ : ⁇ 45-155; Palkova (1996) Biotechniques 21 :982.
  • DNA encoding an IAP or CTE IAP can be identified in either cDNA or genomic libraries by hybridization with nucleic acid probes of the invention.
  • a probe containing 10 to 20 to 50 or more contiguous nucleotides of SEQ ID NO:l or 3 is used in Southern blots to identify Once identified, these DNA regions are isolated by standard methods familiar to those of skill in the art.
  • RNA may be identified by hybridization to nucleic acid probes in Northern blots or other formats; see, e.g., Sambrook, Ausubel, for general procedures.
  • Oligonucleotides for use as, e.g., probes, templates for further amplification, and the like, can be chemically synthesized, as described below.
  • Synthetic nucleic acids including oligonucleotide probes and primers, CTEL ⁇ and IAP coding sequences, can be prepared by a variety of solution or solid phase methods. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available. For example, the solid phase phosphoramidite triester method of Beaucage and Carruthers using an automated synthesizer is described in Itakura, U.S. Pat. No.
  • oligonucleotides 14:5399-5407 (1986); Sinha (1983) Tetrahedron Lett. 24:5843-5846; and Sinha ( 1984) Nucl. Acids Res. 12:4539-4557.
  • Methods to purify oligonucleotides include native acrylamide gel electrophoresis, anion-exchange HPLC, as described in Pearson (1983) J. Chrom. 255:137-149.
  • the sequence of the synthetic oligonucleotide can be verified using any chemical degradation method, for example, see Maxam (1980) Methods in Enzymology 65:499-560, Xiao (1996) Antisense Nucleic Acid Drug Dev 6:247-258, or for solid-phase chemical degradation procedures, Rosenthal (1987) Nucleic Acids Symp Ser 18:249-252.
  • the present invention provides oligonucleotide primers and probes that can hybridize specifically to nucleic acids having CTE ⁇ p and IAP nucleic acid sequences.
  • Such reagents can be used to identify further species, such as polymorphisms alleles and other variations, of CTE IAP and IAP.
  • PCR primers and amplification methods are described.
  • Amplification of CTE j ⁇ and IAP sequences which are conserved amongst different CTE ⁇ p and IAP species are preferred reagents of the invention for use as hybridization probes to identify and isolate additional species from various organisms.
  • These oligonucleotides can also be used as primers to directly amplify nucleic acids sequences, as described below.
  • Oligonucleotides can be used to identify and detect additional CTE j ⁇ species using a variety of hybridization techniques and conditions. Suitable amplification methods include, but are not limited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N. Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.
  • the invention provides for amplification and manipulation or detection of the products from each of the above methods to prepare DNA encoding CT ⁇ IAP and IAP nucleic acid.
  • oligonucleotide primers complementary to the two borders of the DNA region to be amplified are synthesized and used (see, Innis).
  • PCR can be used in a variety of protocols to amplify, identify, quantify, isolate and manipulate nucleic acids encoding CTE [AP and IAP.
  • primers and probes for amplification and hybridization are generated that comprise all or any portion of the DNA sequences listed herein
  • An illustrative primer pair that can amplify the CTE ⁇ of the invention under appropriate conditions is: sense: 5'-CAACTGTTTCTTGGCATGCTAGAGAAGTAGT-3' (SEQ ID NO:4); and antisense 5'-CTGTCTTTACTTATTGGCAGAGAGAA-3' (SEQ ID NO:5).
  • PCR-amplified sequences can also be labeled and used as detectable oligonucleotide probes, but such nucleic acid probes can be generated using any synthetic or other technique well known in the art, as described above.
  • the labeled amplified DNA or other oligonucleotide or nucleic acid of the invention can be used as probes to further identify and isolate CTEL ⁇ J , and IAP species from various cDNA or genomic libraries.
  • Another useful means of obtaining nucleic acids of the invention, such as large genomic clones, is to screen YAC, BAC or PI genomic libraries.
  • BACs, bacterial artificial chromosomes, are vectors that can contain 120+ Kb inserts. BACs are based on the E. coli F factor plasmid system and simple to manipulate and purify in microgram quantities.
  • BAC vectors can include marker genes for luciferase and green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Yeast artificial chromosomes, or YACS can also be used for contain inserts ranging in size from 80 to 700 kb, see, e.g., Tucker (1997) Gene 199:25-30; Adam (1997) Plant J.l 1:1349-1358.
  • PI is a bacteriophage that infects E. coli that can contain 75-100 Kb DNA inserts (Mejia
  • the invention also provides CTE IAP - and IAP-encoding expression vectors to produce large quantities of full or partial length (e.g., a single RNA loop stmcture) nucleic acid molecules of the invention.
  • the expression vector, including expression cassettes include, e.g., those used in bacterial, yeast, plant, insect, in vitro, or mammalian systems.
  • generation of CTE ⁇ p in this manner is useful for assaying for CTE L ⁇ P activity modulators, analysis of the activity of newly isolated species of CTE, ⁇ , identifying and isolating compounds which specifically associate with CTE tAp , such as binding proteins, analysis which has been site-specifically mutated, as described below.
  • Nucleic acid so produce can be used the the invention's method to screening for a NCTE binding proteins using a single loop A or a loop B, or both loops as in Figure 1A, of a CTE IAP of the invention.
  • the nucleic acids of the invention can also be used as immunogens, as a few examples. See, e.g., Radic (1994) Annu. Rev. Immunol. 12:487-520; Cabral (1997) Curr. Opin. Rheumatol.
  • Sequencing of newly isolated DNA will identify and characterize CTE Laj ,- and IAP-encoding nucleic acid of the invention. Sequencing of isolated CTE L ⁇ - and IAP- encoding nucleic acid can be used to identify, in addition to functional criteria, new CTE ⁇ p - and IAP-encoding species or allelic variations. Secondary stmctures can be identified. For example, sequencing of CTE IAP and subsequent computer predicted analysis of its secondary stmcture revealed a loop A with a "bulge" (e.g., an "AAGA" bulge), a loop B, a stem stmcture separating the loops and a hairpin turn, see Figure 1, SEQ ID NO: 1.
  • a "bulge" e.g., an "AAGA" bulge
  • a nucleic acid is a CTE 1AP specie within the scope of the claimed invention if the sequence of its loop domains has at least 90% nucleic acid-sequence identity to the loop A and loop B domains of CTE 1AP illustrated in Figure 1 , SEQ ID NO: 1 (the DNA subsequence encoding CTE 1AP is located between nucleotides 3029-3204 of Figure 4, SEQ ID NO:3).
  • CTE L ⁇ -encoding nucleic acid sequences can be sequenced as inserts in vectors, as inserts released and isolated from the vectors or in any of a variety of other forms (i.e., as amplification products).
  • CTE L ⁇ ,- and IAP-encoding inserts can be released from the vectors by restriction enzymes or amplified by PCR or transcribed by a polymerase.
  • primers based on the N- or C- terminus, or based on insertion points in the original phage or other vector can be used. Additional primers can be synthesized to provide overlapping sequences.
  • nucleic acid sequencing techniques are well known and described in the scientific and patent literature, e.g., see Rosenthal (1987) supra; Arlinghaus (1997) Anal. Chem. 69:3747-3753, for use of biosensor chips for sequencing; Pastinen (1996) Clin. Chem. 42:1391-1397; Nyren (1993) Anal Biochem. 208:171-175. 5. Nucleic Acid Hybridization Techniques The hybridization techniques disclosed herein can be utilized to identify, isolate and characterize genes and gene products (i.e., mRNA) encoding IAP and CTE IAP - encoding nucleic acid of the invention, including different species, isoforms, alleles and polymorphisms.
  • mRNA genes and gene products
  • nucleic acid probes can be designed based on conserved nucleic acid sequences amongst the class of proteins. Preferably nucleic acid probes are 20 bases or longer in length (see, e.g., Sambrook for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization). Visualization of the hybridized portions allows the qualitative determination of the presence or absence of DNA encoding IAP or CTE IAP .
  • RNA containing IAP or CTE 1AP sequences can be used for the detection of RNA containing IAP or CTE 1AP sequences.
  • RNA is isolated from a given cell sample using an acid guanidinium-phenol-chloroform extraction method. The RNA is then electrophoresed to separate different species and transferred from the gel to a nitrocellulose membrane.
  • labeled probes or PCR can be used to identify the presence or absence of IAP or CTE i ⁇ nucleic acid.
  • Sandwich assays are commercially useful hybridization assays for detecting or isolating protein or nucleic acid. Such assays utilize a "capture" nucleic acid or protein that is often covalently immobilized to a solid support and a labeled "signal" nucleic acid, typically in solution. A clinical or other sample provides the target nucleic acid or protein. The "capture” nucleic acid or protein and “signal” nucleic acid or protein hybridize with or bind to the target nucleic acid or protein to form a "sandwich” hybridization complex. To be effective, the signal nucleic acid or protein cannot hybridize or bind substantially with the capture nucleic acid or protein.
  • oligonucleotide probes are labeled signal nucleic acids that are used to detect hybridization.
  • Complementary probe nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. Methods of detection can use labels for autoradiography or autofluorography, such as 3 H, 125 1, 35 S, 14 C, or 32 P-labeled probes or the like (see definition of label, above).
  • Other labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal, i.e., antibody-antigen or complementary nucleic acid binding.
  • the label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzymatic molecules to the antibodies or, in some cases, by attachment of a radioactive label.
  • the sensitivity of the hybridization assays may be enhanced through use of a target nucleic acid or signal amplification system which multiplies the target nucleic acid or signal being detected.
  • a target nucleic acid or signal amplification system which multiplies the target nucleic acid or signal being detected.
  • In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known, as described above. These systems can be used to directly identify IAP or variations, polymorphisms, or mutated sequences where the PCR or LCR primers or other reagents are designed to be extended or ligated only when a specific sequence is present.
  • the specific sequences can be generally amplified using, for example, more generic PCR primers and the amplified target region later probed or sequenced to identify a specific sequence indicative of the variant, polymorphism or mutation.
  • Nucleic acid hybridization assays for the detection of isoforms, mutations and for sequencing can also be performed in an array-based format. Arrays are a multiplicity of different "probe” or "target” nucleic acids (or other compounds) are hybridized against a target nucleic acid. In this manner a large number of different hybridization reactions can be run essentially "in parallel”. This provides rapid, essentially simultaneous, evaluation of a wide number of reactants.
  • in situ hybridization An alternative means for determining the level of expression of a gene is in situ hybridization.
  • In situ hybridization assays are well known (e.g., Angerer (1987) Methods Enzymol 152:649).
  • an in situ hybridization assay cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein.
  • the probes are typically labeled, i.e., with radioisotopes or fluorescent reporters.
  • FISH fluorescence in situ hybridization Another well-known in situ hybridization technique is the so-called FISH fluorescence in situ hybridization, as described by Macechko (1997) J. Histochem. Cytochem. 45:359-363; and, Raap (1995) Hum. Mol. Genet. 4:529-534.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known, as described above.
  • Nucleic acid sequence-based amplifications are also used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a select sequence is present.
  • the select sequences can be generally amplified using nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • Oligonucleotides for use as probes are typically synthesized chemically; e.g., such as by the solid phase phosphoramidite triester method described by Beaucage and Caruthers, supra, or, using an automated synthesizer, as described in Needham-VanDevanter, supra.
  • Purification of oligonucleotides, where necessary, is typically performed by native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier. The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (Maxam (1980) supra).
  • nucleic acid hybridization assays can also be performed in an array-based format.
  • arrays bearing a multiplicity of different "probe" nucleic acids are hybridized against a target nucleic acid.
  • a large number of different hybridization reactions can be run essentially "in parallel”.
  • Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Jackson (1996) Nature Biotechnol. 14:1685, and Chee (1995) Science 274:610).
  • sequence Comparison Analysis of the CTE IAP and IAP 0RG IAP and NCTE-encoding nucleic acid sequences of the invention include both genes (genomic sequences, including introns and 5' and 3' non-coding regions) and gene transcription products (mRNA) identified and characterized by analysis of IAP 0RG and CTE IAP sequences. Optimal alignment of sequences for comparison can be conducted as described above (see definitions).
  • a CTE ⁇ p sequence of the invention has a secondary stmcture with at least a loop A domain, a stem stmcture separating the loops, and a loop B domain, where the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1.
  • the invention also provides for CTEi ⁇ j , and IAP 0RG that have been modified in a site-specific manner to modify, add to, or delete some or all of the nucleic acid's functions.
  • specific base pairs can be modified to alter, increase or decrease the affinity of binding proteins, thus modifying the relative level of attenuation of the CTE; or, modifications can change the stability of the secondary stmcture of the nucleic acid; or, base pair changes can augment expression of the nucleic acid in a cell, such as a bacteria; as a few illustrative examples.
  • Site-specific mutations can be introduced into CTE IAP and IAP ORG -encoding nucleic acid by a variety of conventional techniques, well described in the scientific and patent literature.
  • Illustrative examples include: site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR), as in Urban (1997) Nucleic Acids Res. 25:2227-2228; Ke (1997) Nucleic Acids Res 25:3371-3372, and Chattopadhyay (1997) Biotechniques 22:1054-1056, describing PCR-based site-directed mutagenesis
  • Modified CTE IAP and IAP 0RG of the invention can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.
  • the invention provides for methods and reagents the expression of novel CTE ⁇ and IAP 0RG of the invention in any prokaryotic, eukaryotic, yeast, fungal, plant, insect, human or animal cell. Antisense, in addition to sense, sequences are provided for by the invention. To create cell-based and in vitro assay systems to screen for novel
  • NCTEs using CTE ⁇ p a variety of in vivo and in vitro expression systems are provided by the invention.
  • the invention provides for methods and reagents for expressing the novel CI ytf and IAP 0RG nucleic acids described above as sense or antisense coding sequences.
  • inventions provide methods and reagents for identifying, isolating and using CTE L ⁇ and IAP 0RG cz ' sacting transcriptional control elements and transacting NCTE binding proteins.
  • CTE IAP and IAP 0RG cz After the coding region of a CTE IAP and IAP 0RG gene has been identified, the expression of natural or synthetic CTEL ⁇ , and IAP ORG -encoding or other (i.e., antisense, ribozyme) nucleic acids can be achieved by operably linking the coding region to transcriptional regulatory elements, such as promoters and enhancers.
  • promoter sequence elements typically include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. Promoters can be tissue- specific or not, constitutive or inducible. Promoters that drive expression continuously under physiological conditions are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Typical expression systems, such as expression cassettes and vectors, also contain transcription and translation terminators, transcription and translation initiation sequences.
  • TATAAT TATA box consensus sequence
  • Promoters can be tissue- specific or not, constitutive or inducible. Promoters that drive expression continuously under physiological conditions are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
  • Typical expression systems such as expression cassettes and vectors, also contain transcription and translation terminators, transcription and translation initiation sequences.
  • Generic expression cassettes typically contain at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • eukaryotes or prokaryotes, or both
  • shuttle vectors selection markers for both prokaryotic and eukaryotic systems.
  • Product information from- manufacturers of biological reagents and experimental equipment also provide information regarding known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia Biotech (Piscataway, NJ), Clontech Laboratories, Inc.
  • promoters and vectors used in this invention can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic methods, as described herein.
  • the CTE j ⁇ a j , and IAP 0RG sense or antisense sequences of the invention can be expressed in vectors which are transiently expressed in cells using, e.g., episomal vectors such as vaccinia vims, see Cooper (1997) Proc Natl Acad Sci USA 94:6450-6455.
  • CTE ⁇ and IAP 0RG coding sequences can be inserted into the host cell genome becoming an integral part of the host chromosomal DNA, using for example, retroviral vectors such as SIV or HIV, see for example, Naldini (1996) Science 272:263-267.
  • Expression vectors can contain selection markers that confer a selectable phenotype on transformed cells and sequences coding for episomal maintenance and replication such that integration into the host genome is not required.
  • the marker may encode antibiotic resistance, particularly resistance to chloramphenicol, kanamycin, G418, bleomycin and hygromycin, to permit selection of those cells transformed with the desired DNA sequences, see for example, Blondelet-Rouault (1997) Gene 190:315-317. Because selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture, chemoresistance genes are also used as selectable markers in vitro and in vivo.
  • chemoresistance genes encoding, e.g., P-glycoprotein, multidmg resistance-associated protein-transporter, dihydrofolate reductase, glutathione -S-transferase, O 6-alkylguanine DNA alkyltransferase, or aldehyde reductase (Licht (1997) Stem Cells 15:104-1 1 1) and the like.
  • chemoresistance genes encoding, e.g., P-glycoprotein, multidmg resistance-associated protein-transporter, dihydrofolate reductase, glutathione -S-transferase, O 6-alkylguanine DNA alkyltransferase, or aldehyde reductase (Licht (1997) Stem Cells 15:104-1 1 1) and the like.
  • Illustrative vectors incorporating CTE ⁇ p and IAP 0RG genes include, for example, adenovims-based vectors (Cantwell (1996) Blood 88:4676-4683; Ohashi (1997) Proc Natl Acad Sci USA 94:1287-1292), Epstein-Barr vims-based vectors (Mazda (1997) J Immunol Methods 204:143-151), adenovims-associated vims vectors, Sindbis vims vectors (Strong (1997)
  • Herpes simplex vims vectors (Kennedy (1997) Brain 120: 1245-1259) and retroviral vectors (Schubert (1997) Curr Eye Res 16:656-662).
  • Epstein- Barr vims episomal vectors (Horlick (1997) Protein Expr. Purif. 9:301-308, and plasmid DNA (Lowrie (1997) Vaccine 15: 834-838); all of which can be used to express the nucleic acids of the invention in vivo or ex vivo
  • the invention further provides for nucleic acids complementary to, i.e., antisense sequences to, the CTE L ⁇ J. and IAP 0RG of the invention.
  • Antisense sequences are capable of inhibiting the transport, splicing or transcription of CTE IAP and IAP ORG - encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
  • One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind CTE j ⁇ and IAP ORG gene or message, in either case preventing or inhibiting the production or function of CTE ⁇ and IAP 0RG .
  • the association can be though sequence specific hybridization.
  • Such inhibitory nucleic acid sequences can, for example, be used to completely inhibit or further depress the replicative potential of an attenuated vims.
  • Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of CTE, ⁇ and IAP 0RG message.
  • the oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes.
  • the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid.
  • an enzyme or composition capable of cleaving the complementary nucleic acid.
  • the invention provides for with antisense oligonucleotides capable of binding CTE L ⁇ J. and IAP 0RG message which can inhibit IAP 0RG activity by targeting mRNA.
  • antisense oligonucleotides capable of binding CTE L ⁇ J. and IAP 0RG message which can inhibit IAP 0RG activity by targeting mRNA.
  • " Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such CTE j ⁇ and IAP 0RG oligonucleotides using the novel reagents of the invention.
  • naturally occurring nucleic acids used as antisense oligonucleotides may need to be relatively long (18 to 40 nucleotides) and present at high concentrations.
  • a wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem.
  • peptide nucleic acids containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.
  • Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).
  • Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
  • Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense CTE j ⁇ and IAP 0RG sequences of the invention (for general background information, see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
  • the invention provides for with ribozymes capable of binding CTE ⁇ and
  • IAP 0RG message which can inhibit CTE ⁇ and IAP 0RG activity by targeting mRNA.
  • Strategies for designing ribozymes and selecting the CTC ⁇ and IAP 0RG -specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such CTE j ⁇ and IAP 0RG ribozymes using the novel reagents of the invention.
  • Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
  • the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence, or, as with CTE ⁇ p, prevent viral message from binding to NCTE binding protein, thus preventing subsequent transport of the message to the cytoplasm. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
  • a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule, as with CTE ⁇ p) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
  • antisense technology where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule, as with CTE ⁇ p
  • This potential advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
  • the enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta vims, group I intron or RNaseP-like RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
  • RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
  • the invention provides for an attenuated retrovims and vaccine comprising the CTEL A J. of the invention.
  • One means to genetically engineer a wild-type, vimlent vims to a hybrid, attenuated vims involves constructing a vims which either lacks or has a non- functional endogenous post-transcriptional RNA nucleo-cytoplasmic transport elements
  • NCTEs The endogenous NCTE is subsequently replaced by the exogenous NCTE of the invention which functions less efficiently in vivo than its wild-type counterpart, thus effecting the attenuation.
  • the CTE IAP of the invention when inserted in a RRE(-) and/or Rev(-) HIV-1, is capable of functioning as a less efficient, "attenuated" NCTE in place of the wild-type RRE/Rev NCTE system.
  • This level of attenuation can be measured; when the CTE Ly .-containing hybrid HIV-1 vims infects activated huPBMCs, the level of expression of HIV- 1 p24 ⁇ g is between about 50 fold and about 200 fold less than levels of p24 g ⁇ g expression when HIV-1 wild type virus, utilizing wild-type NCTE, infects activated huPBMCs.
  • additional elements of the retrovims which are essential for its replication and/or pathogenicity can also be disabled or eliminated, such as Nef, as explained below.
  • RNA present in the cell as ribonucleoprotein (RNP) complexes
  • RNP ribonucleoprotein
  • all retrovimses have evolved a mechanism that allows the nuclear export of unspliced form of viral RNAs which are necessary for the production of structural proteins and essential for viral replication. This mechanism involves the highly structured NCTE c/sacting RNA element and its corresponding transacting RNA binding proteins, as discussed above.
  • NCTE In simian type retrovimses, the NCTE is termed “CTE” (see Bray (1994) supra; Zolotukhin (1994) supra), and binds to endogenous cellular RNA binding proteins.
  • HIV-l's NCTE does not bind cellular NCTE-binding proteins, it encodes its own NCTE binding protein, called “Rev.”
  • Rev interacts with a specific HIV-1 NCTE sequence, designated the “Rev-responsive element,” or “RRE,” contained in its gag/pol and env encoding transcript. Rev interacts directly with RRE as part of the RNA export machinery which transports RRE-containing transcripts to the cytoplasm from the nucleus. As a result, HIV-1 needs both RRE and Rev to produce infectious vims.
  • Disabling either produces a non-replicative, non-vimlent vims.
  • Replacing i.e., reconstituting
  • HIV-l's RRE/Rev RNA transport mechanism with a less efficient NCTE, such as the CTE IAP of the invention, produces an attenuated, avimlent hybrid vims.
  • RRE and Rev sequences are well known in the art, e.g., see databases, such as the NCBI database at http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html or http://www.ncbi.nlm.nih.gov/ Entrez/protein.html. Further description and sequence of HIV-1 Rev can be found in, e.g.,
  • Salminen (1997) J Virol. 71 :2647-2655, Accession U86770; Theodore (1996) AIDS Res. Hum. Retroviruses 12:191-194, Accession AF004394; Fang, et al, Accession AF003887; Howard (1996) AIDS Res. Hum. Retroviruses 12:1413-1425, Accession L39106; to name only a few. Further description and sequence of HIV- 1 RRE can be found in, e.g., Salminen (1996) JOURNAL AIDS Res. Hum.
  • Retroviruses 12:1329-1339 Accession U46016; WO 9202228-A5 20-FEB-1992, Accession A20711; Battiste (1994) Biochemistry 33:2741-2747; Battiste (1995) J. Biomol. NMR 6:375-389; Battiste (1996) Science 273:1547-1551; to name just a few.
  • additional elements in addition to endogenous NCTE which are essential for the vims' replication and or pathogenicity can also be disabled or eliminated.
  • genetic engineering of a Nef-negative retrovims can produce recombinant hybrid which can replicate in vivo, yet is less vimlent.
  • a functional Nef gene is important for development of high viremia and AIDS. Animals infected with Nef-deleted attenuated viruses are resistant to subsequent challenge with pathogenic wild-type vimses.
  • Nef Newcastle disease virus
  • a critical role for Nef in development of AIDS in humans has been suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection (persons who show no clinical or immunological signs of immunodeficiency despite being HIV seropositive for over a decade) are infected with vimses carrying naturally occurring Nef deletions.
  • the mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell protein kinases.
  • In vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis.
  • Recombinant soluble Nef protein binds to cell surfaces of various murine lymphoid cell lines, including T and B lymphocytes and macrophages; cross-linking of the cell-bound
  • Nef protein with anti -Nef antibodies induces apoptotic cytolysis of the cells.
  • Saksela (1997) supra; Greenberg (1997) supra; Luo (1997) J. Virol. 71 :9531-9537; Luo (1997) J. Virol. 71 :9524-9530; Okada (1997) supra.
  • the vaccine and retrovims of the invention comprising a non-function Nef has incorporated an additional level of attenuation.
  • the skilled artisan can delete and or mutate any portion of the Nef coding sequence.
  • Nef sequences are well known in the art, e.g., see databases, such as the NCBI databases described above.
  • databases such as the NCBI databases described above.
  • HIV- 1 Nef nucleic acid and polypeptide sequences see, e.g., Accession Nos. Y15123, U88826, Y15121, Y15120, Y15116, to name only a few.
  • the nucleic acids and oligonucleotides of the invention can be delivered into cells in culture, tissues and organisms for synthesis, mutation, screening and the like.
  • the invention provides for a method for screening for a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) binding protein by contacting a CTE IAP of the invention with a test compound and measuring the ability of the test compound to bind the NCTE; and this screening technique can be used in intact cells.
  • Inhibitory oligonucleotides of the invention, and vectors capable of expressing these sequences are also transferred into intact cells in cell culture, tissues or intact organisms.
  • oligonucleotides of the invention can be transferred into a cell using a variety of techniques well known in the art.
  • oligonucleotides can be delivered into the cytoplasm spontaneously, without specific modification. Altematively, they can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis.
  • a DNA binding protein e.g., HBGF-1, is known to transport oligonucleotides into a cell.
  • the procedures for delivering the nucleic acids and oligonucleotides of the invention to cells are useful in vivo.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ
  • Huwyler 1997) J. Pharmacol. Exp. Ther. 282:1541-1546, describing receptor mediated delivery using immunoliposomes.
  • cells may be permeabilized to enhance transport of oligonucleotides into the cell, without injuring the host cells. See, e.g., Verspohl (1997) Cell. Biochem. Fund. 15:127-134; Kang (1997) Pharm. Res. 14:706-712; Bashford (1994) Methods Mol. Biol.
  • Live, CTE IAP -attenuated HIV-1 vims can be grown and harvested from activated human peripheral mononuclear cells or from a variety of tissue culture cells, such as human 293 cell line, as described herein; see also, e.g., Eberlein (1991) Virus Res. 19:153-161; Parente (1996) Gene Ther. 3:756-760; Margolis (1997) AIDS Res. Hum.
  • Retroviruses 13:1411-1420 Virion-containing supernatants are collected, and, typically, filtered. CTE IAP sequences in the harvested, attenuated vims for use in vaccine formulations can be confirmed by conventional sequencing.
  • the attenuated vims can be further purified, for example, by ultrafiltration or ultra-centrifugation.
  • the live, attenuated vims can be stored by refrigeration, or on a long-term basis, by freezing in liquid nitrogen.
  • a formulation for administering the vims as a vaccine is prepared using, for example, any physiologically acceptable buffer, such as saline or phosphate buffered saline (PBS). This can be stored in a frozen state.
  • PBS phosphate buffered saline
  • the formulation can also be freeze-dried, stored at room temperature, and reconstituted by adding appropriate volume of buffer.
  • the vaccine pharmaceutical formulation can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptabie diluent or solvent, such as a solution of 1,3-butanediol.
  • the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables.
  • the live attenuated viral vaccine of the invention can be administered using any acceptable route, as, e.g., by application to a mucosal surface, by injection, by inhalation or by ingestion.
  • inhalants see Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.
  • Injection of vaccine can be intravenous or intramuscular; see, e.g., Groswasser (1997) Pediatrics 100:400-403, as example of injection techniques for efficient intramuscular vaccine delivery.
  • Administration by application to any mucosal surface including, e.g., intraoral (sublingual, buccal, and the like), intranasal, intrarectal, intravaginal, or ocular.
  • mucosal administration methods see, e.g., Pope (1997) AIDS Res Hum Retroviruses 13:945-952; Okada (1997) J. Immunol.
  • the amount of vims (number of virions) per dose will vary depending on results of different titrations used in clinical trials. The range can range, for example, from only a few infectious units, to about 10 to 10 infectious units (i.e., virions) per dose. Protocols and means to determine safety and efficacy used for other attenuated vaccines can be adapted and used with the novel reagents provided by the invention; see, e.g.,
  • the vaccine After the vaccine has formulated in an acceptable carrier, it can be placed in an appropriate container and labeled.
  • labeling would include, e.g., instructions concerning the amount, frequency and method of administration.
  • the invention provides for a kit and instmctional material teaching the indications, dosage and schedule of administration of the vaccine. Selection of individuals who would benefit from receiving the live, attenuated vaccine of the invention include, but are not limited to, individuals who have a high risk of being exposed to HIV, such as intravenous dmg users, individuals who may been exposed, as through a needle stick or transfusion, and individuals whose exposure to the vims has been confirmed, e.g., by a positive blood test.
  • the vaccine can be administered in conjunction with other treatment regimens, e.g., it can be coadministered or administered before or after any anti-viral pharmaceutical or a non-attenuated anti-HIV vaccine.
  • the vaccine can be administered in any form of schedule regimen, e.g., in a single dose, or, using several doses (e.g., boosters) at dosages and time intervals to be determined by clinical trials.
  • the attenuated vaccine of the invention is considered efficacious, i.e., immunoprotective, if it elicits any humoral or cell-mediated anti-HIV response without causing clinically significant pathology, such as significant T cell cytotoxicity.
  • the anti- HIV response can be assessed, for example, by measuring the levels of anti-viral antibodies or HlV-specific T cells, the amount of HIV in the blood or lymph nodes (see, e.g. Brown (1997) Transfusion 37:926-929), and or levels of circulating helper (CD4 + ) T cells. See also, O'Brien (1997) "Changes in plasma HIV RNA levels and CD4 + lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure ' nn. Intern. Med.
  • the invention provides for cell-based and in vitro assay systems to screen for novel NCTE-binding proteins using the CTE ⁇ of the invention.
  • the full-length, two looped CTE ⁇ can be utilyzed, or, altematively, a portion of a CTEL ⁇ ., such as a single loop A or a single loop B, can be used to assay for RNA binding proteins.
  • One embodiment of the invention provides for a method of screening for an NCTE binding protein by contacting a CTE ⁇ of the invention with a test compound and measuring the ability of the test compound to bind the NCTE.
  • Many assays are available that screen for nucleic acid binding proteins and all can be adapted and used with the novel reagents provided for by the invention. A few illustrative example are set forth below.
  • One technique for isolating co-associating proteins includes use of UV crosslinking or chemical cross- linkers, including cleavable cross-linkers dithiobis(succinimidylpropionate) and 3,3'-dithiobis (sulfosuccinimidyl- propionate); see, e.g., McLaughlin (1996) Am. J. Hum. Genet. 59:561-569; Tang (1996) Biochemistry 35:8216-8225; Lingner (1996) Proc. Natl. Aca. Sci. U.S.A. 93:10712; Chodosh (1986) Mol. Cell. Biol 6:4723-4733.
  • CTE IAP -affinity columns can be generated to screen for potential CTE ⁇ -binding proteins.
  • the CTE IAP -containing nucleic acid is biotinylated, reacted with a solution suspected of containing a CTE L ⁇ p-binding protein, and then reacted with a strepavidin affinity column to isolate the CTE L ⁇ -containing nucleic acid/binding protein complex (see, e.g., Grabowski (1986 " ) Science 233:1294-1299; Chodosh (1986) supra). The protein can then be conventionally eluted and isolated.
  • Mobility shift DNA-protein binding assay using nondenaturing polyacrylamide gel electrophoresis is an extremely rapid and sensitive method for detecting specific polypeptide binding to DNA (see, e.g., Chodosh (1986) supra, Carthew (1985) Cell 43:439-448; Trejo (1997) J. Biol. Chem. 272:2741 1-27421 ; Bayliss (1997) Nucleic Acids Res. 25:3984-3990).
  • Interference assays and DNase and hydroxy radical footprinting can be used to identify specific residues in the nucleic acid protein-binding site, see, e.g.,
  • Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions, see, e.g., Lundblad (1996) o/. Endocrinol. 10:607-612..
  • Proteins identified in by these techniques can be further separated on the basis of their size, net surface charge, hydrophobicity and affinity for ligands.
  • antibodies raised against such proteins can be conjugated to column matrices and the proteins immunopurified. All of these general methods are well known in the art. See
  • Chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
  • Example 1 Identification, Isolation and Characterization of CTE p
  • RNA secondary stmcture of this NCTE element is a stem-loop stmcture with two internal loops, A and B.
  • Queries were designated to search for homology to the loop motifs A and B, which are separated by spacers to match the lengths of the interloop stem (15 to 35 nucleotides) and the hairpin loop: AANGACNGGT (2,1,1,) (SEQ ID NO:6); AACCTAAGACAGG (1,1,1) (SEQ ID NO:7); CNANGACNGG (2,1,1) (SEQ ID NO:8); and, AACCTAAGACAGG (1,1,1) (SEQ ID NO:9).
  • the numbers in parentheses indicate the number of allowed nucleotide changes, deletions, and insertions, respectively.
  • the searches were performed with the PatScan program (http://www.mcs.anl.gov/home/ papka/RO SS/patscan. html) .
  • This novel 176 bp sequence was analyzed by computer program (using MFold, University of Wisconsin Genetics Computer Group Package) for its ability to form secondary stmctures (MFold is an adaptation of the MFold package by Zuker and Jaeger, see Zuker (1989) Science 244:48-52; Jaeger (1989) Proc. Natl. Acad. Sci. USA 86:7706-7710).
  • MFold is an adaptation of the MFold package by Zuker and Jaeger, see Zuker (1989) Science 244:48-52; Jaeger (1989) Proc. Natl. Acad. Sci. USA 86:7706-7710
  • the analysis predicted that the 176 bp sequence forms an RNA secondary stmcture that shares several important features with the NCTE stmcture predicted for SRV-1 CTE. As shown in Figure 1, two internal loops, designated loop A and loop B, are conserved. The distance between the two loops is also conserved.
  • the region upstream (5') of the novel segment was sequenced from the NCTE-containing plasmid, designated U53820, as described above. A computer sequence identity search using this new sequence data was performed. The analysis revealed that this NCTE is part of a novel IAP retroelement region within the mouse ORG.
  • the novel CTE segment was named "CTE ⁇ " and the novel IAP termed "ORG, ⁇ .”
  • ORG, ⁇ the novel IAP is located in the transcribed ORG between its promoter and transcriptional start site and the ORG protein coding region (open reading frame).
  • ORG IAP is 3,402 nucleotides in length and is flanked by two imperfect direct repeats, see Figure 2.
  • the direct repeats have partial homology to the LTRs of the previously identified "Eker rat-associated IAP" (ERA-IAP) (Xiao (1995) Oncogene 11 :81- 87). Although they do not contain any intact open reading frames, ORG, ⁇ LTRs contain a predicted TATA box, a polyadenylation signal, and a section with sequence identity to putative gag/pol and env regions of other IAPs.
  • the novel CTE ⁇ of the invention is located between the putative env gene and the 3' LTR of ORG ⁇ . This is similar to the location of other NCTEs, as those seen in type D retrovimses.
  • Example 2 CTE P Replacing HIV-1 NCTE Creates an Attenuated Virus
  • this novel NCTE element was inserted into a Rev/RRE- deficient recombinant clone of HIV- 1, which is described in Zolotukhin (1994) supra, and is designated pR(-)Rev(-).
  • the HIV-1 molecular clone designated pNL4-3 was altered to lack functional Rev protein and Rev-responsive element (RRE), as described in Nasioulas (1994) J Virol. 68:2986-2993, and Zolotukhin (1994) supra, respectively.
  • This hybrid HIV-1 was termed "pR(-)Rev(-).IAP CTE.”
  • Stocks were prepared from: this hybrid; wild-type (wt) HIV-1 (the wt designated "NL4-3," Adachi (1986) J. Virol. 59:285-291); and, a R(-)Rev(-) hybrid into which was inserted an SRV-1 NCTE, termed "pR(-)Rev(-).S,” (Zolotukhin (1994) supra).
  • Human 293 cells were transfected by a calcium phosphate coprecipitation technique. Supematants were collected and filtered through a 0.2 um filter. The sequence of CTE L ⁇ , in these replicating recombinant hybrids was confirmed by conventional sequencing techniques.
  • PBMCs peripheral blood mononuclear cells
  • PHA phytohemagglutinin
  • Equal amounts of each of the vimses from the filtered 293 supematants were used to infect 5 x 10 activated PBMCs and 3 x 10 Jurkat cells. Vims propagation was monitored over time using a p24 ⁇ antigen capture assay (using either a commercial assay, Cellular Products (Buffalo, NY) or an in-house p24 g ⁇ g antigen capture assay using standard techniques, as described herein.
  • CTE j ⁇ 's replicative potential was further tested utilizing a syncytium formation assay.
  • Human 293 cells were transfected with SRV-1 (with wild type CTE), a CTE negative SRV-1, and an SRV-1 clone which had had its CTE deleted and replaced with the CTE j ⁇ of the invention.
  • Transfectants were co-cultivated with the human Raji B cell line. The cultures were monitored for syncytium formation.
  • SRV-1 wild type CTE and SRV-1 /CTE negative clones reconstituted with the CTE ⁇ of the invention produced syncytium.
  • CTE negative clones produced no syncytium.
  • hybrid HIV-1 using CTE IAP rather than the wildtype RRE or SRV-1 CTE as the NCTE element has less replicative potential (i.e, is more attenuated), as shown by measuring levels of p24.
  • these experiments demonstrate that the CTE ⁇ of the invention produces a slower-growing, attenuated HIV-1 hybrid.
  • Example 3 CTE-Containing HIV-1 are Attenuated In Vivo
  • NCTEs such as the SRV-1 CTE.
  • Hybrid HIV-1 clones in which the NCTE from SRV-1 ("CTE") was inserted to replace the wild type HIV-1 NCTE (RRE) were used in the SCID-hu mouse model to demonstrate the replication and cytopathic effect of CTE(+) attenuated HIV-1.
  • CTE(+) hybrids were used to infect a Thy/Liv implant (Kollmann (1995) supra) in
  • CTE-attenuated vimses propagate slower than wild-type and Nef-negative (otherwise NCTE wild-type) HIV-1 clones.
  • CTE-attenuated HIV-1 replicate about 10 times less efficiently than the SRV-1 NCTE-containing HIV-1.
  • Levels of circulating CD4 + lymphocytes were monitored for 6 weeks after initial infection. No depletion of CD4 + cells was observed. This demonstrates an attenuated phenotype for cytotoxicity of the CTE(+)/RRE(-)-containing HIV-1 clones.
  • Rev and Nef were compared.
  • the recombinant HIV-1 clone into which the SRV-1 CTE had been inserted termed "pR(-)Rev(-).S,” as described in Example 2, was used.
  • This hybrid is a RRE(-)Rev(-)Nef(-) molecular clone of HIV- 1 that contains the SRV-1 NCTE inserted within the ne/gene.
  • Rev and RRE were destroyed by multiple point mutations designed not to affect the overlapping tat and env open reading frames.
  • proximal portion of nef spanning nucleotides 8782 to 8954 was PCR-amplified from HIV-1 NL4-3 using primers gcatcactcgagATAAGATGGGTGGCAAGTGG (sense) (SEQ ID NO: 10) and
  • GGCACAAGCAGCATTGTTAG antisense (SEQ ID NO:l 1).
  • the regions of complementarity are shown in uppercase, while the lowercase sequence contains Xhol site that was used to insert the PCR fragment in the Xhol site of R(-)Rev(-).S. This resulted in pR(-)Rev(-)Nef(+)CTE that contains an intact ne/gene downstream of the SRV-1 CTE. To assure nef expression, all AUG codons within the remaining ne/region upstream of
  • CTE were converted to CUG by PCR mutagenesis.
  • Two AUG codons within the 241 base pair SRV-1 CTE fragment that do not belong to the known minimal 173 base pair CTE were also mutated to CUG.
  • the Hpal-Xbal fragment was PCR-amplified from pR(-)Rev(-)Nef(+)CTE as two overlapping fragments, using primer pairs ATAGTGCTGTTAACTTGCTCAATGCCACAGCC (SEQ ID NO: 12) and
  • the mutated nucleotides are shown in lowercase, and a flanking region containing the Xbal restriction site is italicized.
  • Wild type HIV-1 pNL4-3) and the CTE(+) clones pR(-)Rev(-)Nef(-)CTE (also called pR(-)Rev(-).S), and, pR(-)Rev(-)Nef(+)CTE (i.e., pR(-)Rev(-).S with the Nef reinserted)) were transfected into human 293 cells by calcium phosphate coprecipitation.
  • HIV-1 their infectivity in PHA-activated human PBMCs was determined by standard end-point dilutions (see McDougal (1985) J Immunol. Methods 76:171-183). Briefly, viral aliquots were diluted in medium using 5 -fold dilution steps starting with a 1:10 dilution. 100 microliters of Jurkat cell suspension was seeded in 96-well plates at 7.5x10 cells per well. 100 microliters of vims dilution was added to 12 parallel wells. The culture medium was changed twice a week for the next two weeks and on day 14 a 120 microliter sample from each well was analyzed using the p24 ⁇ g antigen ELISA assay described above.
  • the ID 50 values were defined as the reciprocal of the vims dilution resulting in 50% positive wells using a Reed-Munch calculation (see Albert (1990) AIDS 4:107-112; McDougal (1985) J. Immunol. Methods 76:171-183).
  • the ID 50 values for the CTE(+) clones were about 5-fold lower as compared to wild-type HIV-1. No significant difference in the infectivity was observed for the Nef(-) and Nef(+) variants of the CTE(+) viruses, indicating that RRE replacement, rather than Nef, is responsible for this slow-growing phenotype.
  • propagation of the Nef(-) and the Nef(+) HIV-1 clones in human primary cells was determined.
  • Human PBMCs were infected by equal infectious units of the different vimses, using the viral stocks described above. Vims propagation was monitored over time by measuring extracellular Gag production (as described above). Wild-type HIV-1 (NL4-3) and NL4-3/Nef-negative clones had similar growth properties in activated PBMCS': The Nef(-) vims propagated approximately 5- 10-fold less efficiently, whereas the replicative capacity of its Nef(+) counterpart was further decreased by about
  • the attenuation of the CTE-containing HIV-1 was further demonstrated by measuring its lower replicative capacity in vivo in an art-recognized animal model, the SCID-hu mouse (see Aldrovandi (1993) Nature 363:732-736; Bonyhaki (1993) Nature 363:728-732).
  • the SCID-hu mice are produced by surgical implantation of human fetal liver and thymus under the kidney capsule of severe combined immunodeficient (SCID) mice. Normal T-cell differentiation has been shown to occur in the Thy/Liv implant. This is an art-recognized system for the study of HIV infection and cytotoxicity in human lymphopoietic tissue. Normally, infection by wt HIV-1 results in depletion of CD4 + T cells. Significantly, infection with CTE-containing HIV-1, resulting in low levels of viral replication, did not cause CD4 + cell depletion in vivo.
  • Vims stocks of wt HIV-1 (NL4-3), NL4-3/Nef negative, CTE(+) Nef(-), and CTE(+) Nef(+) HIV clones were tested. 1000 infectious units/mouse are typically used to establish good SCID-hu infection (i.e., at least 50% to 90% infected) by attenuated strains of HIV- 1 , see Aldrovandi ( 1996) J. Virol.
  • mice were infected by injection of vims into the Thy/Liv implant at 500 to 850 TCID 50 in a final volume of 100 ⁇ l. Sequential biopsies of the implants were performed 3 and 6 weeks postinfection, and the samples were analyzed for vims replication by quantitative DNA-PCR and for the number of CD4 + thymocytes by flow cytometry (PCR and cytometry as described in Aldrovandi (1996) supra). All 7 mice infected by wt HIV-1 scored positive for HIV proviral sequences at 3 weeks postinfection.
  • CD4 + thymocytes defined as measurement of less than 55% CD4 + cells in the thymocyte population
  • CD4 + thymocyte depletion None of the mock-infected mice showed any sign of HIV infection or CD4 + thymocyte depletion.
  • Replication of the NL4-3/Nef-negative was detectable in 2 of 10 mice after 3 weeks and in 8 of 9 mice at 6 weeks post-infection.
  • CD4 + thymocytes were found in 3 of 9 mice infected with NL4-3 /Nef-negative recombinants at 6 weeks postinfection (which is typical, as reported in Aldrovandi (1996) supra, and Jamieson (1994) J. Virol. 68:3478-3485).
  • Biopsies were analyzed at 3 and 6 weeks postinfection. At 3 weeks postinfection, no vims could be detected in the 6 mice infected with the either of the CTE(+) vimses. At 6 weeks postinfection, in all 4 mice infected by the CTE(+)/Nef(-) hybrid and in all 7 mice infected by the CTE(+)/Nef(+) hybrid, HIV-1 replication was detectable. Although the time necessary to detect the CTE(+) hybrid was still 6 weeks, the viral loads were clearly elevated, due to the higher amount of input vimses.
  • the average proviral copies per 10 5 cells was 15,000 in the case of CTE(+)/ Nef(-) hybrid, which is similar to that obtained from mice infected with 5-fold lower amount of RRE+/ Nef-negative clone.
  • the vims load in the implants infected by the CTE(+)/Nef(+) variant was at least ten-fold lower than that of the CTE(+)/Nef(-) counterpart.
  • no significant changes were observed in the thymocyte profiles (within the time frame of the experiment, 6 weeks). Therefore, no depletion of CD4 + thymocytes by CTE(+) hybrids was observed.
  • Thy/Liv implant in SCID-hu mice they propagate slower than wt HIV-1 and NL4-3/Nef- negative hybrids. No depletion of CD4 + cells was observed in the CTE(+) hybrids, demonstrating an attenuated phenotype for cytotoxicity. Direct comparison to RRE+/Nef- negative HIV-1 showed that the CTE+ is responsible for attenuation independent of the absence or presence of Nef. Thus, these experiments demonstrate that NCTEs, such as the CTE ⁇ of the invention, can produce a slow-growing, attenuated HIV-1 hybrid in vivo.

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Abstract

The invention provides for a novel post-transcriptional RNA nucleocytoplasmic transport element (NCTE), designated 'CTEIAP'' derived from a novel intracisternal A particle (IAP). The novel IAP was found inserted in a murine osteocalcinrelated gene (ORG) between its promoter and ORG's coding region, and is thus termed 'IAPORG''. The invention also provides for an attenuated HIV-1 hybrid virus for use as a vaccine and a kit incorporating the hybrid virus. The kit also includes instructional material teaching the use of the vaccine, where the instructional material indicates that the vaccine is used for the prophylaxis or amelioration of HIV-1 infection in a mammal; that the vaccine is to be administered to a mammal in a therapeutically effective amount sufficient to express a viral protein; where the vaccine will not cause clinically significant CD4+ cell depletion; and, the expression of the viral protein elicits an immune response to the attenuated HIV-1 virus. The invention further provides for a method for screening for a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) binding protein.

Description

MURINE INTRACISTERNAL A PARTICLE CONSTITUTIVE
TRANSPORT ELEMENTS AND USES THEREOF
FIELD OF THE INVENTION
This invention pertains to the field of virology and vaccine development. In particular, this invention pertains to the discovery that a novel post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) from a novel murine intracisternal-A particle (IAP) can be used to construct an attenuated retrovirus.
BACKGROUND OF THE INVENTION Intracistemal A-type particles (IAPs) are defective endogenous retroviruses found in a variety of mammals. These "particles" were first discovered because some murine IAPs with reverse transcriptase activity and polyadenylated RNA accumulate in the endoplasmic reticulum. Wilson (1974) Biochemistry 13:1087-1094; Leuders (1977) Cell 12:963-972. IAP retroviral genomes share extensive sequence homologies with D-type retroviruses. IAP genes in variable copy number, up to 2000 in a genome, exist in all rodent species studied and are dispersed throughout the genome. Because of this high copy number, IAP elements have been used as genetic markers. Kaushik (1994) Mamm. Genome 5:688-695.
Murine IAPs are constitutively expressed in high levels in many mouse tumors and at basal levels in most normal adult mouse tissues. Kuff (1990) Cancer Cells 2:398-400. While they are not infectious retroviruses, IAPs can be transcriptionally active. Expression of some murine IAPs results in translation products corresponding to retroviral gag and Pol polyproteins. Fehrmann (1997) Virology 235:352-359. Under certain circumstances, IAPs can transpose to different positions in the genome, in some cases acting as insertional mutagens. When these transpositions affect the expression of a gene that can either positively or negatively regulate cell growth, a malignant or transformed cell or tumor may arise. IAP transpositions have been frequently observed in hematopoietic cells, often affecting cytokine gene expression. Wang (1997) Cytokines Cell. Mol. Ther. 3:3-19.
Genetic elements that possess IAP transposable characteristics are also contained in the human genome. Human IAP-like retroviral particles have been detected in a lymphoblastoid cell line exposed to mononuclear cells from a patient with severe
CD4+ T-cell deficiency who was negative for known human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV) infection. Ultrastructurally, the human IAP particle was distinct from HIV and HTLV. Supernatants of activated mononuclear cells showed significant reverse transcriptase activity. While the patient's serum was negative for antibodies to known HIVs and HTLVs, his serum was positive for antibodies against the human IAP. Similar IAP particles were detected in a lymphoblastoid cell line exposed to mononuclear cells from the patient's daughter, who also showed CD4+ T-cell dysfunction. These results suggest that human IAP is associated with CD4+ T-cell immunodeficiency and dysfunction (Gupta (1992) Proc. Natl. Acad. Sci. USA 89:7831-7835).
Some retroviruses, including HIV type 1 (HIV-1), simian retrovirus type 1 (SRV-1), SRV-2, and Mason-Pfizer monkey virus (MPMV) (the later three are type D simian retroviruses), regulate their expression by controlling the nuclear transport of unspliced mRNA encoding structural proteins. They do this using cts-acting post- transcriptional RNA nucleo-cytoplasmic transport elements ("NCTEs," designated "CTEs" for some type D retroviruses). NCTEs fold into highly structured secondary structures, typically stem-loop structures. Trans-acύng proteins bind to the NCTE loop structures to regulate RNA expression at several levels. Interaction of the NCTE with a trαλw-acting RNA binding protein stabilizes certain unspliced viral transcripts. This interaction is necessary for the message's transport from the nucleus to the cytoplasm and is necessary for expression of the transcript. Some retroviruses, such as Simian type D retroviruses, including SRV-1, do not encode their own tr< s-acting, NCTE-binding proteins and instead utilize cellular NCTE binding proteins. Other retroviruses, such as HIV-1, utilize a retrovirally-encoded NCTE RNA binding protein, called "Rev." HIV-1 regulates the expression of its structural proteins encoded by the g g/pol- and ewv-encoding transcript using this NCTE system. HIV-l's NCTE binding protein "Rev" interacts with a specific NCTE sequence, designated the "Rev-responsive element," or "RRE," contained in its gag/pol and env encoding transcript. HIV-l's RRE does not bind cellular NCTE-binding proteins. Rev interacts directly with RRE as part of the RNA export machinery which transports REE-containing transcripts to the cytoplasm from the nucleus. As a result, Rev and RRE are needed to produce infectious virus.
Simian retroviruses contain an NCTE, or "CTE," element located between their env gene and 3' long terminal repeat (LTR). HIV-1 lacking a functional Rev RRE control system is uninfectious, but can be reconstituted with simian retroviral CTE {e.g., CTE from SRV-1, SRV-2, MPMV) to produce transcripts and infectious virions (Bray (1994) Proc. Natl. Acad. Sci. USA 91 :1256-1260; Tabernero (1996) J. Virol. 70:5998-
6011; Zolotukhin (1994) J. Virol. 68:7944-7952). However, reconstitution of RRE/Rev with CTE produces hybrids with lower levels of transcript and productive virion than seen with wild-type HIV-1.
It has been found that some individuals are infected with the HIV-1 virus but do not go on to manifest the symptoms of acquired immune deficiency syndrome
(AIDS). Rev gene mutations that caused viral growth attenuation were found in some of these asymptomatic HIV-1 infected individuals. Thus, it was hypothesized that the resultant production of relatively low levels of virus contributed to their lack of disease progression, an asymptomatic state, and stable CD4+ levels (Iversen (1995) J. of Virology 69:5743-5753; Hua (1996) Virology 222:423-429.
Thus, disruption of either the cis- (e.g., CTE, RRE) or trans-acting (e.g., Rev protein) elements of the RNA post-transcriptional nucleo-cytoplasmic transport regulatory system in retroviruses produces an attenuated virus, decreasing their replicative ability and pathogenicity in vivo. Such attenuated infection, results in an asymptomatic state in the infected individual. Thus, there exists a need for cis- and transacting post- transcriptional RNA regulatory elements capable of attenuating a retrovirus, such as HIV- 1. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION The invention provides for an isolated nucleic acid consisting of a
Constitutive Transport Element derived from an IAP nucleotide sequence, called CTE^. The isolated CTE1AP nucleic acid is defined as having the following properties: (i) the CTEIAP, when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a NCTE in place of wild-type NCTE in the hybrid HIV-1, and when the CTEIAP-containing hybrid HIV-1 virus infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24gag is between about 50 fold and about
200 fold less than levels of p24gαg expression when HIV-1 wild type virus, utilizing wild- type NCTE, infects activated huPBMCs; and, (ii) the secondary structure of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid comprising the loop structure as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
In further embodiments, the invention provides for an isolated nucleic acid CTE1AP comprising a ribonucleic acid comprising a motif of contiguous base pairs consisting of XA 10.500 - GUC AAUGAC GGGU AAGA - XB 16.24 - ACCU AAGACA GG - Xc 6.100- CAA U GUU - XD 6.100- CC GAGGAC AGGU - XE 16.24 - CA ACCU AAGACA GGCA -XFιo-5oo> wherein X is any nucleotide base; and, an isolated nucleic acid CTEIAP comprising a deoxyribonucleic acid comprising a motif of contiguous base pairs consisting of XA 10.500- GTC AATGAC GGGT AAGA - XB 16-24 - ACCT AAGACA GG - Xc 6.100- CAA T GTTO - XD 6.100 - CC GAGGAC AGGT - XE 16.24 - CA ACCT AAGACA GGCA -XF 10.500 .wherein X is any nucleotide base. In other embodiments, the isolated nucleic acid CTEIAP comprises the sequence as set forth in SEQ ID NO:l; and, in the isolated nucleic acid CTEIAP the distance between the loop A domain and the loop B domain is between about 21 to about 26 base pairs in length. The invention also provides for an isolated transcription product of a CTEIAP nucleic acid, the CTEIAP nucleic acid as defined above.
The invention also provides for an expression vector comprising a nucleic acid encoding a CTEIAP and a non-naturally occurring nucleic acid sequence, the
Figure imgf000006_0001
nucleic acid as defined above, and, a transfected cell comprising a polynucleotide encoding a CTELJJ. and a non-naturally occurring nucleic acid sequence, the CTEIAP nucleic acid as defined above. In another embodiment, the invention provides for a recombinant retrovirus either lacking or having a non-functional endogenous NCTE and further comprising a CTE1AP operatively inserted into the retrovirus and capable of acting as a functional exogenous NCTE to reconstitute the lacking or non-functional endogeous NCTE and to reconstitute the infectivity of the retrovirus in a mammalian cell, the CTEjAP as defined above. In alternative embodiments, the recombinant virus can be HIV-1, the NCTE can be RRE, and the HIV-1 can further lack a functional Nef.
The invention further provides for a vaccine for the prophylaxis or amelioration of a viral infection in a mammal comprising an attenuated retrovirus, where the retrovirus, when administered as a vaccine in sufficient amounts, is capable of eliciting an immune response to the retrovirus in a mammal with a functional immune system; and the attenuated retrovirus lacks an endogenous functional NCTE and/or the ability to express an endogenous functional NCTE binding protein, and the attenuated retrovirus further comprises a CTEIAP nucleic acid as defined above. In alternative embodiments, the attenuated retrovirus can HIV- 1 , the attenuated HIV- 1 can also lack a functional Nef, the
NCTE can be RRE, and the NCTE binding protein can be Rev.
Another embodiment provides for a kit for the prophylaxis or amelioration of a virus infection in a mammal, the kit comprising the above described vaccine and a pharmacologically acceptable carrier. The kit can further comprise an instructional material teaching the use of the vaccine, where the instructional material indicates that the vaccine is used for the prophylaxis or amelioration of HIV- 1 infection in a mammal; that the vaccine is to be administered to a mammal in a therapeutically effective amount sufficient to express a viral protein; wherein the vaccine will not cause clinically significant CD4+ cell depletion; and, the expression of the viral protein elicits an immune response to the attenuated HIV-1 virus.
The invention also provides for use of a CTE^ in the manufacture of a medicament for the prophylaxis or amelioration of a viral infection, such as an HIV-1 infection, where the CTEIAP is as described above.
In one embodiment, the invention provides for a method for eliciting an immune response to a virus in a mammal, comprising administering to a mammal a therapeutically effective amount of an attenuated recombinant virus, wherein the virus comprises the CTEIAP as defined above.
In another embodiment, the invention provides for a method for screening for a NCTE binding protein comprising the following steps: a) providing a composition comprising a loop A and/or a loop B of a CTEIA as defined above; b) contacting the composition with a test compound; and, c) measuring the ability of the test compound to bind the NCTE.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates two RNA sequences: the novel CTELAJ. of the invention (SEQ ID NO:l, Figure 1A) and SRV-1 CTE (SEQ ID NO:2, Figure IB). In Figures 1 A and IB the two sequences are shown folded in a secondary structure whose configuration is based on intramolecular base-pairing. This secondary structure includes two loops, A and B, a four nucleotide AAGA "bulge" adjacent to loop A, and a hairpin turn. Loops A
(with "bulge") and B are circled.
Figure 2 illustrates how the novel IAP, including the CTEL^ of the invention, is inserted into the transcribed osteocalcin related gene (ORG) between its promoter (designated "P") and the ORG coding region, thus displacing ORG's functional promoter by 3.4 kilobases. The Figure shows that the murine osteocalcin gene consists of osteocalcin 1 ("mOGl"), osteocalcin 2 ("mOG2"), and ORG. Exons are designated as Roman numerals; exons lb and Ic are non-coding exons. The sequence of the ORG promoter is also shown, with the identified start of transcription 47 nucleotides downstream of the ORG TATA box in the 5' LTR (long terminal repeat) of the ORG^. The location of CTE^ of the invention within the ORG gene, shown as "CTE," and splice sites are indicated (see Figure 4, where the DNA subsequence encoding CTE1AP is between nucleotides 3029-3204 of SEQ ID NO:3).
Figure 3 illustrates data demonstrating that the novel CTE1AP is able to replace Rev-mediated activation of HIV- 1 in both Jurkat and PBMCs, as shown in Figure 3A and 3B, respectively; and explained in detail in below.
Figure 4 illustrates the nucleic sequence of the novel intracistemal A particle of the invention, ORG^, which is 3,402 base pairs in length, and flanking imperfect direct repeats (SEQ ID NO:3). The DNA subsequence encoding the CTE^ of the invention is located between nucleotides 3029-3204, as numerated in this figure.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for a novel post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE), designated "CrE^," derived from a novel intracistemal A particle (IAP). The novel IAP, including the CTE^ of the invention, was found inserted in a murine osteocalcin-related gene (ORG) between its promoter and coding region, and is thus termed "IAPORG."
It was surprisingly found that the novel CTE^^, of the invention can be used functionally replace the NCTE of HIV- 1, or "RRE." It was discovered that when CTE^ is used in place of RRE to construct an HIV-1 hybrid clone, a slower growing, attenuated virus results. Significantly, while capable of producing infectious virions in vivo, this HIV-1 hybrid (a CTEj^ -containing RRE-negative recombinant virus) has lower replicative activity than wild-type vims, resulting in an attenuated HIV-1 strain. The level of attenuation can be quantitated using in vitro or in vivo assays, such as determining the amount of HIV- 1 p24 αg synthesized by hybrid viruses. For example, the CTEIAP of the invention, when inserted in an RRE/Rev deficient HIV-1 , is capable of functionally reconstituting this post-transcriptional RNA nucleo-cytoplasmic transport system. When the CTE^-containing hybrid HIV-1 vims infects activated huPBMCs, the level of expression of HIV- 1 p24gα is between about 50 fold and about 200 fold less than levels of p24gag expression when HIV-1 wild type vims, utilizing wild-type NCTE (i.e., RRE), infects activated huPBMCs. The efficacy of the CTE1AP-recombinant hybrid as an attenuating agent in HIV-1 infection and AIDS pathogenesis can be demonstrated using functionally analogous NCTEs, such as the SRV-1 CTE. Recombinant, hybrid HIV-1 clones in which NCTE from SRV-1 (termed "CTE") functionally replaced wild-type HIV-1 NCTE ("RRE") were used to demonstrate the replicative, yet non-cytopathic, effect of CTE-attenuated HIV- 1 in the SCID-hu mouse model, as discussed in Example 3. CTE(+)/ RRE(-) HIV-1 clones were used to infect Thy/Liv implants (implanted human thymus and liver, see Kollmann (1995) J. Immunol. 154:907-921) in SCID-hu mice. Significantly, these viruses propagated slower than both wild-type and Nef-negative HIV-1 clones, demonstrating that they have lower replicative capacity in human lymphocytes. Furthermore, the
CTE(+) RRE(-) attenuated HIV-1 clones were not lymphocytopathic, no depletion of CD4+-bearing cells was observed. This demonstrates that HIV-1 hybrid clones utilizing exogenous NCTEs, as CTE (of SRV) or CTE^ have an attenuated phenotype for cytotoxicity. Analogously, when the CTE^ -attenuated HIV-1 of the invention infects activated human lymphocytes in vivo, it will also produce low levels of infectious virions without any lymphocytotoxic effects, i.e., levels of CD4+ T cells will not decline. Significantly, the invention's CTEIAP -containing HIV-1 is significantly more attenuated than SRV-1 CTE-attenuated virus, as indicated by that fact that the level of expression of p24gαg by CTEL^. -containing HIV-1 is between about 50 fold less than wild type vims, while SRV-1 NCTE produces a hybrid vims only about 10 fold less than wild type.
Importantly, this CTE^ -attenuated vims will elicit an immune response in the infected, yet asymptomatic, individual.
The finding that an IAP element can be utilized in the attenuation of a retrovirus whose productive infection does not lead to loss of CD4+ cells is especially unexpected in view of past findings that a human IAP has been found to be associated with CD4+ T-cell immunodeficiency and dysfunction. The presence of IAP sequence has also been associated with the occurrence of carcinogenesis, as discussed above.
This invention also provides for a vaccine in the form of a pharmacological compositions and a kit. The pharmacological compositions can comprise a pharmaceutically acceptable carrier and the attenuated vims of the invention. The kit can comprise a container containing a vaccine formulation. DEFINITIONS
The term "CTEIAP" refers to a family, or genus of nucleic acid sequences defined as having the following stmctural and functional properties: the CTEIAP, when inserted in an HIV-1 is capable of functioning as an NCTE in place of wild-type HIV-1 NCTE (i.e., RRE), and when the CTEIAP-containing hybrid HIV-1 vims infects activated huPBMCs, the level of expression of HIV- 1 p24gαg is between about 50 fold to about 200 fold less than levels of p24gαg expression when HIV-1 wild type vims, utilizing wild-type NCTE, infects activated huPBMCs; and, the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1. The nucleic acid of SEQ ID NO:l is an exemplary specie of the CTE1AP family of the invention.
The term "intracistemal A particle" or "IAP" refers to a family of defective endogenous retroviruses found in a variety of mammals. Murine IAPs can have reverse transcriptase activity and can accumulate in the endoplasmic reticulum. IAPs are further described by, e.g., Wilson (1974) supra; Leuders (1977) supra; Kaushik (1994) supra; Kuff (1990) supra; Fehrmann (1997) supra; and, Wang (1997) supra.
The term "IAP0RG" refers to the novel intracistemal A particle (IAP) of the invention, including the TE ^ of the invention, which was found inserted in a murine osteocalcin-related gene (ORG) between its promoter and coding region (thus termed
I PORG )•
The term "constitutive transport element" or "RNA nucleocytoplasmic transport element" or "NCTE" refers to cw-acting, post-transcriptional RNA nucleo- cytoplasmic transport elements. Simian retrovims NCTE is also referred to as "CTE."
These RNA sequences typically have a high degree of secondary stmcture in the form of stem-loop structures, which can interact with tn s-acting NCTE-binding proteins, hairpin rums, and the like. Some retrovimses, including HIV and simian retrovimses, regulate their growth through expression of their RNA using NCTEs and corresponding NCTE- binding proteins. NCTE binding with viral or cellular tram-acting NCTE-binding proteins stabilizes unspliced viral transcripts and allows interaction with cellular machinery to transport the message from the nucleus to the cytoplasm. HIV-l's NCTE is designated the "Rev-responsive element," or "RRE," contained in its gag/pol and env encoding transcript. NCTEs are further described, e.g., by Bray (1994) supra; Tabernero (1996) supra; Zolotukhin (1994) supra; Hua (1996) supra; Grate (1997) Structure 5:7-11. The term "NCTE-binding protein" refers to a trαns-acting polypeptide which binds to RNA NCTE sequences, typically interacting with specific secondary stmctures. This RNA binding protein functions with other cellular proteins in the cellular nuclear export machinery to constitutively transport message nucleic acid (mRNA) from the nucleus to the cytoplasm. An NCTE-binding protein can be encoded by endogenous cellular (eukaryotic) gene or a viral gene. HIV-1 encodes for its own specific, trαrø-acting NCTE-binding polypeptide, termed "Rev." Rev is further described, e.g., Hua (1996) supra; Iversen (1995) supra.
The term "HIV" refers to a lentivims usually called "human immunodeficiency vims" which is believed to the causal agent of acquired immune deficiency syndrome, or AIDS. There are several known subtypes of HIV, including HIV-
1 and HIV-2. HIV and AIDS are well described in the literature and, e.g., are further described by Gottfredsson (1997) Front Biosci. 2: D619-D634; Burton (1997) Proc. Natl. Acad. Sci. USA 94:10018-10023; Barnadas (1997) J. Cutan. Pathol. 24:507-510; Doms (1997) Virology 235:179-190; Cossarizza (1997) AIDS 11:1075-1088; Carpenter (1997) JAMA 277:1962-1969; Klein (1995) Trends Microbiol. 3:386-391.
The term "P24gαg antigen" or "p24 α " refers to the 24 kd HIV-1 polypeptide found associated with the vims' RNA genome in the core of the virion, as described, e.g., by Jones (1996) Nat. Struct. Biol. 3:818-820 (1996); Doe (1996) AIDS 10:793-794; Klenerman (1996) AIDS 10:348-350; Klenerman (1994) Nature 369:403-407. The term "Nef refers to a 27 - 34 kD myristoylated protein unique to primate lentivimses. A functional Nef gene is important for development of high viremia and AIDS. For a description of Nef, see discussion infra and, e.g., Saksela (1997) Front Biosci. 2: D606-D618; Greenberg (1997) EMBO J. 16:6964-6976; Luo (1997) J. Virol. 71:9531-9537; Luo (1997) J. Virol. 71 :9524-9530; Okada (1997) FERSZett. 417:61-64. The term "capable of functioning as a post-transcriptional RNA nucleo- cytoplasmic transport element (NCTΕ) in place of wild-type NCTΕ in a hybrid HIV-1 " means that the NCTE, such as the claimed CTE1AP, when inserted into (is part of the sequence of) an HIV-1 messenger RNA, is capable of interacting with the appropriate transacting polypeptides to effect the splicing and subsequent transfer of the mRNA from the nucleus to the cytoplasm. An NCTE is still considered "capable of functioning as an NCTE in place of wild-type NCTE in a hybrid HIV-1 " even if it is less efficient, less accurate, or less capable, of splicing and/or transferring mRNA (of which it is a part) from the nucleus to the cytoplasm. For example, the CTEIAP of the invention, when inserted in HIV-1, is considered capable of functioning as an NCTE in place of wild-type NCTE (RRE) in the hybrid HIV-1 even though it is less efficient than RRE; i.e., when the CTEIAP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24 α is between about 50 fold and about 200 fold less than levels of p24gαg expression when HIV-1 wild type vims, utilizing wild-type NCTE (RRE), infects activated huPBMCs.
The term "activated" refers to a non-dormant cellular state, for example, as when a lymphocyte has been activated by an antigen, cytokine(s) or other mitogen.
The term "wild-type" refers to any form (e.g., tertiary stmcture), stmcture (e.g., secondary stmcture) or sequence (e.g., primary stmcture) of a composition, as a nucleic acid or polypeptide, as found in nature, versus stmctures or sequences which have been manipulated by the hand of man, i.e., recombinant nucleic acids or polypeptides. The term "secondary stmcture" refers to the stmcture formed by the intramolecular folding of a polypeptide or a nucleic acid, such as the stem and loop stmctures formed by the CTEL^ of the invention.
The term "peripheral blood mononuclear cell" refers to any peripheral mononuclear white blood cell. The term "expression vector" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression "cassettes" which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
The term "isolated," when referring to a molecule or composition, such as, for example, a polypeptide or nucleic acid, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g.,
RNAs), or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, a polypeptide or nucleic acid is considered isolated when it has been isolated from any other component with which it is naturally associated, e.g., cell membrane, as in a cell extract. An isolated composition can, however, also be substantially pure. An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).
The term "polynucleotide," "nucleic acid molecule" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleo tides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired. The term also encompasses nucleic-acid-like stmctures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs, which contain non-ionic backbones, such as N-(2- aminoethyl) glycine units); see Oligonucleotides and Analogues, A Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press) in its entirety and specifically Chapter 15, by Sanghvi. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197. Other synthetic backbones encompasses by the term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev. 6:153-156). The term nucleic acid is used interchangeably with gene, DNA, cDNA, RNA, mRNA, oligonucleotide primer, probe and amplification product.
The term "exogenous" as in "exogenous nucleic acid" refers to a molecule (e.g., nucleic acid or polypeptide) that has been isolated, synthesized, and/or cloned, in a manner that is not found in nature, and/or introduced into and/or expressed in a cell or cellular environment other than or at levels or forms different than the cell or cellular environment in which said nucleic acid or protein can be found in nature. The term encompasses both nucleic acids originally obtained from a different organism or cell type than the cell type in which it is expressed, and also nucleic acids that are obtained from the same organism, cell, or cell line as the cell or organism in which it is expressed. The term "endogenous" refers to a molecule, e.g., a nucleic acid or polypeptide, in a form, stmcture and/or sequence found in nature.
"Sequence identity" in the context of two nucleic acid or polypeptide sequences includes reference to the nucleotides (or residues) in the two sequences which are the same when aligned for maximum correspondence over a specified "comparison window." Sequence identity analysis is used to determine whether a nucleic acid is within scope of the invention. For example, to identify a specie of the CTE^ family of the invention, a nucleic acid must have a secondary stmcture with at least a two loop A domain and/or a loop B domain, where the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid as set forth in Figure 1 , SEQ ID NO: 1. "Sequence identity" can be analyzed by optimal alignment of sequences for comparison using any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed "PILEUP"; by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms, e.g. BLAST, GAP, BESTFIT, FASTA, and TFASTA in, e.g., the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI; or, by inspection. See also Morrison (1997) Mol. Biol. Evol. 14:428-441, as an example of the use of PileUp, ClustalW, TreeAlign, MALIGN, and SAM sequence alignment computer programs. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J.
Mol. Evol. 35:351-360 (1987) and is similar to the method described by Higgins & Sharp CABIOS 5:151-153 (1989). The BLAST algorithm is described in Altschul (1990) J. Mol. Biol. 215: 403-410, and software for performing BLAST analyses is publicly available, e.g., see National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/. See also Corpet (1988) Nucleic Acids Res. 16:10881-90; Huang (1992) Computer
Applications in the Biosciences 8:155-65; and Pearson (1994) Methods in Molec. Biol. 24:307-31.
The term "recombinant," when used with reference to, e.g., a cell, nucleic acid, polypeptide, expression cassette or vector, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell (i.e., "exogenous nucleic acids") or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all. The term "recombinant means" refers to techniques where, e.g., a recombinant nucleic acid such as a cDNA encoding a protein or an antisense sequence, is inserted into an expression cassette, such as an expression vector, the resultant constmct is introduced into a cell, and the cell expresses the nucleic acid, and the protein, if appropriate. "Recombinant means" also encompass the ligation of nucleic acids to coding or promoter sequences from different sources into one expression cassette or vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein.
The term "test compound" refers to any synthetic or natural compound or composition. The term includes all organic and inorganic compounds; including, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids and the like. The term "motif or "domain" refers to a nucleic acid or amino acid sequence pattern, or stmcture, which is shared between related molecules.
The term "ameliorating" or "ameliorate" refers to any indicia of success in the treatment of a'pathology or condition, including any objective or subjective parameter such as abatement, remission or diminishing of symptoms or an improvement in a patient's physical or mental well-being. Amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination and/or a psychiatric evaluation.
The term "prophylaxis" refers to any form of prevention, delay or abatement a pathology or condition or symptom thereof, including any objective or subjective parameter.
The term "attenuated" refers to a state wherein an infectious agent, i.e., a pathogen, such as a microbial or viral agent, has a phenotype manifested by a lessened ability to grow, proliferate, or cause pathogenesis, in a host, i.e., the non-wild type, attenuated phenotype is less vimlent. For example, a CTEL^-attenuated HIN-1 vims is capable of replication, infection and production of infectious virions without causing clinically significant pathology in its host.
The term "immune response" in a host refers to both cellular and humoral (antibody) mediated responses to an immunogen, i.e., a compound or composition capable of eliciting an immune response. The immune response can be elicited by a foreign substance or a pathogen, and the immunogen can be a carbohydrate, a nucleic acid, a polypeptide, a lipid, or a combination of these elements.
The term "vaccine" is used in its ordinary sense, meaning an agent which is capable of eliciting a humoral and/or cell-mediated immunoprotective immune response when administered to an individual with an at least partially functioning immune system.
I. CHARACTERIZATION AND ISOLATION OF NUCLEIC ACIDS
ENCODING IAP and CTE^p
This invention has for the first time provided for the characterization, cloning and expression of a novel NCTE, CTEJAP, derived from a novel intracistemal A particle, IAPORG. The invention also provides for novel means of expressing the IAPORG and CTEιAP of the invention in vitro and in vivo. In a further embodiment, this expression provides for a means to screen for novel NCTEs.
The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature. Therefore, only a few general techniques will be described prior to discussing specific methodologies and examples relative to the novel reagents and methods of the invention. A. General Techniques
Methods of isolating total DNA or RNA encoding the nucleic acids of the invention are well known to those of skill in the art. Techniques for isolation, purification and manipulation of nucleic acids, genes and CTEL^J, sequences, such as generating libraries, subcloning into expression vectors, labeling probes, DNA hybridization, and the like are described, e.g., in Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) ("Sambrook"); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997) ("Ausubel"); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR
BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993) ("Tijssen").
The nucleic acids of this invention, whether RNA, mRNA, DNA, cDNA, genomic DNA, or a hybrid of the genetic recombinations, may be isolated from a variety of sources or may be synthesized in vitro. Nucleic acids of the invention can be expressed in transgenic animals, transformed cells, in a transformed cell lysate, or in a partially purified or a substantially pure form. Sequencing methods typically use dideoxy sequencing (Sequenase, U.S. Biochemical), however, other kits and methods are available and well known to those of skill in the art. Nucleic acids and proteins are detected and quantified in accordance with the teachings and methods of the invention described herein by any of a number of general means well known to those of skill in the art. These include, for example, analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (FIPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISAs), immuno-fluorescent assays, and the like, Southern analysis, Northern analysis,
Dot-blot analysis, gel electrophoresis, RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography, to name only a few.
B. Isolation. Synthesis and Purification of Nucleic Acids Encoding IAPottc and CTEIΛP
In one embodiment, the invention provides for a novel IAP and CTE^ and methods to identify alleles, isoforms and polymorphisms. 1. Preparation and Screening of DNA Libraries
There are numerous methods for isolating the DNA sequences encoding the CTE^ and I AP0RG of the invention. For example, DNA can be isolated from a genomic or cDNA library using labeled oligonucleotide probes having sequences complementary to the sequences or subsequences disclosed herein, such as CTEL^ and IAP0RG, SEQ ID NOS: 1 and SEQ ID NO:3, respectively (the DNA subsequence encoding the CTEL^, of the invention is located between nucleotides 3029-3204 of SEQ ID NO:3). Such probes can be used directly in hybridization assays to isolate DNA encoding IAPORG or CTE^ isoforms and polymorphisms. Alternatively probes can be designed for use in amplification techniques, such as, e.g., PCR, and nucleic acid encoding IAP0RG or CLE^ can be isolated by using methods (see infra).
To prepare a cDNA library, mRNA is isolated, reverse transcribed from the mRNA according to procedures well known in the art and inserted into vectors. The vectors are transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known. See, e.g., Gubler (1983) Gene 25:263-269, Sambrook, Ausubel.
To make a genomic library, total DNA is extracted and purified by well- known methods (see, e.g., Sambrook). DNA of appropriate size is produced by known methods, such as mechanical shearing or enzymatic digestion, to yield DNA fragments, e.g., of about 12 to 20 kb. The fragments are then separated, as for example, by gradient centrifugation, or gel electrophoresis, from undesired sizes. Selected fragments can be inserted in bacteriophage or other vectors. These vectors and phage can be packaged in vitro, as described, e.g., in Sambrook. Recombinant phage can be analyzed by plaque hybridization described, e.g., in Benton (1977) Science 196:180; Chen (1997) Methods Mol Biol 62:199-206. Colony hybridization can be carried out as generally described in, e.g., Grunstein (1975) Proc. Natl. Acad. Sci. USA 72:3961-3965; Yoshioka (1997) J. Immunol Methods-20\ :\45-155; Palkova (1996) Biotechniques 21 :982. DNA encoding an IAP or CTEIAP can be identified in either cDNA or genomic libraries by hybridization with nucleic acid probes of the invention. For example, a probe containing 10 to 20 to 50 or more contiguous nucleotides of SEQ ID NO:l or 3 (see Figure 1 A and Figure 4) is used in Southern blots to identify
Figure imgf000020_0001
Once identified, these DNA regions are isolated by standard methods familiar to those of skill in the art. Alternatively, RNA may be identified by hybridization to nucleic acid probes in Northern blots or other formats; see, e.g., Sambrook, Ausubel, for general procedures.
Oligonucleotides for use as, e.g., probes, templates for further amplification, and the like, can be chemically synthesized, as described below. Synthetic nucleic acids, including oligonucleotide probes and primers, CTEL^ and IAP coding sequences, can be prepared by a variety of solution or solid phase methods. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available. For example, the solid phase phosphoramidite triester method of Beaucage and Carruthers using an automated synthesizer is described in Itakura, U.S. Pat. No. 4,401,796; Carruthers, U.S. Pat. Nos. 4,458,066 and 4,500,707; Carruthers (1982) Genetic Engineering 4:1-17. See also Needham-VanDevanter (1984) Nucleic Acids Res. 12:6159-6168; Beigelman (1995) Nucleic Acids Res 23: 3989-3994; OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, Gait (ed.), IRL Press, Washington D.C. (1984), see Jones, chapt 2, Atkinson, chapt 3, and Sproat, chapt 4; Froehler (1986) Tetrahedron Lett. 27:469-472; Froehler, Nucleic Acids Res. 14:5399-5407 (1986); Sinha (1983) Tetrahedron Lett. 24:5843-5846; and Sinha ( 1984) Nucl. Acids Res. 12:4539-4557. Methods to purify oligonucleotides include native acrylamide gel electrophoresis, anion-exchange HPLC, as described in Pearson (1983) J. Chrom. 255:137-149. The sequence of the synthetic oligonucleotide can be verified using any chemical degradation method, for example, see Maxam (1980) Methods in Enzymology 65:499-560, Xiao (1996) Antisense Nucleic Acid Drug Dev 6:247-258, or for solid-phase chemical degradation procedures, Rosenthal (1987) Nucleic Acids Symp Ser 18:249-252.
2. Amplification of Nucleic Acids Encoding CTE P and IAP
The present invention provides oligonucleotide primers and probes that can hybridize specifically to nucleic acids having CTE^p and IAP nucleic acid sequences.
Such reagents can be used to identify further species, such as polymorphisms alleles and other variations, of CTEIAP and IAP. For illustrative purposes, PCR primers and amplification methods are described.
Amplification of CTEj^ and IAP sequences which are conserved amongst different CTE^p and IAP species are preferred reagents of the invention for use as hybridization probes to identify and isolate additional species from various organisms. These oligonucleotides can also be used as primers to directly amplify nucleic acids sequences, as described below.
Oligonucleotides can be used to identify and detect additional CTEj^ species using a variety of hybridization techniques and conditions. Suitable amplification methods include, but are not limited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N. Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y. (Innis )), ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc.
Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see Berger (1987) Methods Enzymol. 152:307-316, Sambrook, and Ausubel, as well as Mullis (1987) U.S. Patent Nos. 4,683,195 and 4,683,202; Arnheim (1990) C &EN 36-47; Lomell
J. Clin. Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleic acids are described in Wallace, U.S. Pat. No. 5,426,039.
The invention provides for amplification and manipulation or detection of the products from each of the above methods to prepare DNA encoding CTΕIAP and IAP nucleic acid. In PCR techniques, oligonucleotide primers complementary to the two borders of the DNA region to be amplified are synthesized and used (see, Innis). PCR can be used in a variety of protocols to amplify, identify, quantify, isolate and manipulate nucleic acids encoding CTE[AP and IAP. In these protocols, primers and probes for amplification and hybridization are generated that comprise all or any portion of the DNA sequences listed herein
An illustrative primer pair that can amplify the CTE^ of the invention under appropriate conditions is: sense: 5'-CAACTGTTTCTTGGCATGCTAGAGAAGTAGT-3' (SEQ ID NO:4); and antisense 5'-CTGTCTTTACTTATTGGCAGAGAGAA-3' (SEQ ID NO:5). PCR-amplified sequences can also be labeled and used as detectable oligonucleotide probes, but such nucleic acid probes can be generated using any synthetic or other technique well known in the art, as described above. The labeled amplified DNA or other oligonucleotide or nucleic acid of the invention can be used as probes to further identify and isolate CTEL^J, and IAP species from various cDNA or genomic libraries. Another useful means of obtaining nucleic acids of the invention, such as large genomic clones, is to screen YAC, BAC or PI genomic libraries. BACs, bacterial artificial chromosomes, are vectors that can contain 120+ Kb inserts. BACs are based on the E. coli F factor plasmid system and simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs, which can also be employed in the present methods, are eliminated. BAC vectors can include marker genes for luciferase and green fluorescent protein (GFP). (Baker (1997) Nucleic Acids Res 25:1950-1956). Yeast artificial chromosomes, or YACS, can also be used for contain inserts ranging in size from 80 to 700 kb, see, e.g., Tucker (1997) Gene 199:25-30; Adam (1997) Plant J.l 1:1349-1358. PI is a bacteriophage that infects E. coli that can contain 75-100 Kb DNA inserts (Mejia
(1997) Genome Res 7:179-186; Ioannou (1994) Nat Genet 6:84-89) , and are screened in much the same way as lambda libraries. 3. Cloning of CTEIAP- and IAP-Encoding Inserts
The invention also provides CTEIAP- and IAP-encoding expression vectors to produce large quantities of full or partial length (e.g., a single RNA loop stmcture) nucleic acid molecules of the invention. The expression vector, including expression cassettes, include, e.g., those used in bacterial, yeast, plant, insect, in vitro, or mammalian systems. For example, generation of CTE^p in this manner is useful for assaying for CTEL^P activity modulators, analysis of the activity of newly isolated species of CTE,^, identifying and isolating compounds which specifically associate with CTEtAp, such as binding proteins, analysis
Figure imgf000023_0001
which has been site-specifically mutated, as described below. Nucleic acid so produce can be used the the invention's method to screening for a NCTE binding proteins using a single loop A or a loop B, or both loops as in Figure 1A, of a CTEIAP of the invention. The nucleic acids of the invention can also be used as immunogens, as a few examples. See, e.g., Radic (1994) Annu. Rev. Immunol. 12:487-520; Cabral (1997) Curr. Opin. Rheumatol. 9:387-392; Pisetsky (1997) Methods 11:55-61; Marion (1997) Methods 11 :3-11, for general discussion on anti-DNA antibodies; for discussion on generation of anti-RNA antibodies using combinatorial phage display libraries see Marchbank (1995) Nucleic Acids Symp. Ser. 33:120-122.
There are several well-known methods of introducing nucleic acids into bacterial and other cells, a process often called "transforming," any of which may be used in the methods of the present invention (see, e.g., Sambrook). Techniques for transforming a wide variety of animal and plant cells are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, for plant cells and Sambrook for animal and bacterial cells. 4. Sequencing of CTEιAP- and IAP-Encoding Nucleic Acid
Sequencing of newly isolated DNA will identify and characterize CTELaj,- and IAP-encoding nucleic acid of the invention. Sequencing of isolated CTEL^- and IAP- encoding nucleic acid can be used to identify, in addition to functional criteria, new CTE^p- and IAP-encoding species or allelic variations. Secondary stmctures can be identified. For example, sequencing of CTEIAP and subsequent computer predicted analysis of its secondary stmcture revealed a loop A with a "bulge" (e.g., an "AAGA" bulge), a loop B, a stem stmcture separating the loops and a hairpin turn, see Figure 1, SEQ ID NO: 1. In terms of primary sequence criteria, a nucleic acid is a CTE1AP specie within the scope of the claimed invention if the sequence of its loop domains has at least 90% nucleic acid-sequence identity to the loop A and loop B domains of CTE1AP illustrated in Figure 1 , SEQ ID NO: 1 (the DNA subsequence encoding CTE1AP is located between nucleotides 3029-3204 of Figure 4, SEQ ID NO:3).
CTEL^-encoding nucleic acid sequences can be sequenced as inserts in vectors, as inserts released and isolated from the vectors or in any of a variety of other forms (i.e., as amplification products). CTEL^,- and IAP-encoding inserts can be released from the vectors by restriction enzymes or amplified by PCR or transcribed by a polymerase. For sequencing of the inserts to identify full length CTEL^J, or IAP coding sequences, primers based on the N- or C- terminus, or based on insertion points in the original phage or other vector, can be used. Additional primers can be synthesized to provide overlapping sequences. A variety of nucleic acid sequencing techniques are well known and described in the scientific and patent literature, e.g., see Rosenthal (1987) supra; Arlinghaus (1997) Anal. Chem. 69:3747-3753, for use of biosensor chips for sequencing; Pastinen (1996) Clin. Chem. 42:1391-1397; Nyren (1993) Anal Biochem. 208:171-175. 5. Nucleic Acid Hybridization Techniques The hybridization techniques disclosed herein can be utilized to identify, isolate and characterize genes and gene products (i.e., mRNA) encoding IAP and CTEIAP- encoding nucleic acid of the invention, including different species, isoforms, alleles and polymorphisms. A variety of methods for specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art. See. e.g., NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH, Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1985; Sambrook..
One method for evaluating the presence or absence of DNA encoding IAP or CTEj^ in a sample involves a Southern transfer. Briefly, the digested bacterial genomic DNA is run on agarose slab gels in buffer and transferred to membranes. Hybridization is carried out using nucleic acid probes. The nucleic acid probes can be designed based on conserved nucleic acid sequences amongst the class of proteins. Preferably nucleic acid probes are 20 bases or longer in length (see, e.g., Sambrook for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization). Visualization of the hybridized portions allows the qualitative determination of the presence or absence of DNA encoding IAP or CTEIAP. Similarly, a Northern transfer can be used for the detection of RNA containing IAP or CTE1AP sequences. For example, RNA is isolated from a given cell sample using an acid guanidinium-phenol-chloroform extraction method. The RNA is then electrophoresed to separate different species and transferred from the gel to a nitrocellulose membrane. As with the Southern transfers, labeled probes or PCR can be used to identify the presence or absence of IAP or CTEi^ nucleic acid.
Sandwich assays are commercially useful hybridization assays for detecting or isolating protein or nucleic acid. Such assays utilize a "capture" nucleic acid or protein that is often covalently immobilized to a solid support and a labeled "signal" nucleic acid, typically in solution. A clinical or other sample provides the target nucleic acid or protein. The "capture" nucleic acid or protein and "signal" nucleic acid or protein hybridize with or bind to the target nucleic acid or protein to form a "sandwich" hybridization complex. To be effective, the signal nucleic acid or protein cannot hybridize or bind substantially with the capture nucleic acid or protein.
Typically, oligonucleotide probes are labeled signal nucleic acids that are used to detect hybridization. Complementary probe nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. Methods of detection can use labels for autoradiography or autofluorography, such as 3H, 1251, 35S, 14C, or 32P-labeled probes or the like (see definition of label, above). Other labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal, i.e., antibody-antigen or complementary nucleic acid binding. The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzymatic molecules to the antibodies or, in some cases, by attachment of a radioactive label. 'The sensitivity of the hybridization assays may be enhanced through use of a target nucleic acid or signal amplification system which multiplies the target nucleic acid or signal being detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known, as described above. These systems can be used to directly identify IAP or
Figure imgf000026_0001
variations, polymorphisms, or mutated sequences where the PCR or LCR primers or other reagents are designed to be extended or ligated only when a specific sequence is present. Alternatively, the specific sequences can be generally amplified using, for example, more generic PCR primers and the amplified target region later probed or sequenced to identify a specific sequence indicative of the variant, polymorphism or mutation. Nucleic acid hybridization assays for the detection of isoforms, mutations and for sequencing can also be performed in an array-based format. Arrays are a multiplicity of different "probe" or "target" nucleic acids (or other compounds) are hybridized against a target nucleic acid. In this manner a large number of different hybridization reactions can be run essentially "in parallel". This provides rapid, essentially simultaneous, evaluation of a wide number of reactants. Methods of performing hybridization reactions for detection and sequencing in array based formats are well known to those of skill in the art, e.g., Pastinen (1997) Genome Res. 7:606-614; (1997) Jackson (1996) Nature Biotechnology 14:1685; Chee (1995) Science 274:610.
An alternative means for determining the level of expression of a gene is in situ hybridization. In situ hybridization assays are well known (e.g., Angerer (1987) Methods Enzymol 152:649). In an in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The probes are typically labeled, i.e., with radioisotopes or fluorescent reporters. Another well-known in situ hybridization technique is the so-called FISH fluorescence in situ hybridization, as described by Macechko (1997) J. Histochem. Cytochem. 45:359-363; and, Raap (1995) Hum. Mol. Genet. 4:529-534.
The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known, as described above. Nucleic acid sequence-based amplifications are also used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a select sequence is present. Alternatively, the select sequences can be generally amplified using nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
Oligonucleotides for use as probes, e.g., in vitro amplification methods, as gene probes in diagnostic methods, or as inhibitor components (see below) are typically synthesized chemically; e.g., such as by the solid phase phosphoramidite triester method described by Beaucage and Caruthers, supra, or, using an automated synthesizer, as described in Needham-VanDevanter, supra. Purification of oligonucleotides, where necessary, is typically performed by native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier. The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (Maxam (1980) supra).
It will be appreciated that nucleic acid hybridization assays can also be performed in an array-based format. In this approach, arrays bearing a multiplicity of different "probe" nucleic acids are hybridized against a target nucleic acid. In this manner a large number of different hybridization reactions can be run essentially "in parallel". This provides rapid, essentially simultaneous, evaluation of a wide number of reactants. Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Jackson (1996) Nature Biotechnol. 14:1685, and Chee (1995) Science 274:610).
6. Sequence Comparison Analysis of the CTEIAP and IAP0RG IAP and NCTE-encoding nucleic acid sequences of the invention include both genes (genomic sequences, including introns and 5' and 3' non-coding regions) and gene transcription products (mRNA) identified and characterized by analysis of IAP0RG and CTEIAP sequences. Optimal alignment of sequences for comparison can be conducted as described above (see definitions).
Sequence identity analysis, in addition to functional analysis, is used to determine whether a nucleic acid is within scope of the invention. For example, in one embodiment, a CTE^p sequence of the invention has a secondary stmcture with at least a loop A domain, a stem stmcture separating the loops, and a loop B domain, where the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity to a loop A and a loop B domain of a nucleic acid as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1.
II. MUTAGENESIS OF CTE,AP and IAPORG NUCLEIC ACID
The invention also provides for CTEi^j, and IAP0RG that have been modified in a site-specific manner to modify, add to, or delete some or all of the nucleic acid's functions. For example, specific base pairs can be modified to alter, increase or decrease the affinity of binding proteins, thus modifying the relative level of attenuation of the CTE; or, modifications can change the stability of the secondary stmcture of the nucleic acid; or, base pair changes can augment expression of the nucleic acid in a cell, such as a bacteria; as a few illustrative examples. Site-specific mutations can be introduced into CTEIAP and IAPORG-encoding nucleic acid by a variety of conventional techniques, well described in the scientific and patent literature. Illustrative examples include: site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR), as in Urban (1997) Nucleic Acids Res. 25:2227-2228; Ke (1997) Nucleic Acids Res 25:3371-3372, and Chattopadhyay (1997) Biotechniques 22:1054-1056, describing PCR-based site-directed mutagenesis
"megaprimer" method; Bohnsack (1997) Mol. Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques 22:624-626, describing site-directed mutagenesis using a PCR-based staggered re-annealing method without restriction enzymes; Nicolas (1997) Biotechniques 22:430-434, site-directed mutagenesis using long primer-unique site elimination and exonuclease III. Modified CTEIAP and IAP0RG of the invention can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.
III. EXPRESSION OF CTE1AP and IAPORG NUCLEIC ACID
The invention provides for methods and reagents the expression of novel CTE^ and IAP0RG of the invention in any prokaryotic, eukaryotic, yeast, fungal, plant, insect, human or animal cell. Antisense, in addition to sense, sequences are provided for by the invention. To create cell-based and in vitro assay systems to screen for novel
NCTEs using CTE^p, a variety of in vivo and in vitro expression systems are provided by the invention.
A. Vectors and Transcriptional Control Elements
The invention provides for methods and reagents for expressing the novel CI ytf and IAP0RG nucleic acids described above as sense or antisense coding sequences.
Other embodiments of the invention provide methods and reagents for identifying, isolating and using CTEL^ and IAP0RG cz'sacting transcriptional control elements and transacting NCTE binding proteins. After the coding region of a CTEIAP and IAP0RG gene has been identified, the expression of natural or synthetic CTEL^, and IAPORG-encoding or other (i.e., antisense, ribozyme) nucleic acids can be achieved by operably linking the coding region to transcriptional regulatory elements, such as promoters and enhancers. These sequences have characteristic subsequences, for instance, promoter sequence elements typically include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. Promoters can be tissue- specific or not, constitutive or inducible. Promoters that drive expression continuously under physiological conditions are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Typical expression systems, such as expression cassettes and vectors, also contain transcription and translation terminators, transcription and translation initiation sequences. Generic expression cassettes typically contain at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. See, e.g., Roberts (1987) Nature 328:731 ; Berger (1987) supra; Schneider (1995) Protein Expr. Purif. 6435:10; Sambrook and Ausubel. Product information from- manufacturers of biological reagents and experimental equipment also provide information regarding known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia Biotech (Piscataway, NJ), Clontech Laboratories, Inc. (Palo Alto, CA), Aldrich Chemical Company (Milwaukee, WI), GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill. The promoters and vectors used in this invention can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic methods, as described herein.
The CTEj^aj, and IAP0RG sense or antisense sequences of the invention can be expressed in vectors which are transiently expressed in cells using, e.g., episomal vectors such as vaccinia vims, see Cooper (1997) Proc Natl Acad Sci USA 94:6450-6455. Alternatively, CTE^ and IAP0RG coding sequences can be inserted into the host cell genome becoming an integral part of the host chromosomal DNA, using for example, retroviral vectors such as SIV or HIV, see for example, Naldini (1996) Science 272:263-267. Expression vectors can contain selection markers that confer a selectable phenotype on transformed cells and sequences coding for episomal maintenance and replication such that integration into the host genome is not required. For example, the marker may encode antibiotic resistance, particularly resistance to chloramphenicol, kanamycin, G418, bleomycin and hygromycin, to permit selection of those cells transformed with the desired DNA sequences, see for example, Blondelet-Rouault (1997) Gene 190:315-317. Because selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture, chemoresistance genes are also used as selectable markers in vitro and in vivo. Various target cells are rendered resistant to anticancer dmgs by transfer of chemoresistance genes encoding, e.g., P-glycoprotein, multidmg resistance-associated protein-transporter, dihydrofolate reductase, glutathione -S-transferase, O 6-alkylguanine DNA alkyltransferase, or aldehyde reductase (Licht (1997) Stem Cells 15:104-1 1 1) and the like. Illustrative vectors incorporating CTE^p and IAP0RG genes include, for example, adenovims-based vectors (Cantwell (1996) Blood 88:4676-4683; Ohashi (1997) Proc Natl Acad Sci USA 94:1287-1292), Epstein-Barr vims-based vectors (Mazda (1997) J Immunol Methods 204:143-151), adenovims-associated vims vectors, Sindbis vims vectors (Strong (1997)
Gene Ther 4: 624-627), Herpes simplex vims vectors (Kennedy (1997) Brain 120: 1245-1259) and retroviral vectors (Schubert (1997) Curr Eye Res 16:656-662). Epstein- Barr vims episomal vectors (Horlick (1997) Protein Expr. Purif. 9:301-308, and plasmid DNA (Lowrie (1997) Vaccine 15: 834-838); all of which can be used to express the nucleic acids of the invention in vivo or ex vivo
IV. INHIBITING EXPRESSION OF CTEIAP and IAPORG NUCLEIC ACID
The invention further provides for nucleic acids complementary to, i.e., antisense sequences to, the CTEL^J. and IAP0RG of the invention. Antisense sequences are capable of inhibiting the transport, splicing or transcription of CTEIAP and IAPORG- encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage. One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind CTEj^ and IAPORG gene or message, in either case preventing or inhibiting the production or function of CTE^ and IAP0RG. The association can be though sequence specific hybridization. Such inhibitory nucleic acid sequences can, for example, be used to completely inhibit or further depress the replicative potential of an attenuated vims. Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of CTE,^ and IAP0RG message. The oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes. The oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity. 1. Antisense Oligonucleotides
The invention provides for with antisense oligonucleotides capable of binding CTELΛJ. and IAP0RG message which can inhibit
Figure imgf000032_0001
IAP0RG activity by targeting mRNA. "Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such CTEj^ and IAP0RG oligonucleotides using the novel reagents of the invention. In some situations, naturally occurring nucleic acids used as antisense oligonucleotides may need to be relatively long (18 to 40 nucleotides) and present at high concentrations. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense CTEj^ and IAP0RG sequences of the invention (for general background information, see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
2. Inhibitory Ribozymes The invention provides for with ribozymes capable of binding CTE^ and
IAP0RG message which can inhibit CTE^ and IAP0RG activity by targeting mRNA. Strategies for designing ribozymes and selecting the CTC^ and IAP0RG-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such CTEj^ and IAP0RG ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence, or, as with CTE^p, prevent viral message from binding to NCTE binding protein, thus preventing subsequent transport of the message to the cytoplasm. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
In some circumstances, the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule, as with CTE^p) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide. This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta vims, group I intron or RNaseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis delta vims motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The recitation of these specific motifs is not intended to be limiting; those skilled in the art will recognize that an enzymatic RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
V. CONSTRUCTION OF ATTENUATED VIRUS AND VIRAL VACCINE
The invention provides for an attenuated retrovims and vaccine comprising the CTELAJ. of the invention. One means to genetically engineer a wild-type, vimlent vims to a hybrid, attenuated vims involves constructing a vims which either lacks or has a non- functional endogenous post-transcriptional RNA nucleo-cytoplasmic transport elements
(NCTEs). The endogenous NCTE is subsequently replaced by the exogenous NCTE of the invention which functions less efficiently in vivo than its wild-type counterpart, thus effecting the attenuation. The CTEIAP of the invention, when inserted in a RRE(-) and/or Rev(-) HIV-1, is capable of functioning as a less efficient, "attenuated" NCTE in place of the wild-type RRE/Rev NCTE system. This level of attenuation can be measured; when the CTELy.-containing hybrid HIV-1 vims infects activated huPBMCs, the level of expression of HIV- 1 p24 αg is between about 50 fold and about 200 fold less than levels of p24gαg expression when HIV-1 wild type virus, utilizing wild-type NCTE, infects activated huPBMCs. Furthermore, in constmcting the attenuated retrovims of the invention, additional elements of the retrovims which are essential for its replication and/or pathogenicity can also be disabled or eliminated, such as Nef, as explained below.
In normal mammalian cells, message RNA, present in the cell as ribonucleoprotein (RNP) complexes, is only exported from the nucleus to the cytoplasm after splicing is completed. To circumvent the requirement of splicing prior to export from the nucleus, all retrovimses have evolved a mechanism that allows the nuclear export of unspliced form of viral RNAs which are necessary for the production of structural proteins and essential for viral replication. This mechanism involves the highly structured NCTE c/sacting RNA element and its corresponding transacting RNA binding proteins, as discussed above. In simian type retrovimses, the NCTE is termed "CTE" (see Bray (1994) supra; Zolotukhin (1994) supra), and binds to endogenous cellular RNA binding proteins. In contrast, HIV-l's NCTE does not bind cellular NCTE-binding proteins, it encodes its own NCTE binding protein, called "Rev." Rev interacts with a specific HIV-1 NCTE sequence, designated the "Rev-responsive element," or "RRE," contained in its gag/pol and env encoding transcript. Rev interacts directly with RRE as part of the RNA export machinery which transports RRE-containing transcripts to the cytoplasm from the nucleus. As a result, HIV-1 needs both RRE and Rev to produce infectious vims. Disabling either produces a non-replicative, non-vimlent vims. Replacing (i.e., reconstituting) HIV-l's RRE/Rev RNA transport mechanism with a less efficient NCTE, such as the CTEIAP of the invention, produces an attenuated, avimlent hybrid vims.
To engineer a non-functional RRE and/or Rev, the skilled artisan can delete and or mutate any portion of the RRE or Rev coding sequence. Means to delete or mutate nucleic acid sequence are described herein, and are well known in the art. Constmction of exemplary, attenuated retrovimses are also discussed in the Examples, below. RRE and Rev sequences are well known in the art, e.g., see databases, such as the NCBI database at http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html or http://www.ncbi.nlm.nih.gov/ Entrez/protein.html. Further description and sequence of HIV-1 Rev can be found in, e.g.,
Salminen (1997) J Virol. 71 :2647-2655, Accession U86770; Theodore (1996) AIDS Res. Hum. Retroviruses 12:191-194, Accession AF004394; Fang, et al, Accession AF003887; Howard (1996) AIDS Res. Hum. Retroviruses 12:1413-1425, Accession L39106; to name only a few. Further description and sequence of HIV- 1 RRE can be found in, e.g., Salminen (1996) JOURNAL AIDS Res. Hum. Retroviruses 12:1329-1339, Accession U46016; WO 9202228-A5 20-FEB-1992, Accession A20711; Battiste (1994) Biochemistry 33:2741-2747; Battiste (1995) J. Biomol. NMR 6:375-389; Battiste (1996) Science 273:1547-1551; to name just a few.
In constmcting the attenuated retrovimses and vaccines of the invention, additional elements in addition to endogenous NCTE which are essential for the vims' replication and or pathogenicity can also be disabled or eliminated. For example, genetic engineering of a Nef-negative retrovims can produce recombinant hybrid which can replicate in vivo, yet is less vimlent. In the case of HIV- 1, a functional Nef gene is important for development of high viremia and AIDS. Animals infected with Nef-deleted attenuated viruses are resistant to subsequent challenge with pathogenic wild-type vimses. A critical role for Nef in development of AIDS in humans has been suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection (persons who show no clinical or immunological signs of immunodeficiency despite being HIV seropositive for over a decade) are infected with vimses carrying naturally occurring Nef deletions. The mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell protein kinases. In vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. Recombinant soluble Nef protein binds to cell surfaces of various murine lymphoid cell lines, including T and B lymphocytes and macrophages; cross-linking of the cell-bound
Nef protein with anti -Nef antibodies induces apoptotic cytolysis of the cells. For a further description of Nef, see, e.g., Saksela (1997) supra; Greenberg (1997) supra; Luo (1997) J. Virol. 71 :9531-9537; Luo (1997) J. Virol. 71 :9524-9530; Okada (1997) supra. Thus, the vaccine and retrovims of the invention comprising a non-function Nef has incorporated an additional level of attenuation. To engineer a non-functional Nef, the skilled artisan can delete and or mutate any portion of the Nef coding sequence. Nef sequences are well known in the art, e.g., see databases, such as the NCBI databases described above. For examples of HIV- 1 Nef nucleic acid and polypeptide sequences, see, e.g., Accession Nos. Y15123, U88826, Y15121, Y15120, Y15116, to name only a few.
VI. DELIVERY OF NUCLEOTIDES INTO CELLS
The nucleic acids and oligonucleotides of the invention, including vectors expressing CTE^ and IAP0RG, can be delivered into cells in culture, tissues and organisms for synthesis, mutation, screening and the like. For example, the invention provides for a method for screening for a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) binding protein by contacting a CTEIAP of the invention with a test compound and measuring the ability of the test compound to bind the NCTE; and this screening technique can be used in intact cells. Inhibitory oligonucleotides of the invention, and vectors capable of expressing these sequences, are also transferred into intact cells in cell culture, tissues or intact organisms. The nucleic acids and oligonucleotides of the invention can be transferred into a cell using a variety of techniques well known in the art. For example, oligonucleotides can be delivered into the cytoplasm spontaneously, without specific modification. Altematively, they can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. For example, a DNA binding protein, e.g., HBGF-1, is known to transport oligonucleotides into a cell. See, e.g., Tseng (1997) J. Biol. Chem. 272:25641-25647; Satoh (1997) Biochem. Biophys. Res. Commun. 238:795-799, describing efficient gene transduction by Epstein-Barr-virus-based vectors coupled with cationic liposome and HVJ-liposome.
The procedures for delivering the nucleic acids and oligonucleotides of the invention to cells are useful in vivo. For example, by using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one may provide for the introduction of the oligonucleotides into the target cells in vivo. See, e.g., Huwyler (1997) J. Pharmacol. Exp. Ther. 282:1541-1546, describing receptor mediated delivery using immunoliposomes. Altematively, cells may be permeabilized to enhance transport of oligonucleotides into the cell, without injuring the host cells. See, e.g., Verspohl (1997) Cell. Biochem. Fund. 15:127-134; Kang (1997) Pharm. Res. 14:706-712; Bashford (1994) Methods Mol. Biol.
27, 295-305, describing use of bacterial toxins for membrane permeabilization; and for general principles of membrane permeabilization, see, e.g., Hapala (1997) Crit. Rev. Biotechnol. 17:105-122.
VII. PREPARATION, FORMULATION AND ADMINISTRATION OF ATTENUATED VIRAL VACCINES
Live, CTEIAP-attenuated HIV-1 vims can be grown and harvested from activated human peripheral mononuclear cells or from a variety of tissue culture cells, such as human 293 cell line, as described herein; see also, e.g., Eberlein (1991) Virus Res. 19:153-161; Parente (1996) Gene Ther. 3:756-760; Margolis (1997) AIDS Res. Hum.
Retroviruses 13:1411-1420. Virion-containing supernatants are collected, and, typically, filtered. CTEIAP sequences in the harvested, attenuated vims for use in vaccine formulations can be confirmed by conventional sequencing. The attenuated vims can be further purified, for example, by ultrafiltration or ultra-centrifugation. The live, attenuated vims can be stored by refrigeration, or on a long-term basis, by freezing in liquid nitrogen. A formulation for administering the vims as a vaccine is prepared using, for example, any physiologically acceptable buffer, such as saline or phosphate buffered saline (PBS). This can be stored in a frozen state. The formulation can also be freeze-dried, stored at room temperature, and reconstituted by adding appropriate volume of buffer. The vaccine pharmaceutical formulation can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptabie diluent or solvent, such as a solution of 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. The live attenuated viral vaccine of the invention can be administered using any acceptable route, as, e.g., by application to a mucosal surface, by injection, by inhalation or by ingestion. For examples of inhalants, see Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111; Fernandez-de Castro (1997) "Measles vaccination by the aerosol method in Mexico," Salud Publica Mex 39:53-60. Injection of vaccine can be intravenous or intramuscular; see, e.g., Groswasser (1997) Pediatrics 100:400-403, as example of injection techniques for efficient intramuscular vaccine delivery. Administration by application to any mucosal surface, including, e.g., intraoral (sublingual, buccal, and the like), intranasal, intrarectal, intravaginal, or ocular. For examples of mucosal administration methods, see, e.g., Staats (1997) AIDS Res Hum Retroviruses 13:945-952; Okada (1997) J. Immunol. 159:3638-3647; Wu (1997) AIDS Res Hum Retroviruses 13:1187-1194. The amount of vims (number of virions) per dose will vary depending on results of different titrations used in clinical trials. The range can range, for example, from only a few infectious units, to about 10 to 10 infectious units (i.e., virions) per dose. Protocols and means to determine safety and efficacy used for other attenuated vaccines can be adapted and used with the novel reagents provided by the invention; see, e.g.,
Gmber (1997) "Evaluation of bivalent live attenuated influenza A vaccines in children 2 months to 3 years of age: safety, immunogenicity and dose-response," Vaccine 15:1379-1384; Tingle (1997) "Randomised double-blind placebo-controlled study on adverse effects of rubella immunisation in seronegative women," Lancet 349:1277-1281; Varis (1996) "Efficacy of high-titer live attenuated varicella vaccine in healthy young children," J. Infect. Dis. 174:S330-S334; Gmber (1996) "Evaluation of live attenuated influenza vaccines in children 6-18 months of age: safety, immunogenicity, and efficacy," J. Infect. Dis. 173:1313-1319.
After the vaccine has formulated in an acceptable carrier, it can be placed in an appropriate container and labeled. For administration of the vaccine, such labeling would include, e.g., instructions concerning the amount, frequency and method of administration. In one embodiment, the invention provides for a kit and instmctional material teaching the indications, dosage and schedule of administration of the vaccine. Selection of individuals who would benefit from receiving the live, attenuated vaccine of the invention include, but are not limited to, individuals who have a high risk of being exposed to HIV, such as intravenous dmg users, individuals who may been exposed, as through a needle stick or transfusion, and individuals whose exposure to the vims has been confirmed, e.g., by a positive blood test.
The vaccine can be administered in conjunction with other treatment regimens, e.g., it can be coadministered or administered before or after any anti-viral pharmaceutical or a non-attenuated anti-HIV vaccine. The vaccine can be administered in any form of schedule regimen, e.g., in a single dose, or, using several doses (e.g., boosters) at dosages and time intervals to be determined by clinical trials.
The attenuated vaccine of the invention is considered efficacious, i.e., immunoprotective, if it elicits any humoral or cell-mediated anti-HIV response without causing clinically significant pathology, such as significant T cell cytotoxicity. The anti- HIV response can be assessed, for example, by measuring the levels of anti-viral antibodies or HlV-specific T cells, the amount of HIV in the blood or lymph nodes (see, e.g. Brown (1997) Transfusion 37:926-929), and or levels of circulating helper (CD4+) T cells. See also, O'Brien (1997) "Changes in plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure ' nn. Intern. Med.
126:939-945; Hughes (1997) "Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response," Ann. Intern. Med. 126:929-938; Burgisser (1997) "Monitoring responses to antiretroviral treatment in human immunodeficiency vims type 1 (HΙV-l)-infected patients by serial lymph node aspiration," J. Infect. Dis. 175 : 1202- 1205.
VIII. SCREENING FOR NCTE BINDING PROTEINS USING CTE p
The invention provides for cell-based and in vitro assay systems to screen for novel NCTE-binding proteins using the CTE^ of the invention. The full-length, two looped CTE^ , can be utilyzed, or, altematively, a portion of a CTEL^., such as a single loop A or a single loop B, can be used to assay for RNA binding proteins. One embodiment of the invention provides for a method of screening for an NCTE binding protein by contacting a CTE^ of the invention with a test compound and measuring the ability of the test compound to bind the NCTE. Many assays are available that screen for nucleic acid binding proteins and all can be adapted and used with the novel reagents provided for by the invention. A few illustrative example are set forth below.
A variety of well-known techniques can be used to identify polypeptides which specifically bind to CTEL^,, for example, mobility shift DNA-binding assays, methylation and uracil interference assays, DNase and hydroxy radical footprinting analysis, fluorescence polarization, and UV crosslinking or chemical cross-linkers. For a general overview, see, e.g., Ausubel (chapter 12, DNA-Protein Interactions).
One technique for isolating co-associating proteins, including nucleic acid and DNA/RNA binding proteins, includes use of UV crosslinking or chemical cross- linkers, including cleavable cross-linkers dithiobis(succinimidylpropionate) and 3,3'-dithiobis (sulfosuccinimidyl- propionate); see, e.g., McLaughlin (1996) Am. J. Hum. Genet. 59:561-569; Tang (1996) Biochemistry 35:8216-8225; Lingner (1996) Proc. Natl. Aca. Sci. U.S.A. 93:10712; Chodosh (1986) Mol. Cell. Biol 6:4723-4733. If a specific protein is believed to bind to CTEIAP, and an antibody is available or can be generated for that protein, co-immunoprecipitation analysis can be used. Altematively, CTEIAP-affinity columns can be generated to screen for potential CTE^-binding proteins. In a variation of this assay, the CTEIAP-containing nucleic acid is biotinylated, reacted with a solution suspected of containing a CTEL^p-binding protein, and then reacted with a strepavidin affinity column to isolate the CTEL^-containing nucleic acid/binding protein complex (see, e.g., Grabowski (1986") Science 233:1294-1299; Chodosh (1986) supra). The protein can then be conventionally eluted and isolated. Mobility shift DNA-protein binding assay using nondenaturing polyacrylamide gel electrophoresis is an extremely rapid and sensitive method for detecting specific polypeptide binding to DNA (see, e.g., Chodosh (1986) supra, Carthew (1985) Cell 43:439-448; Trejo (1997) J. Biol. Chem. 272:2741 1-27421 ; Bayliss (1997) Nucleic Acids Res. 25:3984-3990). Interference assays and DNase and hydroxy radical footprinting can be used to identify specific residues in the nucleic acid protein-binding site, see, e.g.,
Bi (1997) J. Biol. Chem. 272:26562-26572; Karaoglu (1991) Nucleic Acids Res. 19:5293-5300. Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions, see, e.g., Lundblad (1996) o/. Endocrinol. 10:607-612..
Proteins identified in by these techniques can be further separated on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against such proteins can be conjugated to column matrices and the proteins immunopurified. All of these general methods are well known in the art. See
Scopes, R. K., Protein Purification: Principles and Practice, 2nd ed., Springer Verlag, (1987). Chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention. Example 1: Identification, Isolation and Characterization of CTE p
The following example details the identification, isolation and
Figure imgf000042_0001
To identify elements similar to SRV-1 CTE, as described by Tabemero
(1996) supra and Zolotukhin (1994) supra, a search of the sequence databases for elements with nucleotide similarities to the 173 base pair SRV-1 NCTE was conducted. The RNA secondary stmcture of this NCTE element is a stem-loop stmcture with two internal loops, A and B. Queries were designated to search for homology to the loop motifs A and B, which are separated by spacers to match the lengths of the interloop stem (15 to 35 nucleotides) and the hairpin loop: AANGACNGGT (2,1,1,) (SEQ ID NO:6); AACCTAAGACAGG (1,1,1) (SEQ ID NO:7); CNANGACNGG (2,1,1) (SEQ ID NO:8); and, AACCTAAGACAGG (1,1,1) (SEQ ID NO:9). The numbers in parentheses indicate the number of allowed nucleotide changes, deletions, and insertions, respectively. The searches were performed with the PatScan program (http://www.mcs.anl.gov/home/ papka/RO SS/patscan. html) .
This search resulted in identification of a novel 176 base pair (bp) segment which has sufficient sequence identity to the submitted SRV NCTE element. This 176 bp sequence was a portion of a larger sequence which had been previously submitted as GenBank accession number U53820. The novel element is located upstream of the coding region in the mouse osteocalcin related gene (ORG), as shown in Figure 2. ORG is a member of the murine osteocalcin gene cluster (see Desbois (1994) J. Biol Chem. 269:1183-1190; Rahman (1993) Endocrinology 133:3050-3053).
This novel 176 bp sequence was analyzed by computer program (using MFold, University of Wisconsin Genetics Computer Group Package) for its ability to form secondary stmctures (MFold is an adaptation of the MFold package by Zuker and Jaeger, see Zuker (1989) Science 244:48-52; Jaeger (1989) Proc. Natl. Acad. Sci. USA 86:7706-7710). The analysis predicted that the 176 bp sequence forms an RNA secondary stmcture that shares several important features with the NCTE stmcture predicted for SRV-1 CTE. As shown in Figure 1, two internal loops, designated loop A and loop B, are conserved. The distance between the two loops is also conserved. The AAGA "bulge" adjacent to loop A is conserved. To a lesser extent, the sequence of the hairpin loop is also conserved. Sequence comparison shows that the four stretches of imperfect direct repeats spanning internal loops A and B have significant sequence identity. In contrast, the stem stmctures show no sequence identity. Previous work has shown that the changing the sequence of the stem stmcture in SRV-1 's NCTE does not affect function (Tabemero,
1996, supra). This finding, together with the lack of sequence identity between the stems of SRV and the 176 bp segment predicts that the stem stmcture serves a primarily structural, loop-separating role in the overall secondary stmcture.
The region upstream (5') of the novel segment was sequenced from the NCTE-containing plasmid, designated U53820, as described above. A computer sequence identity search using this new sequence data was performed. The analysis revealed that this NCTE is part of a novel IAP retroelement region within the mouse ORG. Thus, the novel CTE segment was named "CTE^" and the novel IAP termed "ORG,^." As shown in Figure 2, the IAP is located in the transcribed ORG between its promoter and transcriptional start site and the ORG protein coding region (open reading frame).
The ORGIAP is 3,402 nucleotides in length and is flanked by two imperfect direct repeats, see Figure 2. The direct repeats have partial homology to the LTRs of the previously identified "Eker rat-associated IAP" (ERA-IAP) (Xiao (1995) Oncogene 11 :81- 87). Although they do not contain any intact open reading frames, ORG,^ LTRs contain a predicted TATA box, a polyadenylation signal, and a section with sequence identity to putative gag/pol and env regions of other IAPs. The novel CTE^ of the invention is located between the putative env gene and the 3' LTR of ORG^. This is similar to the location of other NCTEs, as those seen in type D retrovimses. Example 2: CTE P Replacing HIV-1 NCTE Creates an Attenuated Virus
The following example details the production of a Rev(-)/RRE(-) CTE^p- containing HIV-1 hybrid recombinant, attenuated vims.
To demonstrate that the CTEL^ of the invention can produce an attenuated, immunoprotective HIV-1 vaccine, this novel NCTE element was inserted into a Rev/RRE- deficient recombinant clone of HIV- 1, which is described in Zolotukhin (1994) supra, and is designated pR(-)Rev(-). Briefly, the HIV-1 molecular clone designated pNL4-3 (Adachi (1986) J. Virol. 59:284-291) was altered to lack functional Rev protein and Rev-responsive element (RRE), as described in Nasioulas (1994) J Virol. 68:2986-2993, and Zolotukhin (1994) supra, respectively. 37 point mutations were introduced into the RRE that did not affect the overlapping env reading frame (Nasioulas (1994) supra). Two mutations that eliminated Rev were constmcted. The first mutation eliminated the Rev initiation codon. The second mutation introduced a stop codon in the 23 rd amino acid of the Rev protein (Zolotukhin (1994) supra). A unique Xhol site was used to insert CTEL^, into the RRE and Rev deficient clone (pR(-)Rev(-)) at nucleotide 8887 of NL4-3, as described in Zolotukhin (1994) supra. This hybrid HIV-1 was termed "pR(-)Rev(-).IAP CTE." Stocks were prepared from: this hybrid; wild-type (wt) HIV-1 (the wt designated "NL4-3," Adachi (1986) J. Virol. 59:285-291); and, a R(-)Rev(-) hybrid into which was inserted an SRV-1 NCTE, termed "pR(-)Rev(-).S," (Zolotukhin (1994) supra). Human 293 cells were transfected by a calcium phosphate coprecipitation technique. Supematants were collected and filtered through a 0.2 um filter. The sequence of CTEL^, in these replicating recombinant hybrids was confirmed by conventional sequencing techniques.
The wild-type NL4-3 and recombinant HIV-1 clones were used to infect Jurkat cells and in vitro activated human peripheral blood mononuclear cells (PBMCs) using a cell-free protocol, as described by Zolotukhin (1994) supra. PBMC cell cultures were obtained and activated as described in Zolotukhin (1994) supra. Briefly, PBMC cell cultures were obtained from healthy, HIV-seronegative blood donors, isolated by density centrifugation and stimulated for three days with phytohemagglutinin (PHA) at 5 to 10 micrograms per ml. Equal amounts of each of the vimses from the filtered 293 supematants were used to infect 5 x 10 activated PBMCs and 3 x 10 Jurkat cells. Vims propagation was monitored over time using a p24 α antigen capture assay (using either a commercial assay, Cellular Products (Buffalo, NY) or an in-house p24gαg antigen capture assay using standard techniques, as described herein.
Replacement of HIV-l's wild type NCTE (RRE) by CTEIAP resulted in a slow-growing hybrid in both Jurkat and activated PBMCs, as demonstrated by Figure 3A and 3B, respectively. In PBMCs, the CTE^p-containing hybrid, RRE(-)Rev(-).IAP CTE, as measured by p24 levels, replicated about 100 times less efficiently than the wild-type HIV-1 (NL4-3) and about 10 times less efficiently than the SRV-1 NCTE-containing hybrid, RRE(-) Rev(-).S. In Jurkat cells, the CTEL^p-containing hybrid replicated about 10 times less efficiently than the wild-type HIV-1 and the SRV-1 containing hybrid. This data demonstrates that CTE1AP attenuates HIV-1. Significantly, hybrid vims attenutated using the novel CTEj^ of the invention has a greater degree of attenuation than SRV-1 NCTE- attenuated HIV-1; CLE^ -attenuated HIV-1 replicates about 10 times less efficiently than SRV-1 CTE-attenuated hybrids. The role of the loop A stmcture was then investigated. A single nucleotide change was generated in CIE^'s stem stmcture immediately adjacent (51 end) to loop A. In CTEL^,, the GUC/GGC stem stmcture preceding loop A was changed to GCC/GG.C, this new hybrid termed "pR(-)Rev(-).IAP CTEm." Creation of this G-C base pair (from the CTEj^ wild type U-G base pair) generates a stronger stem stmcture (three, rather than two, hydrogen bonds with the G-C bp). The SRV-1 CTE has the GCC/GGC stem stmcture. As seen in Figure 3, the "stronger" stem resulted in a hybrid which is less attenuated (i.e., having less replicative potential), generating p24 antigen in amounts similar to the SRV-1 NCTE-containing hybrid.
CTEj^'s replicative potential was further tested utilizing a syncytium formation assay. Human 293 cells were transfected with SRV-1 (with wild type CTE), a CTE negative SRV-1, and an SRV-1 clone which had had its CTE deleted and replaced with the CTEj^ of the invention. Transfectants were co-cultivated with the human Raji B cell line. The cultures were monitored for syncytium formation. SRV-1 wild type CTE and SRV-1 /CTE negative clones reconstituted with the CTE^ of the invention produced syncytium. CTE negative clones produced no syncytium. In conclusion, hybrid HIV-1 using CTEIAP rather than the wildtype RRE or SRV-1 CTE as the NCTE element has less replicative potential (i.e, is more attenuated), as shown by measuring levels of p24. Thus, these experiments demonstrate that the CTE^ of the invention produces a slower-growing, attenuated HIV-1 hybrid.
Example 3: CTE-Containing HIV-1 are Attenuated In Vivo
The following example details use of CTE-containing post-transcriptional regulatory agents in place of the wild-type Rev/RRE post-transcriptional regulatory system as attenuating agents in HIV-1 infection and AIDS pathogenesis. The efficacy CTE^ as an HIV-1 attenuating agent in vivo can be demonstrated using functionally analogous
NCTEs, such as the SRV-1 CTE.
Hybrid HIV-1 clones in which the NCTE from SRV-1 ("CTE") was inserted to replace the wild type HIV-1 NCTE (RRE) were used in the SCID-hu mouse model to demonstrate the replication and cytopathic effect of CTE(+) attenuated HIV-1. CTE(+) hybrids were used to infect a Thy/Liv implant (Kollmann (1995) supra) in
SCID-hu mice. Significantly, CTE-attenuated vimses propagate slower than wild-type and Nef-negative (otherwise NCTE wild-type) HIV-1 clones. Significantly, as measured by HIV-1 proviral DNA levels (as measured by PCR in sequential biopsies), CTE-attenuated HIV-1 replicate about 10 times less efficiently than the SRV-1 NCTE-containing HIV-1. Levels of circulating CD4+ lymphocytes were monitored for 6 weeks after initial infection. No depletion of CD4+ cells was observed. This demonstrates an attenuated phenotype for cytotoxicity of the CTE(+)/RRE(-)-containing HIV-1 clones. Direct comparison to a Nef-negative HIV-1 clone showed that the CTE(+)/RRE(-)- containing vimses are more attenuated, independent of the absence or presence of Nef. Therefore, the replacement of HIV- 1 's wild-type NCTE (RRE) with SRV-1 NCTE is responsible for the distinct, non-cytotoxic (non-CD4+ lymphocyte depleting) phenotype of the slowly replicating hybrid HIV-1.
To distinguish between the effects of Rev and Nef on the HIV-1 viral replicative capacity and pathogenicity, the effects of Rev (-)and Nef(-) mutations on HIV-1 were compared. The recombinant HIV-1 clone into which the SRV-1 CTE had been inserted, termed "pR(-)Rev(-).S," as described in Example 2, was used. This hybrid is a RRE(-)Rev(-)Nef(-) molecular clone of HIV- 1 that contains the SRV-1 NCTE inserted within the ne/gene. As discussed above, Rev and RRE were destroyed by multiple point mutations designed not to affect the overlapping tat and env open reading frames. To further distinguish between the effects of Rev and Nef on the viral phenotype, a Nef(+) version of the CTE-containing clone, R(-)Rev(-).S, was generated. For this, the N-terminal portion of ne/was reinserted downstream of the SRV-1 CTE and the AUGs preceding the Nef initiation codon were destroyed. Specifically, the ne/coding sequence was reconstmcted in R(-)Rev(-).S. The proximal portion of nef spanning nucleotides 8782 to 8954 was PCR-amplified from HIV-1 NL4-3 using primers gcatcactcgagATAAGATGGGTGGCAAGTGG (sense) (SEQ ID NO: 10) and
GGCACAAGCAGCATTGTTAG (antisense) (SEQ ID NO:l 1). The regions of complementarity are shown in uppercase, while the lowercase sequence contains Xhol site that was used to insert the PCR fragment in the Xhol site of R(-)Rev(-).S. This resulted in pR(-)Rev(-)Nef(+)CTE that contains an intact ne/gene downstream of the SRV-1 CTE. To assure nef expression, all AUG codons within the remaining ne/region upstream of
CTE were converted to CUG by PCR mutagenesis. Two AUG codons within the 241 base pair SRV-1 CTE fragment that do not belong to the known minimal 173 base pair CTE were also mutated to CUG. The Hpal-Xbal fragment was PCR-amplified from pR(-)Rev(-)Nef(+)CTE as two overlapping fragments, using primer pairs ATAGTGCTGTTAACTTGCTCAATGCCACAGCC (SEQ ID NO: 12) and
CTGCTCCCACCCCAgCTGCTGCTGGCTCAGCTCGTCTCAgTCTTTCCCTTACAGC AGGCCAgCCAATCACACTACTTTTTGACCACTTGCCACCCAgCTTATAGCAAAA TC (SEQ ID NO: 13) for the 5'-portion; and GAGACGAGCTGAGCCAGCAGCAGcTGGGGTGGGAGCAG (SEQ ID NO: 14) and gcαtcαgctctαgαCCAAGACAgCAgCCGGGCAGCACGG (SEQ ID NO: 15) for the 3 '-portion. The mutated nucleotides are shown in lowercase, and a flanking region containing the Xbal restriction site is italicized. These two PCR fragments were mixed and reamplified with primers ATAGTGCTGTTAACTTGCTCAATGCCACAGCC (SEQ ID NO: 16) and gcαtcαgctctαgαCCAAGACAgCAgCCGGGCAGCACGG (SEQ ID NO: 17), and the resultant fragment was used to replace the corresponding Hpal-Xbal fragment of Rev(-)RRE(-)Nef(+)CTE(+). The stmcture of all recombinant regions was verified by sequencing on both strands. All viral stocks and cultured cells were generated and prepared as described in Example 2.
Wild type HIV-1 (pNL4-3) and the CTE(+) clones pR(-)Rev(-)Nef(-)CTE (also called pR(-)Rev(-).S), and, pR(-)Rev(-)Nef(+)CTE (i.e., pR(-)Rev(-).S with the Nef reinserted)) were transfected into human 293 cells by calcium phosphate coprecipitation.
One day after the transfection, the cells were harvested and analyzed for viral protein expression. Western immunoblots (see Hadzopoulou (1989) J. Virol. 63:1265-1274) were incubated with HIV-1 patient serum and anti -Nef antibody. Nef protein was detectable only in cells transfected with the Rev-independent Nef(+) plasmid and wt HIV-1 (NL4-3). Both CTE(+) clones produced lower amounts of Env and Gag as compared to wild type vims. Quantitation of Gag protein expression using a p24gαg antigen capture assay (see above) revealed about a 5- 10-fold lower than wild-type levels of production by both of the CTE-containing clones. Thus, use of SRV-1 NCTE in place of wt HIV-1 NCTE results in a slow-growing vims, i.e., a hybrid with a lower replicative capacity. To further characterize the lower replicative potential of CTE-containing
HIV-1, their infectivity in PHA-activated human PBMCs was determined by standard end-point dilutions (see McDougal (1985) J Immunol. Methods 76:171-183). Briefly, viral aliquots were diluted in medium using 5 -fold dilution steps starting with a 1:10 dilution. 100 microliters of Jurkat cell suspension was seeded in 96-well plates at 7.5x10 cells per well. 100 microliters of vims dilution was added to 12 parallel wells. The culture medium was changed twice a week for the next two weeks and on day 14 a 120 microliter sample from each well was analyzed using the p24 αg antigen ELISA assay described above. The ID50 values were defined as the reciprocal of the vims dilution resulting in 50% positive wells using a Reed-Munch calculation (see Albert (1990) AIDS 4:107-112; McDougal (1985) J. Immunol. Methods 76:171-183). The ID50 values for the CTE(+) clones were about 5-fold lower as compared to wild-type HIV-1. No significant difference in the infectivity was observed for the Nef(-) and Nef(+) variants of the CTE(+) viruses, indicating that RRE replacement, rather than Nef, is responsible for this slow-growing phenotype. Next, propagation of the Nef(-) and the Nef(+) HIV-1 clones in human primary cells was determined. Human PBMCs were infected by equal infectious units of the different vimses, using the viral stocks described above. Vims propagation was monitored over time by measuring extracellular Gag production (as described above). Wild-type HIV-1 (NL4-3) and NL4-3/Nef-negative clones had similar growth properties in activated PBMCS': The Nef(-) vims propagated approximately 5- 10-fold less efficiently, whereas the replicative capacity of its Nef(+) counterpart was further decreased by about
5-fold. Protein production was also measured by Western blot analysis. Both p24 ag and Western immunoblot measurements were comparable. Infection by the CTE(+) vimses in PBMCs resulted in lower levels of Gag and Env protein production, further demonstrating that replacement of HIV- 1 RRE with a CTE affected both the infectivity and the replicative capacity of HIV- 1.
The attenuation of the CTE-containing HIV-1 was further demonstrated by measuring its lower replicative capacity in vivo in an art-recognized animal model, the SCID-hu mouse (see Aldrovandi (1993) Nature 363:732-736; Bonyhaki (1993) Nature 363:728-732). The SCID-hu mice are produced by surgical implantation of human fetal liver and thymus under the kidney capsule of severe combined immunodeficient (SCID) mice. Normal T-cell differentiation has been shown to occur in the Thy/Liv implant. This is an art-recognized system for the study of HIV infection and cytotoxicity in human lymphopoietic tissue. Normally, infection by wt HIV-1 results in depletion of CD4+ T cells. Significantly, infection with CTE-containing HIV-1, resulting in low levels of viral replication, did not cause CD4+ cell depletion in vivo.
To further assess the attenuation of the CTE-containing HIV-1 , vims load and cytotoxicity after infection in SCID-hu mice was measured. Vims stocks of wt HIV-1 (NL4-3), NL4-3/Nef negative, CTE(+) Nef(-), and CTE(+) Nef(+) HIV clones were tested. 1000 infectious units/mouse are typically used to establish good SCID-hu infection (i.e., at least 50% to 90% infected) by attenuated strains of HIV- 1 , see Aldrovandi ( 1996) J. Virol.
70: 1505-1511. Since the infectivity and replicative capacity of CTE(+) vimses are reduced (as demonstrated by the experiments described above), the amount of input vims was increased. The SCID-hu mice were infected by injection of vims into the Thy/Liv implant at 500 to 850 TCID50 in a final volume of 100 μl. Sequential biopsies of the implants were performed 3 and 6 weeks postinfection, and the samples were analyzed for vims replication by quantitative DNA-PCR and for the number of CD4+ thymocytes by flow cytometry (PCR and cytometry as described in Aldrovandi (1996) supra). All 7 mice infected by wt HIV-1 scored positive for HIV proviral sequences at 3 weeks postinfection. Depletion of CD4+ thymocytes (defined as measurement of less than 55% CD4+ cells in the thymocyte population) was detected in 2 of 7 mice at this time point, and in another 2 of 2 mice analyzed after 6 weeks. None of the mock-infected mice showed any sign of HIV infection or CD4+ thymocyte depletion. Replication of the NL4-3/Nef-negative was detectable in 2 of 10 mice after 3 weeks and in 8 of 9 mice at 6 weeks post-infection. Depletion of CD4+ thymocytes was found in 3 of 9 mice infected with NL4-3 /Nef-negative recombinants at 6 weeks postinfection (which is typical, as reported in Aldrovandi (1996) supra, and Jamieson (1994) J. Virol. 68:3478-3485).
In contrast, after infection with the CTE(+) Nef(-) and CTE(+) Nef(+) hybrids, provims DNA was detectable only at the 6 week time point in 4 of 8 and 1 of 5 mice, respectively. The viral loads in the mice infected by the CTE(+) vimses were significantly lower than that of the RRE+/Nef-negative hybrids (i.e., wt except for lack of Nef). While implants infected by the RRE+/Nef-negative hybrids showed 3,000 to 28,000 provims copies per 105 cells, the CTE(+) Nef(-) vims contained about 500 to 2,400 copies per 105 cells, resulting in about a 20-fold decrease in average provims copies in the CTE(+) infected mice. In CTE(+) Nef(+) vims only 1 of 5 mice was provims positive after 6 weeks. Analysis of the implants of all the mice infected by CTE(+) vimses showed normal thymocyte profiles, i.e., no cytotoxicity. The vims' ability to produce Nef did not affect pathogenicity. These findings suggest that replacement of wt RRE with an NCTE, in this case NCET from SRV-1, is primarily responsible for the slower growing, attenuated non-cytotoxic phenotype in the SCID-hu mice.
To address the question whether the lower vims load is responsible for the lower cytotoxicity of the attenuated, CTE(+) hybrids, the experiments were repeated using about 5 -fold higher amounts of input vimses (approximately 2500 infectious units/mouse). Infection with these larger amounts of vims also does not lead to CD4+ thymocyte depletion. New stocks for CTE(+) Nef(-) and CTE(+) Nef(+) HIV clones with higher titers were generated yielding 5x104 and 4.7x104 TCID50/ml, respectively. The Thy/Liv implants in SCID-hu mice were injected with 100 μl of these high titer stocks. Biopsies were analyzed at 3 and 6 weeks postinfection. At 3 weeks postinfection, no vims could be detected in the 6 mice infected with the either of the CTE(+) vimses. At 6 weeks postinfection, in all 4 mice infected by the CTE(+)/Nef(-) hybrid and in all 7 mice infected by the CTE(+)/Nef(+) hybrid, HIV-1 replication was detectable. Although the time necessary to detect the CTE(+) hybrid was still 6 weeks, the viral loads were clearly elevated, due to the higher amount of input vimses. The average proviral copies per 105 cells was 15,000 in the case of CTE(+)/ Nef(-) hybrid, which is similar to that obtained from mice infected with 5-fold lower amount of RRE+/ Nef-negative clone. The vims load in the implants infected by the CTE(+)/Nef(+) variant was at least ten-fold lower than that of the CTE(+)/Nef(-) counterpart. Interestingly, even with these differences in viral load, no significant changes were observed in the thymocyte profiles (within the time frame of the experiment, 6 weeks). Therefore, no depletion of CD4+ thymocytes by CTE(+) hybrids was observed. Parallel infections using wt HIV-1 resulted in high levels of vims load accompanied by loss of CD4+ cells. In conclusion, increasing the vims inocula did not increase the low cytotoxicity of the CTE(+) vimses in vivo, as observed in this SCID-hu mice model.
Therefore, replacement of wt HIV-1 RRE with CTE generated hybrids that are less cytotoxic, more attenuated, than Nef-negative (otherwise wt) HIV-1.
In conclusion, using an NCTE functionally analogous to the CTE^ of the invention, these data demonstrate the efficacy of NCTEs as attenuating agents in HIV-1 infection and AIDS pathogenesis in vivo. While CTE(+) HIV-1 hybrids can infect a
Thy/Liv implant in SCID-hu mice, they propagate slower than wt HIV-1 and NL4-3/Nef- negative hybrids. No depletion of CD4+ cells was observed in the CTE(+) hybrids, demonstrating an attenuated phenotype for cytotoxicity. Direct comparison to RRE+/Nef- negative HIV-1 showed that the CTE+ is responsible for attenuation independent of the absence or presence of Nef. Thus, these experiments demonstrate that NCTEs, such as the CTE^ of the invention, can produce a slow-growing, attenuated HIV-1 hybrid in vivo.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid consisting of a Constitutive Transport Element derived from an Intracistemal A Particle (CTE1AP) nucleotide sequence, the isolated CTEIAP nucleic acid defined as having the following properties: (i) the CTEj^sj,, when inserted in a recombinant, hybrid HIV- 1, is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1, and when the CTE[AP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24g╬▒g is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and,
(ii) the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO:l; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
2. The isolated nucleic acid CTEIAP of claim 1 , comprising a ribonucleic acid comprising a motif of contiguous base pairs consisting of:
XA╬╣o-5oo- GUC AAUGAC GGGU AAGA - XB 16.24- ACCU AAGACA GG - Xc 6.100- CAA U GUU - XD 6.100 - CC GAGGAC AGGU - XE 16.24 - CA ACCU AAGACA GGCA -XF 10.500 wherein X is any nucleotide base.
3. The isolated nucleic acid CTE^ of claim 1, comprising a deoxyribonucleic acid comprising a motif of contiguous base pairs consisting of:
XA╬╣o-5oo- GTC AATGAC GGGT AAGA - XB 16.24- ACCT AAGACA GG - Xc 6.l00- CAA T GTT0 - XD 6.100- CC GAGGAC AGGT - XE l6.24- CA ACCT AAGACA GGCA -XF 10-50o wherein X is any nucleotide base.
4. The isolated nucleic acid CTEIAP of claim 1 comprising the sequence as set forth in SEQ ID NO: 1.
5. The isolated nucleic acid CTEIAP of claim 1 wherein the distance between the loop A domain and the loop B domain is between about 21 to about 26 base pairs in length.
6. An isolated transcription product of a CTEIAP nucleic acid, the CTEIAP nucleic acid defined as having the following properties:
(i) the CTEIAP, when inserted in a recombinant, hybrid HIV-1 , is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1, and when the CTEIAP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24 ╬▒g is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and,
(ii) the secondary structure of the CTE^ comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
7. An expression vector comprising a nucleic acid encoding a CTEj^ nucleic acid and a non-naturally occurring nucleic acid sequence, the CTEL^J, nucleic acid defined as having the following properties:
(i) the CTEj^j), when inserted in a recombinant, hybrid HIV-1 , is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1, and when the CTELajj-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV-1 p24g╬▒ is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and,
(ii) the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
8. A transfected cell comprising a polynucleotide encoding a CTEIAP nucleic acid and a non-naturally occurring nucleic acid sequence, the CTEIAP nucleic acid defined as having the following properties:
(i) the CTEIAP, when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV- 1 , and when the CTEj ^-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24g╬▒g is between about 50 fold and about 200 fold less than levels of p24 ag expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and, (ii) the secondary stmcture of the CTEIAP comprises at least a two loop
A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
9. A recombinant retrovims, wherein the retrovims either lacks or has non-functional endogenous post-transcriptional RNA nucleo-cytoplasmic transport elements (NCTEs), further comprising a Constitutive Transport Element from an Intracistemal A Particle sequence (CTELS ,) operatively inserted into the retrovims, the CTEj^ capable of acting as an exogenous functional NCTE to reconstitute the lacking or non-functional endogeous NCTE and to reconstitute the infectivity of the retrovirus in a mammalian cell, the CTEIAP defined as having the following properties:
(i) the CTEIAP, when inserted in a recombinant, hybrid HIV-1 , is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1 , and when the CTEIAP-containing hybrid HIV- 1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24 ╬▒g is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and, (ii) the secondary stmcture of the CTEIAP comprises at least a two loop
A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
10. The recombinant vims of claim 9, wherein the vims is HIV- 1.
11. The recombinant vims of claim 10, wherein the NCTE is RRE.
12. The recombinant vims of claim 10, wherein the HIV-1 further lacks a functional Nef.
13. A vaccine for the prophylaxis or amelioration of a viral infection in a mammal comprising an attenuated retrovims, wherein the attenuated retrovims, when administered as a vaccine in sufficient amounts is capable of eliciting an immune response to the retrovims in a mammal with a functional immune system, wherein the attenuated retrovims lacks an endogenous functional post- transcriptional RNA nucleo-cytoplasmic transport element (NCTE) and/or the ability to express an endogenous functional NCTE binding protein, and the attenuated retrovims further comprises a CTE1AP nucleic acid defined as having the following properties:
(i) the CTE1AP, when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1, and when the CTEIAP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells (huPBMCs), the level of expression of HIV- 1 p24g╬▒g is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild-type NCTE, infects activated huPBMCs; and,
(ii) the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
14. The vaccine of claim 13, wherein the attenuated retrovims is HIV-1.
15. The vaccine of claim 14, wherein the attenuated HIV-1 further lacks a functional Nef.
16. The vaccine of claim 14, wherein the NCTE is RRE and the NCTE binding protein is Rev.
17. A kit for the prophylaxis or amelioration of a vims infection in a mammal, the kit comprising the vaccine of claim 13 and a pharmacologically acceptable carrier.
18. The kit of claim 17, further comprising an instructional material teaching the use of the vaccine, wherein the instmctional material indicates that the vaccine is used for the prophylaxis or amelioration of HIV- 1 infection in a mammal; that the vaccine is to be administered to a mammal in a therapeutically effective amount sufficient to express a viral protein; wherein the vaccine will not cause clinically significant CD4+ cell depletion; and, the expression of the viral protein elicits an immune response to the attenuated HIV- 1 vims.
19. The use of a CTEIAP in the manufacture of a medicament for the prophylaxis or amelioration of a viral infection wherein the CTEIAP is defined as having the following properties:
(i) the CTEIAP, when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV- 1, and when the CTEIAP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells
(huPBMCs), the level of expression of HIV- 1 p24g╬▒g is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and,
(ii) the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
20. The use of claim 19, wherein the viral infection is an HIV-1 infection.
21. A method for eliciting an immune response to a vims in a mammal, comprising administering to a mammal a therapeutically effective amount of an attenuated recombinant vims, wherein the vims comprises a CTE^ - defined as having the following properties.
(i) the CTEj^ ., when inserted in a recombinant, hybrid HIV-1, is capable of functioning as a post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) in place of wild-type NCTE in the hybrid HIV-1, and when the CTEIAP-containing hybrid HIV-1 vims infects activated human peripheral blood mononuclear cells
(huPBMCs), the level of expression of HIV- 1 p24g╬▒ is between about 50 fold and about 200 fold less than levels of p24g╬▒g expression when HIV-1 wild type vims, utilizing wild- type NCTE, infects activated huPBMCs; and,
(ii) the secondary stmcture of the CTEIAP comprises at least a two loop A domain and a loop B domain, wherein the nucleic acid primary sequence of the loops has at least 90% nucleic acid sequence identity, to a loop A and a loop B domain of a nucleic acid comprising the loop stmcture as set forth in Figure 1 and the sequence as set forth in SEQ ID NO: 1 ; and, the distance between the loop A domain and the loop B domain is between about 11 to about 36 base pairs in length.
22. A method for screening for a post-transcriptional RNA nucleocytoplasmic transport element (NCTE) binding protein comprising the following steps: a) providing a composition comprising a loop A and/or a loop B of a
GTEIΛP ; b) contacting the composition with a test compound; and, c) measuring the ability of the test compound to bind the NCTE.
PCT/US1998/027792 1997-12-31 1998-12-29 Murine intracisternal a particle constitutive transport elements and uses thereof WO1999033992A1 (en)

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Cited By (2)

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EP1364071A2 (en) * 2000-11-10 2003-11-26 Bioalliance Pharma (S.A.) Novel method for analysing phenotypic characteristics of human immunodeficiency virus (hiv)
WO2004060304A2 (en) * 2002-12-27 2004-07-22 Sagres Discovery, Inc. Novel compositions and methods in cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TABERNERO C ET AL: "Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of Human Immunodeficiency Virus type 1", JOURNAL OF VIROLOGY, vol. 71, no. 1, January 1997 (1997-01-01), AMERICAN SOCIETY FOR MICROBIOLOGY US, pages 95 - 101, XP002104206 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1364071A2 (en) * 2000-11-10 2003-11-26 Bioalliance Pharma (S.A.) Novel method for analysing phenotypic characteristics of human immunodeficiency virus (hiv)
WO2004060304A2 (en) * 2002-12-27 2004-07-22 Sagres Discovery, Inc. Novel compositions and methods in cancer
WO2004060304A3 (en) * 2002-12-27 2007-12-06 Sagres Discovery Inc Novel compositions and methods in cancer

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