CA2107789A1 - Retrovirus inhibition with antisense nucleic acids complementary to packaging sequences - Google Patents

Retrovirus inhibition with antisense nucleic acids complementary to packaging sequences

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Publication number
CA2107789A1
CA2107789A1 CA002107789A CA2107789A CA2107789A1 CA 2107789 A1 CA2107789 A1 CA 2107789A1 CA 002107789 A CA002107789 A CA 002107789A CA 2107789 A CA2107789 A CA 2107789A CA 2107789 A1 CA2107789 A1 CA 2107789A1
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Prior art keywords
rna
antisense
dna
packaging
sequence
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CA002107789A
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French (fr)
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Thomas E. Wagner
Lei Han
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Ohio University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/105Murine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/02Animal zootechnically ameliorated
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • A01K2267/0337Animal models for infectious diseases
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Abstract

Antisense nucleic acid molecules are provided which hybridize essentially only to the packaging sequence of a retrovirus and thereby inhibit the packaging of the retroviral genomic RNA. The antisense molecules may be DNA, RNA, or analogues thereof. The antisense molecules may be administered as drugs, or the individual to be protected may be genetically manipulated to express an antisense RNA.

Description

. WO92/1721l PCT/US92/02911 :~ 2~ 977~q `
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RETROVIRUS }N~IBITION WITH ANTISENSE NUCLEIC ACIDS
~ COMPLEMENTARY TO PACKAGING 6EQUENCES
-~ BACRGROUND OF T~E INVEN~ION

Field of the Invention ~ This invention, which is in the field of virology -~ and medicine, relates to DNA sequences encoding an antisense RNA molecule capable of hybridizing with a retrovirus packaging sequence and thereby inhibiting a retrovirus infection, the antisense RNA sequences, ~¢ hosts transfected with the DNA sequences, and methods -for rendering cells and animals resistant to retrovirus infection.
. ~ _ Descri~tion of the Backqround Art A. RETROVIRUSES
Retroviruses are a major threat to the health of humans and animals. This is-most dramatically demonstrated by the role of HIV-1 in the worldwide AIDS epidemic. To date, no effective treatment or cure for retroviral infections has been found. It is widely accepted that the damage caused (both directly and indirectly) by retroviruses as infectious agents has become the most serious globaL challenge to medical science in recent times. Hence, the need for effective methods and compositions for prevention or treatment of retrovirus injections is abundantly clear.
Retroviruses comprise a large class of RNA
viruses which have the property of "reverse transcribing" their genomic RNA into DNA which can integrate into the host cell genome. Many members of this virus class are tumorigenic. These viruses require only a limited number of genes and genetic regulatory sequences to complete their cycle of infection in host cells or organisms (1-4). This SUBSTITUTE SHEET
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~'' WO92/17211 2 1 ~ 7 7 g 9 PCT/US92/02911 remarkable molecular efficiency partially explains the effectiveness of retroviruses, such as human immunodeficiency virus (HIV), as pathogens. The existence of distinct genes and gene products also suggests targets for molecular attenuation of retroviral replication in host organisms.
Retroviruses are small, single-stranded positive-sense RNA viruses. Their genomes contain, among other things, the sequence for the RNA-dependent DNA
polymerase, reverse transcriptase. Many molecules of reverse transcriptase are found in close association with the genomic RNA in the mature viral particle.
Upon entering a cell, this reverse transcriptase i l5 produces a double-stranded DNA copy of the viral genome, which is inserted into the host cell's , .
chromatin. Once inserted, the viral sequence is called a provirus. In some ways retroviraI
integration resembles that of various eucaryotic mobile genetic elements such as Copia and 412 of Drosophila or Ty-l in yeast. In the case of these transposable elements and retroviruses, long stretches of highly conserved "sequence are" flanked by inverted repeats. Also, integration of any of these entities results in the production of short, direct repeats of the host cell's chromatin.
Although the complete details of the integration of retroviral DNA into the genome of its host cell (formation of proviral DNA) have not been worked out, certain facts about it have been established.
Retroviral integration is directly dependent upon viral proteins. Linear viral DNA termini (the LTRs) form the structure allowing integration of the proviral DNAo There is a characteristic duplication of short stretches of the hosts DNA at the site of integration.
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~ W092~17211 3 2~ ~7 PCT/USg2~0291l ` -- The retroviral protein directly involved in inserting the viral DNA into the host DNA is called the i~tegrase protein (IN). The sequence of the IN
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protein is encoded in the 3' part of the viral polymerase gene. After translation it is proteolytically processed from the larger precursor molecule to yield an active protein (of 46kd in Mo-;n MLV; in avian viruses and the human immunodeficiency virus it is 32kd). During integration the IN proteinremoves bases from the 3' hydroxyl termini of both strands of the reverse transcriptase produced viral ~` DNA. These 3' ends are covalently attached to 5'-phosphoryl ends of the host cells' DNA. The IN
`~ ~5 protein of all retroviruses is thought to have an endonuclease activity which results in the production ~i of a staggered cut in the host DNA at the site of '- integration. The filling in of this staggered cut by ~ cellular enzymes after the ligation of the viral DNA
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to the host DNA results in the duplication of the ,i short sequences of the host DNA.
Progeny viral genomes and mRNAs are transcribed from the inserted proviral sequence by host cell RNA
~, polymerase II in response to transcriptional, regulatory signals in the terminal regions of the proviral sequence, the long terminal repeats or LTRs.
".j The host cell's protein production machinery is used to produce viral proteins, many of which are inactive until processed by virally encoded proteases.
Typically, progeny viral particles bud from the cell surface in a non-lytic manner. Retroviral infection does not necessarily interfere with the normal life cycle of an infected cell or organism. While most ~; classes of DNA viruses may be implicated in , 35 tumorogenesis, retroviruses are the only taxonomic , group of RNA viruses that are oncogenic. Various "~

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retroviruses such as the Human Immunodeficiency virus (HIV), which is the etiological agent responsible for ~'! acquired immune deficiency syndrome in humans, are , 5 also responsible for several very unusual diseases of ~i the immune systems of higher animals.
-. All retroviruses share common morphological - characteristics. They are enveloped viruses typically around lOOnm in diameter. The envelope is derived ; lO from the cytoplasmic membrane of the host cell as the ~;~ maturing virus buds from that cell. It is covered by qlycoprotein spikes, coded for by the viral genome.
I' The cytoplasmic membranes of infected cells actively ~ transcribing the proviral sequences and processing ; ~5 viral proteins have viral envelope glycoproteins inserted into them. In some cases, these glycoproteins have been shown to cluster on certain regions of the cell membrane. Furthermore, viruses tend to bud preferentially from these regions.
The envelope encloses an icosahedral capsid, or ~ nucleoid, composed of proteins coded for by the virus.
-j, The capsid contains a ribonucleoprotein complex that includes the genomic RNA, reverse transcriptase, the ^,9 integrase protein and certain other factors necessary for the production of the double-stranded DNA copy of ^;~ the viral genome. It is not uncommon for the capsid ;,~. to contain small amounts of non-viral RNA other than ~'~ the cellular tRNAs which are always present. The ;~ cellular tRNAs are base-paired to specific regions on ~- 30 the viral genome and play an important role in reverse transcription as will be described later. There is also an inner coat composed of core proteins found ;~ between the nucleocapsid and the envelope.
'~i There are several classification schemes applicable to retroviruses. What is, perhaps, the ` simplest classification scheme is based on morphology.

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. .~, . , ,~ 21~77~q :; This morphological classification scheme of retroviruses is based on structural similarities apparent in electron micrographs. In this scheme, retroviruses are divided into four groups or types of particles designated as A-type, B-type, c-type and D-type.
The A-type particles are non-infectious and are i found only within cells. They range in size from 60-`'! 10 90nm. They do not have an encapsulating membrane.
They may be found intracisternally or intracytoplasmically. Their classification is further subdivided on this basis. The role of the intracisternal A-type particles is unknown, but the intracytoplasmic particles appear to be immature, or precursor forms, to B-type mouse mammary tumor ~i- virions. It has also been speculated that they might be retrotransposons.
B-type particles exhibit very prominent spikes on their envelope surface, and their nucleoids are eccentrically located. Mouse mammary tumor virus is the primary example of this type of retrovirus.
C-type particles represent the largest morphological class of retroviruses. The envelope spikes of the C-type viral particles vary greatly in size and quantity, but all viruses of this type have a ~; centrally located core in the mature virion. Moloney murine leukemia virus is a typical C-type virus.
-~ The D-type particles exhibit the same eccentrically located nucleoid as the B-type.
~ However, the spikes of the D-type are noticeably '~ shorter than those of the B-type. Examples of this type have only been found in primates.
To understand many of the concepts presented 3S herein, it is necessary to understand the organization of a typical retrovirus. The Moloney murine leukemia .~

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virus (Mo-MLV) will serve as an example. The sequence of the entire virus is known, see Shimnick, et al., Nature, 293:543-48 ~1981) and Miller and Verma, J.
Virol., 49:214-22 (1984). As with all retroviruses, Mo-MLV carries two copies of its genomic RNA in the mature viral particle. The diploid RNA genome of ~i retroviruses is unique among viruses and is a necessary component of the reverse transcription process. The identical subunits of the viral genomic ~` RNA exhibit several characteristics of a eucaryotic . mRNA molecule. The 5' end of the molecule carries a typical in ~WA cap structure (m7 G5 'ppp5 'Gm). A poly-`~!, A tail of about 200 residues is attached to the 3' ~,~ 15 end, and several internal adenosine residues are - methylated.
~;3 Besides the cap and the poly-A tail, three primary coding regions and six functional regions can be identified on the viral RNA. A copy of the highly conserved LTR, or long terminal repeat, region is found at both ends of the molecule. Like the diploid ~ genome, these are needed for successful reverse ,~j transcription. The 5' LTR region includes sequences :~ having promoter and enhancer activity. The LTR region also contains a poly-A addition signal. The 5' LTR
region is followed by the U5 region. Next, is the L, or untranslated leader,~ sequence. The L region ;~ includes the primer binding (PB) site for the ` initiation of negative strand DNA synthesis, the PB-;'` 30 site. A molecule of tRNA, which is used as a primer in the initiation of negative strand DNA synthesis, is base-paired to the PB-site. It also includes the site, which is required for encapsidation of the viral . .,~
genome.
~ 35 Next are the three major coding regions: qaa, or s the group-specific antigen gene, which code for the .~ .

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~ W092/17211 ~ 7`~ 1 ~ 7 7 ~9 PCT/US92/02911 -viral core proteins; ~ol, which encodes the viral ymerase (or reverse transcriptase); and env, which encodes the envelope proteins and glycoproteins.
` 5 These are followed by the PB+ site, which binds a primer used in positive strand DNA synthesis. Next, ; is the U3 region, which contains the viral enhancer and promoter. The U3 region is followed by the second ~:~ copy of the LTR region.
`~s lO The mature viral particle of MLV contains nine major proteins. These are produced by the post-translational processing of primary translational products. These proteins are typically named according to a system introduced in 1974. In this system, the symbol "p" (for protein) or "gp"
(glycoprotein) is followed by a number showing the j~ approximate molecular weight of the protein in kDa as ;~j determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) (23). Four internal -~
structural proteins are products of the viral qaq -~ gene. They are: p30, the major capsid protein; pl5, a hydrophobic matrix protein; plO, a basic protein found in the nucleocapsid; and pl2, an acidic phosphoprotein often designated as ppl2, whose virion location and function have yet to be determined.
There are two major envelope-associated proteins encoded from thé env gepe. They are the glycoprotein, gp70, and the nonglycosylated plS protein. The pl5 protein is a transmembrane protein which attaches to gp70 and anchors it to the membrane. The gp70 protein attaches the mature virion to cell surface viral receptors.
The viral polymerase protein is also referred to as p70. In Mo-MLV, this protein appears to be a dimer held together by noncovalent and disulfide bonds (26).
Besides acting as an RNA-dependent DNA polymerase, SUBSTITUTE SHEET

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WO92~17211 2 1 0 7 7~ 9 -- 8 ~ PsCT/US92J02911 reverse transcriptase also has RNase H activity, which results in digestion of the RNA in a RNA-DNA hybrid into short oligonucleotides. Two other proteins are thought to be products of the ~ol gene (in at least some retroviruses). A viral protease, pl4, is responsible for maturation of other viral proteins.
An integrase protein, p46 (in MuLV), acts to join or insert the double-stranded DNA copy of the viral genome into the host cell DNA (27, 28, 29, 30).
Other retroviruses feature additional retroviral genes. ThUs, in the HTLV-I retrovirus, we find tax and rex. The product of tax, essential for viral replication, is a transacting transcription-activating factor which enhances viral gene expression (Seiki, M.
et al., Proc. Natl. Acad. Sci. USA 80:3618-3622 (1983); Sodorski, J.G. et al., Science 225:381-385 (1984); Chen, I. et al., Science 229:54-58 (1987)).
The rex gene, also required for viral replication, is a posttranscriptional regulator of viral gene expression (Hidaka, M. et al. EMBO J. 7:519-523 (1988); Inoue, J. et al., Proc. Natl. Acad. Sci._USA
84:3652-3657 (1988)).
The HIV-1 retrovirus also possesses genes modulating viral replication, including v~ F~_tatl rev vpu, and nef (Haseltine, W.A., J. Acquired Immune DeficieDçy_Syndrome 1:217240 (1988)).
Another required element for retroviral replication is the cis-acting viral genomic sequences necessary for the specific encapsidation of the genomic viral RNA molecules into virus particles (4-10). These packaging sequences, termed Psi, have been identified and exploited in the construction of retroviral vectors designed for gene transfer (10-13).
Functional packaging sequences are absolutely required for retroviral replication in host cells. When ,;
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~ WO92/172~1 PCT/US92/0291l packaging sequences were deleted ~rom the retroviral genome (to prevent viral multiplication) and the DNA
was transfected into host cells, the cells could produce all the viral proteins and viral RNA, but could not package the viral RNA genome into an infectious particle. However, such cells could serve as "helper" cells and complement a DNA vector which contained only the retroviral LTRs and the packaging - 10 sequences. The "helper" cell provided: (a) the g3g=
encoded proteins needed to package the vector RNA; (b) the envelope protein to form the capsid; and (3) the ~ reverse transcriptase to convert the RNA genome into n~i a DNA copy upon arrival into the infected cell. The sequences required for RNA packaging into virions have been defined and Shown t~ reside between the 5' LTR
and the beginning of the early portion of the g~
~-- gene.
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B- ANTISENSE RNA
Gene expression involves the transcription of pre-messenger RNA from a DNA template, the processing of the pre-messenger RNA into mature messenger RNA, ;~ and the translation of the messenger RNA into one or more polypeptides. The use of antisense RNA to inhibit RNA function within cells and whole organism has generated much recent interest (14-16). Antisense RNA can bind in a highly specific manner to its complementary sequences ("sense RNA"). This blocks the processing and translation of the sense RNA and may even disrupt interactions with sequence-specific RNA binding proteins (17-20). For example, a plasmid was constructed leaving a promo~er which directed the transcription of a RNA complementary to the normal thymidine kinase (TK) mRNA. When such plasmids, together with plasmids containing a normally expressed ,~ .
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2~ ~7r~q - TK gene, were injected into mutant mouse L cells lacking TK, the presence of the antisense gene substantially reduced expression of TK from the normal ~ 5 plasmid (Izant et al., Cell 36:1007 (1984).
- Antlsense oligonucleotides have been shown to be -~ inhibitory in various viral systems. ZamecniX and Stephensen, Biochemistry, 75:280-84 (197B) inhibited Rous sarcoma virus (a retro~irus) production in `~ 10 cultured CEF cells by adding an oligodeoxynucleotide, complementary to 13 nucleotides of the 3' and 5' LTRS, - to the culture medium. The DNA was terminally blocked to reduce its susceptibility to exonucleases. They ~ speculated that this antisense DNA might act by - ~5 blocking circulati~atoin, DNA integration, DNA
transcription, translation initiation or ribosomal association. Note the conspicuous absence of any reference to interference with packaging.
Chang and Stollzfus, J. Virol., 61:921-24 (1987) inhibited the same virus by means of antisense RNA, ' which they hybridized to the coding region or to the ~`~r'' 5' or 3' flanking regions of the env gene.
Gupta, J. Biol. Chem., 262:7492-96 (1987) inhibited translation of the Sendai virus nucleocapsid ~?,` 25 protein (NP) and phosphoprotein (P.C) mRNAs by means of antisense DNAs complementary to the 5' fla~king region. Oligon~cleotides complementary to the coding i region had no effect on translation. They were unable to explain this difference in effectiveness.
Based on the evidence presented above, a number ~ of laboratories have tried to block transmission of x~ HIV-1, which is the causative agent of AIDS, by blocking viral gene expression with antisense RNA.
~` Thus far, none of these efforts has succeeded.
Antisense experiments have been devised and undertaken by the NIH. S. Amini, Mol. Cell. Biol.

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(1986) 6(7):2305; M. Matsukura, PNAS (1987) 84:7706;
J. Holt, MCB (1988) 8 (2):963; K. Croen, Science News ~-- (1989) 132:356. such experiments are typically directed to testing the ability of antisense agents to block retroviral replication. The current focus in the art emphasizes the notion that blocking - replication means the blocking of the expression of - viral proteins, such as the Rev protein. Because of 10 this emphasis, the assays used to test antisense sequence for antiviral activity measure the expression of a HIV-l gene product. See S. Gott, J. Virol (1981) s 38(1):239 (RT Test); J. McDougal, J. Imniunol. Meth.
-~; (1985) 76:171 (antigen detection). RNA northern blot 15 analysis, the expression assay that is most typically used to test antisense nucleic acid inhibition of HIV-1 (See Thomas, PNAS, 77:3201, 1980), annot detect useful antisense sequences that are homologous to the viral packaging sequence.
Ruden and Gilboa, J. Virol., 63:677-682 (Feb.
1989) inhibited HTLV-I replication in primary human T
cells engineered to express an antisense RNA. One antisense RNA was directed against the first kilobase of the tax gene CDNA. The other was a 1.1 kilobase HindIII-Pst I fragment from the 5' end of the proviral DNA. The latter target is said to include the 5' splice site, the tRNA Rrimer binding site, and "possibly" signals for packaging of genomic virus RNA.
Antisense-encoding DNAs were operably linked to either the SV40 early promoter or the cytomegalovirus immediate early promoter. Only the vectors expressing the antisense RNA under the control of the CMV
~; promoter exerted an inhibitory effect on cell proliferation, though the SV40 early promoter/anti-tax gene also depressed viral production.
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Large antisense molecules of the sort advocated - by Ruden and Gilboa have several disadvantages. They `~ are difficult to synthesize (particularly if abnormal bases are incorporated as discussed below). They conceivably could recombine with the original virus, another virus, or an oncogene. They are also more liable to form secondary structures which interfere with their binding to the viral target. Additionally, . 10 they are more prone to hybridize to cellular DNA, ^r , thereby possibly blocking expression of essential genes.
In addition to the use of antisense oligonucleotides containing normal bases, various investigators have utilized sequences containing nucleoside or nucleotide analogues to block gene , expression. Such analogues have the advantageous properties of resistance to nuclease hydrolysis and ~r, improved penetration of mammalian cells in culture (Miller, P.S. et al., Biochemistry 20:1874-1880 (1981)). For example, an oligo(deoxyribonucleoside phosphonate) complementary to the Shine-Dalgarno sequences of 16S rRNA inhibited protein synthesis in E. coli but not mammalian cells (Jayaraman, K. et al., Proc. Natl. Acad. Sci. USA 78:1537-1541 (1983)). Such oligomers complementary to initiation codon regions of ; rabbit globin mRNA inhibited translation in cell-free systems. (Blake, K.R. et al.. Biochemistrv 24:6139-6145 (1985)) while oligomers complementary to the initiation codons of vesicular stomatitis virus mRNAs inhibited viral but not cellular protein synthesis in infected L cells (Miller, P. et al., Feder. Proc. 43, abstr. 1811 (1984)). More recently, an ,,f;, oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5 was shown to .,, t: SUBSTITUTE SHEET

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selectively inhibit viral infection (Smith, c.c.
et al., Proc. Natl. Acad. Sci. USA 83:2787-279 (1986)).

8UMMA~Y OF T~E IN~ENTION
An object of the present invention is to overcome the deficiencies noted above.
..... .
~ More particularly, the presen~ inventors have "~.~ l0 taken a novel approach to the inhibition of retrovirus replication based on the blockade of virus packaging through hybridization of antisense RNA essentially ~; only to packaging sequences of the viral genome. In one embodiment, the inventors have constructed recombinant plasmids in which murine leukemia virus . proviral Psi (packaging) sequences, under the ~ transcriptional regulation of lymphotropic virus .~ promoter/regulatory elements from the Moloney-MuLV LTR
or the cytomegalovirus immediate early region, were inserted in reverse orientation. This gives rise to production of antisense RNA complementary to Psi, which achieves complete inhibition of productive virus infection.
When these antisense sequences.were also introduced into cells in vitro and stably transformed .~ çell lines isolated, the cells were resistant to M-MuLV infection, and pro~duced only virus devoid of packaqed viral RNA.
^~;, When linear fragments containing the antisense Psi and the appropriate transcriptional regulatory sequences from these plasmids were introduced into the ~-~ mouse germ line by zygote microinjection, the presence of the antisense Psi RNA was detected in the lymphocytes of these transgenic mice. Upon challenge with the appropriate retrovirus (M-MuLV) none of the .~ .

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: WO 92tl721 I PCr~US92/0291 1 ,~ 210778q antisense Psi transgenic mice developed any symptoms of leukemia.
More generally, the present invention is directed to an antisense molecule capable of specifically hybridizing to the packaging sequence of a retrovirus ;~ and thereby inhibiting essentially only the packaging ~- of the genomic RNA of the retrovirus. Preferably, the -~ antisense molecule is less than about 100 bases, and ` lo more preferably less than about 60 bases, and it may be DNA, RAN, or an analogue thereof. The antisense molecule may be administered directly like a drug or, ~,: if an RNA, it may be generated in vivo in the subject through expression of an introduced gene. Where the -~: 15 antisense molecule is administered directly, it may be composed of a nuclease-resistance RNA or DAN analogue that penetrates the cell membrane.
~ The invention includes recombinant DAN molecules .~ comprising a DNA sequence which is transcribable into such an antisense molecule and hosts transformed or transfected with the above DNA sequence, preferably a ~' mammalian cell host, most preferably a human cell.
The invention further relates to a method for rendering a cell resistant to productive infection by ~, 25 a retrovirus comprising inserting into the genome of the cell a DNA sequence, operably linked to a promoter, wherein the DNA sequence is transcribable ;j, into an antisense RNA molecule which hybridizes to the packaging sequence and thereby inhibits the packaging of the genomic RNA of the retrovirus, thus rendering the cell resistant to productive infection.
`~. The present invention includes a method for rendering a vertebrate animal, such as a mammal, resistant to productive infection by a retrovirus comprising inserting into the genome of essentially all of the germ cells and somatic cells of the mammal '~:

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a DNA sequence containing the packaging sequence of i the retrovirus or a segment thereof in reverse `~ orientation operably linked to a promoter and regulatory elements, wherein the DNA sequence or segment is transcribable into an antisense RNA
molecule capable of inhibiting the packaging of the genomic RNA of the retrovirus, thereby rendering the mammal resistant to infection. The DNA se~uence is ~; 10 preferably introduced into the mammal or its ancestor at an embryonic stage.
~,~ The invention therefore relates also to a transgenic non-human mammal essentially all of whose germ cells and somatic cells contain the above DNA
~' 15 sequence, a transgenic in which said DNA seauence has been introduced into the mammal or its ancestor of ~, said mammal at an embryonic stage. Also intended is a chimeric animal, including a human, at least some of whose cells contain the above DNA sequence.
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; WO92/17211 PCT/US92/02911 ',` 210'77~q BRIEF DESCRIPTION OF ~E DRAWING8 Fiqure 1 is a schematic diagram showing the ~ construction of plasmids pLPPsias (A) and pCPPslas i 5 (B)-Fiqure 2 is a partial sequence (SEQIDNO:l) of the ~ Moloney-Murine leukemia virus (M-MuLV) in the region .' including the packaging sequence. The CAP, primer ~;, binding site, splice donor site, core packaging sequence, and the beginning of the qiq gene are "~; marked.
We have found that antisense molecules ~ complementary to the primer binding site or to the ,~ splice donor site, but not to the packaging sequence ~- 15 of MoMLV, did not show significant inhibition of the virus infection while tha best inhibition observed was with anti-sense oligos complementary to the open bold ~;~ sequence, i.e., the core (bases 301-350) of packaging . r' site.
Fiqure 3 is a partial sequence (SEQIDNO:2j of the "~,, genomic RNA of bovine leukosis virus, with the region "~ (341-415) expected to include the packaging signal indicated by open bold letters.
Figure 4 shows the results of the plaque assay.
Figure 5 is a partial sequence (SEQIDNO:3) of HIV-1 in the region including the packaging sequence.

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DE~;CRIPTION OF THE PREFERRED ENBODIMENq~13 Interference with the specific interactions - between the Psi sequences of viral genomic RNA
~' 5 necessary for packaging and virion capsid proteins will block the retroviral replication cycle. This interference may be accomplished through the use of i~ antisense nucleic acids (DNA or RNA) complementary to ,~ a part of the packaging sequences, which hybridizes lo thereto and thereby inhibits replication.
Antisense sequence directed against a retroviral ` gene would not block replication as effectively as the '~ antisense molecules of the present invention. An antisense RNA molecule directed against a retroviral ` 15 gene competes with normal messenger RNA for binding to -~ ribosomal RNA, while one directed against the packaging sequence competes with a qaa-encoded core protein for binding to genomic RNA. RNA-RNA
-i interactions are stronger than RNA-protein 20 interactions.
In order to test the efficacy of such antisense RNA in blocking retroviral replication in cells and in whole animals, the present inventors constructed transgenic mice expressing RNA sequences complementary 25 to the Psi sequences of Moloney murine leukemia virus '~r~. (M-MuLV). It was discovered that such animals completely resisted challenge with this leukemia virus.
The present invention is therefore directed to ~;~ 30 the use of antisense RNA and DNA molecules s complementary to retroviral packaging sequences as agents for the prevention and treatment of diseases in which the causative agent is a retrovirus.
The antisense RNA and genes coding therefor are ` 35 intended to encompass sequences capable of hybridizing to the packaging sequence of a retrovirus. The .

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~1 ~77.~Cl preferred retroviruses of the present invention include both human and other animal retroviruses.
Preferred human retroviruses, include Human Immunodeficiency Virus-1 (HIV-1) (or Human T-Cell Lymphotropic Virus-3 or Lymphadenopathy Associated Virus), Human Immunodeficiency Virus-2 (HIV-2), Human T-Cell Lymphotropic Virus-I (HTLV-1), and Huma~ T-Cell Lymphotropic Virus-2 (HTLV2). Also intended within the scope of the present invention are additional retroviruses of other animal species, most particularly agriculturally important animals such as cows and chickens, and pets such as dogs and cats.
A non-limiting list of additional retroviruses included within the scope of the present invention is provided in Table I, below. Retroviruses are described in detail in weiss, R. et al. (eds), RNA
Tumor Viruses. "Molecular Biology of Tumor Viruses,"
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, which is hereby incorporated by reference.
The target packaging sequence to which the antisense RNA of the present invention is to hybridize in order to inhibit retrovirus infection is be selected based on examination of the sequence of the retroviral genome. Since the packaging sequences of all retroviruses studied are located between the primer binding site just 3' of the 5' LTR and the initiation sequences of the first coded viral protein (usually the qaa protein coding sequences), sequences within this region (such as, for example bases 200-340 in the proviral genome of HIV-l, isolate ELI) are preferred candidates for antisense targeting. Because of the homology in this region among various retroviruses, an antisense sequence designed for one particular virus may be used successfully to inhibit replication of another virus.
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` ;` W092/1721l PCT/US92/02911 2 1 1~ 7 ~ ~ q i..~, ~ Thus, for example, preferred antisense ; aligonucleotides are specific for the region between ;- bases 280-330 of the Mo-MuLV sequence since it is homologous to HIV-l. since different isolates of HIV-1 have the same packaging sequences, but at slightly ~ different positions within the genome, the same -, sequences would be effective in all HIV-1 strains.
',$~ One preferred anti-sense sequence is one which is - 10 substantially complementary to all retrovirus ; packaging sequences, thus serving as a "universal"
; inhibitor. A sequence complementary to the core packaging sequence from HIV-I (see J.Virol., 63:4085, ,~ 1989), may be worth considering in this regard.
Alternatively, oligonucleotide sequences may be ~-~ produced that are "consensus" packaging sequences -~ having a high degree of complementarity to the packaging sequences of a set of retroviruses, or which are specific to a particular retroviruses.
Shown below is an exemplary oligonucleotide : sequence according to the present invention, useful for inhibition of HIV-1, which are complementary to viral genomic RNA, viral mRNA, as well as the viral DNA sequences. For the packaging sequence of HIV-1, 25 see (Lever J. Virol., 63(9):4085-4097 (1989); Rorman, et al., (Proc. Natl. Acad. Scl. USA 84:2150-2154 (1987).
SEQ ID NO: 4 is a 44mer sequence shown below, which is complementary to the HIV-1 packaging ;~i30 sequence.

CTCATGCGGTTTTAAAACTGATCGCCTCCGATCTTCCTCTCTC
The sequence begins (5') immediately after the S.D site, and ends (3') just prior to the gag/Met site. The underlined 19 bases are complementary to the "core" of the HIV packaging sequence. A 19-mer ....
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antisense molecule complementary to this core region, and a 27-mer complementary to the core and to the four flanking bases on either side, likewise are useable in the present invention. The invention is not limited to any of these packaging site-targeting sequences, but rather includes shorter and longer sequences.
Table IV identifies a number of packaging sequences of interest.
The antisense molecule (e.g., RNA) of the present invention preferably has 100% complementarity to at least a significant subsequence of the packaging sequence (on one strand of the viral DNA) for which it is targeted. Thus, the DNA strand encoding this RNA
should be 100% homologous to the DNA strand which is complementary to the packaging sequence. In another embodiment, the sequence may have a lower degree of - homology, such as at least about 60 or 80%. The homology must be sufficient such that the antisense RNA hybridizes to the target packaging sequences with sufficient affinity to achieve its purpose, i.e.
inhibition of viral packaging.
The efficiency of such hybridization is a function of the length and structure of the hybridizing sequences. The longer the sequence and the closer the complementarity to perfection, the stronger the interaction. As the number of base pair mismatches increases, the hybridization efficiency will fall off. Furthermore, the GC content of the packaging sequence DNA or the antisense RNA will also affect the hybridization efficiency due to the additional hydrogen bond present in a GC base pair compared to an AT (or AU) base pair. Thus, a target subsequence richer in GC content is preferable as a target.
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It is desirable to avoid sequences of antisense RNA which would form secondary structure due to intramolecular hybridization, since this would render the antisense RNA less active or inactive for its intended purpose. One of ordinary skill in the art will readily appreciate whether a sequence has a tendency to form a secondary structure. Secondary structures may be avoided by selecting a different target subsequence within the packaging site.
An oligonucleotide, between about 15 and about 100 bases in length and complementary to the target subsequence of the retroviral packaging region may be synthesized from natural mononucleosides or, , ~, ~r15 alternatively, from mononucleosides having substitutions at the non-bridging phosphorous bound oxygens. A preferred analogue is a methylphosphonate ianalogue of the naturally occurring mononucleosides.
More generally, the mononucleoside is any analogue ~J~20 whose use results in oligonucleotides which have the ~,~advantages of (a) an improved ability to diffuse . ~,., through cell membranes and/or (b) resistance to nuclease digestion within the body of a subject ~(Miller, P.S. et al., Biochemistry 20:1874-1800 -~25 (i981)). Such nucleoside analogues are well-known in ~;the art, and their use in the inhibition of gene expression are detailed~in a number of references (Miller, P.S. et al., supra; Jayaraman, K. et al., supra; Blake, K.R. et al., supra; Miller, P. et al., feder. Proc. 43, abstr. 1811 (1984); Smith, C.C. et al., su~ra).
`~J' Basic procedures for constructing recombinant DNA
and RNA molecules in accordance with the present invention are disclosed by Sambrook, J. et al., In:
Molecular Clonina: A LaboratorY Manual, Second 'Edition, Cold Spring Harbor Press, Cold Spring Harbor, .`~.SU8ST1TUTE SHEET

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NY (1989), which reference is herein incorporated by ~;~ reference.
r,~ Oligonucleotide molecules having a strand which -~ 5 encodes antisense RNA complementary to the target ~ retrovirus packaging sequences can be prepared using .¢ procedures which are well known to those of ordinary skill in the art (Belagaje, R., et al., J. Biol. Chem.
254:5765-5780 (1979); Maniatis, T., et al., In:
lo Molecular Mechanisms in the Control of Gene Expression, Nierlich, D.P., et al., Eds., Acad. Press, NY (1976); Wu, R., et al., Prog. Nucl. Acid Res.
Molec. Biol. 21:101-141 (1978); Khorana, R.G., science ~,; 203:614-625 (1979)). Additionally, DNA synthesis may -`~ 15 be achieved through the use of automated synthesizers.
'-7' Techniques of nucleic acid hybridization are disclosed ' r, by Sambrook et al. (su~ra), and by Haymes, B.D., et al. (In: Nucleic Acid Hybridization, A Practical ~ Approach, IRL Press, Washington, DC (1985)), which `,,r~ 20 references are herein incorporated by reference.
An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing an RNA
or protein product. Expression of the cloned ~;,3~, sequences occurs when the expression vector is introduced into an appropriate host cell. If a '`n ~ prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell ~'~ capable of expressing the cloned sequences.
~ Similarly, when a eukaryotic expression vector is ,~¦ employed, then the appropriate host cell would be any --~' eukaryotic cell capable of expressing the cloned sequences.
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`: WO92/17211 PCT/US92/02911 21~ ~7 ~9 DNA sequence encoding the antisense RNA of the - present invention may be recombined with vector DNA in accordance with conventional techniques, including 5 blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate ~; termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable ,3' joining, and ligation with appropriate ligases.
lO Techniques for such manipulations are disclosed by Sambrook et al., suDra, and are well known in the art.
~" A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a mRNA if it contains nucleotide sequences which contain transcriptional 15 regulatory information and such sequences are "operably linked" to nucleotide sequences which encode ~; , the RNA. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but in general include a promoter which ;~ 20 directs the initiation of RNA transcription. Such regions may include those 5'-non-coding sequences involved with initiation of transcription such as the TATA box.
If desired, the non-coding region 3' to the gene 25 sequence coding for the desired RNA product may be obtained by the above-described methods. This region ~ may be retained for its transcriptional termination
3 regulatory sequences, such as those which provide for termination and polyadenylation. Thus, by retaining 30 the 3'-region naturally contiguous to the coding sequence, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in t~e host cell may be substituted.
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! Two DNA sequences (such as a promoter region sequence and a coding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the ~- introduction of a frame-shift mutation in the region - sequence to direct the transcription of the desired -~ gene sequence, or (3) interfere with the ability of -~ the gene sequence to be transcribed by the promoter region sequence. A promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. In order to be "operably linked" it is not necessary that ~? two sequences be immediately adjacent to one another.
s 15 For production of the DNA sequences of the present invention in prokaryotic or eukaryotic hosts, the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable ~ii promoters are inducible, repressible, or, more preferably, constitutive. Examples of suitable ~. prokaryotic promoters include promoters capable of ;;, recognizing the T4 polymerases (Malik, S. et al., J.
Biol. Chem. 263:1174-1181 (1984); Rosenberg, A.H. et al., Gene 59:191-200 (1987) Shinedling, S. et al., J.
~ 25 Molec Biol. 195:471-480 (1987) Hu, M. et al., Gene `~? 42-?1-30 (1986), T3, Sp6, and T7 (Chamberlin, M. et al., Nature 228:227-231 (1970); Bailey, J.N. et al., ;~ Proc. Natl. Acad. Sci. (U.S.A.) 80:2814-2818 (1983~;
; Davanloo, P. et al., Proc. Natl. Acad._Sci ~U.S.A.) -~s 30 81:2035-2039 (1984)); the PR and PL promoters of ~ bacteriophage lambda (~ Bac~terl31~g~ Lambda, s Hershey, A.D., Ed., Cold Spring Harbor Press, Cold ~ Spring Harbor, NY (1973); Lambda II, Hendrix, R.W., -~` Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY
~35 (1980)); the trp. recA, heat shock, and lacZ promoters ;~of E. coli.; the int promoter of bacteriophage lambda;

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the bla promoter of the ~-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind.
Microbiol. 1:277-282 (1987)); Cenatiempo, Y.
(siochimie 68:505-516 (1986)); Watson, J.D. et al.
(In: Molecular Biology of the Gene, Fourth Edition, Benjamin Cummins, Menlo Park, CA (1987) and Gottesman, s. (Ann. Rev. Genet. 18:415-442 (1984)).
Eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J.
Mol. Appl. Gen. 1:273-288 (1982)~; the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982));
the SV40 early promoter (senoist~ c., et zl., Nature (London) 290: 304-310 (1981) and the yeast aal4 gene promoter (Johnston, S.A., et_al., Proc. Natl. Acad.
Sci. (USA) 79: 6971-6975 (1982); Silver, P.A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
For preparation of vectors for use in inhibiting retrovirus infection, in susceptible eukaryotic cells or in whole animals, eukaryotic promoters must be utilized, as described above. Preferred promoters and additional regulatory elements, such as polyadenylation signals, are those which should yield maximum expression in the cell type which the retrovirus to be inhibited infects. Thus, for example, HIV-l, HIV-2, HTLV-l and HTLV-2, as well as the Moloney murine leukemia virus, all infect lymphoid cells, and in order to efficiently express an antisense RNA complementary to the packaging sequence of one (or more) of these viruses, a transcriptional control unit (promoter and polyadenylation signal) are selected which provide efficient expression in lymphoid cells (or tissues). As exemplified below, preferred promoters are the cytomegalovirus immediate .

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early promoter t32), optionally used in conjunction ` with the bovine growth hormone polyadenylation signals (33), and the promoter of the Moloney-MuLV LTR, ~or use with a lymphotropic retrovirus. A desirable feature of the Moloney-MuLV LTR promoter is that it has the same tissue tropism as does the retrovirus.
The CMV promoter is likewise expressed primarily in lymphocyte. The metallothionein promoter has the advantage of inducibility. The SV40 early promoter ;'l exhibits high level expression in vitro in bone marrow cells.
-~ An antisense RNA molecule may be injected into the human or other animal subject to be protected or treated by any compatible route of administration, e.g., intravenously, intramuscularly, subcutaneously or intraperitoneally, or administered by ingestion or inhalation. Special dosage forms, such as slow release capsules or implants, may be used when appropriate. Alternatively an antisense DNA molecule ~ may be provided. DNA is more readily synthesized ln -~ vitro than RNA.
The antisense molecule may be an analogue of DNA
or RHA. The present invention is not limited to use 25 f any particular DNA or RNA analogue, provided it is capable of adequate hybridization to the complementary genomic DNA of a packaging sequence, has adequate resistance to nucleases, and adequate bioavailability p and cell take-up. DNA or RNA may be made more resistant to in vivo degradation by enzymes, e.g., ,~ nucleases, by modifying internucleoside linkages (e.g., methylphosphonates or phosphorothioates) or by incorporating modified nucleosides (e.g., 2'-0-methylribose or 1'-alpha anomers).
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05' ~ lt is also possible to replace the 3'0-P-05' with .~ s other linkages such as 3'0-CH2C(0)-05', 3'0-C(0)-NH5', and 3'C-CH2 CH2 S-C5'.
:j The entire antisense molecule may be formed of such modified-linkages, or only certain portions, such ; as the 5' and 3' ends, may be so affected, thereby ~ providing resistance to exonucleases.
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~ Antlsense molecules suitable for use in the ::.~ present invention include but are not limited to .~ 3s dideoxyribonucleoside methylphosphonates, see Mill, et :
~ al., Biochemistry, 18:5134-43 (19i9), .~

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While direct administration of antisense nucleic . acid drugs provides acute protection, the protective pe~iod is dependent on the half-life of the molecule.
Moreover, the supply of antisense nucleic acid can be increased onIy by further administrations. It may therefore be desirable to provide a self-renewing source of antisense RNA by introducing a recombinant ~; DNA molecule, capable of transcribing said antisense i .
~ 20 RNA, into one o~ more cells of the human or animal .l subject, thus creating a transgenic or chimeric animal ~ having enhanced resistance to retroviral infection.
~ The recombinant DNA may be delivered to the animal by, c e.g., microinjection of the expression cassette into 25 the animal at the oocyte stage, retroviral vector transfection of the embryo, or intravenous injection of the retroviral vectQr into the fetal or postnatal animal.
Thus, the present invention is also directed to 30 a transgenic eukaryotic animal (preferably a vertebrate, and more preferably a mammal) the germ cells and somatic cells of which contain recombinant genomic DNA according to the present invention which .. : encodes an antisense RNA capable of hybridizing to a ~j 35 retroviral packaging sequence. "Antisense" DNA is introduced into the animal to be made transgenlc, or , .
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` an ancestor of the animal, at an embryonic stage, preferably the one-cell, or fertilized oocyte, stage, and generally not later than about the 8-cell stage.
~` 5 The term "transgene," as used herein, means a gene -~; which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of the RNA transcript o~ t~e transgene in the ~ transgenic animal.
`'.j? 10 There are several means by which such a gene can be introduced into the genome of the animal embryo so as to ~e chromosomally incorporated and expressed.
The DNA may be microinjected into the male or female pronucleus of fertilized eggs. It may also be microinjected into the cytoplasm of the embryonic cells. The cells may be transfected by a retrovirus carrying the transgene. The use of retroviral ~: transfection is not limited to the embryonic stage;
;~ the vector may be intravenously or intraperitoneally :.; io introduced into the fetal or postnatal animal.
`~s Introduction of the desired gene sequence at the fertilized oocyte stage ensures that the transgene is present in all of the germ cells and somatic cells of the transgenic animal and has the potential to be 25 expressed in all such cells. The presence of the transgene in the germ cells of the transgenic "founder" animal in tur~n means that all its progeny ~; will carry the transgene in all of their germ cells and somatic cells. Introduction of the transgene at a 30 later embryonic stage in a founder animal may result in limited presence of the transgene in some somatic cell lineages of the founder; however, all t~e progeny l~ of this founder animal that inherit the transgene ; conventionally, from the founder's germ cells, will , 35 carry the transgene in all of their germ cells and ~ somatic cells.

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Chimeric mammals in which fewer than all of the ~ somatic and germ cells contain the antisense DNA of ;' the present invention, such as animals produced when fewer than all of the cells of the morula or blastula are transfected in the process of producing the transgenic mammal, are also intended to be within the scope of the present invention. Chimeric animals may ' be created by "gene therapy". in which the transgene is typically introduced after birth.
The techniques which may be used include those disclosed by Wagner, T. et al. (Proc. Natl Acad. Sci.
;~ USA 78:6376-6380 (1981)); Wagner et al., U.S. Patent
4,873,191 (1989); Palmiter, R. et al., Ann. Rev.
~, 15 Genet. 20:465-99 (1986); and Leder, U.S. Patent 4,736,866, the entire contents of which are hereby incorporated by reference. Analysis of the progeny -~ mice produced from the microinjected eggs, as well as offspring of transgenic animals bred conventionally, -.~, 20 is achieved by DNA extraction and slot blot hybridization analysis of the DNA for the presence of ~ the transgene (McGrane, M. et al., J. B ol. Chem.
,'J! 263~ 443 11451 (1988)).
-~ Antisense RNA might be delivered to the lymphocytes of AIDS patients by gene therapy methods.
For example, bone marrow cells may be treated with a recombinant, replicatio~n deficient, retroviral vector containing DNA sequences encoding and expressing anti-:i~ sense RNA complementary to the packaging sequences of HIV. In this procedure bone marrow cells would beremoved from the patient, treated with the recombinant retroviral vector and cells in which the DNA genome of ~ the vector had integrated in their chromosomes .. s selected using FACS cell sorting based upon a light visualization marker also incorporated into the ~ retorviral vector (i.e., the ~-galactosidase gene).
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These vector integrated bone marrow cells could then be reintroduced into the patient after oblation of their other bone marrow cells by irradiation. The resulting patient would then have only lymphocytes which encoded the anti-sense RNA sequences to the HIV
packaging sequences and would be resistant to AIDS
just as the transgenic mice described herein are resistant to M-MuLV.
` 10 For commercial animals the method of choice would be transgenic introduction into a line of animals, but several other methods could be used. It is unlikely that gene therapy approaches like the example given for human AIDS protection would be used in animals ' 15 because the procedure is too complex and expensive.
Recently it has been demonstrated that DNA can be introduced into various tissues by attaching it to proteins which are the ligands for cellular receptors (Wu, et al., J. Biol. Chem., 263:14621; 1988), or by unassisted introduction into muscle cells (Wolff, et al., Science, 247:1465; 1990). Using these methods it ~ would be possible to inject animals with DNA
r~ constructs coding for anti-sense RNA directed against the packaging sequences of retroviruses. These "DNA
injections" would provide protection against viral infection within the tissues targeted by the DNA
, delivery system used. This approach would be very ; appealing both for animals and for humans, because it would involve simply the injection of a DNA molecule coding for the anti-sense RNA, or this DNA molecule bound to a receptor targeted ligand. This injection might have to be given several times a year (for example, when an outbreak of a retroviral disease occurs in a given region) but would provide protection against retroviral infection when needed.

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`~. The antisense-RNA encoding DNA may be introduced when the animal (including human) already is infected, or prior to infection, as a prophylactic measure. In the latter case, use of a regulatable promoter may be desirable.
Having now generally described the invention, the same will be more readily understood through reference '''~'!~ to the following examples which are provided by way of . lo illustration, and are not intended to be limiting of the present invention, unless so specified.
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EXAMPLE I
Plasmids Recombinant plasmids pLPPsias and pCPPsias were constructed as shown in Fig. 1~. Plasmids pLJ(21) was cleaved with SmaI and the 540 bp SmaI fragment containing the M-MuLV sequences isolated. Plasmid p3'-LTR-2, containing the 3'M-MuLV LTR (22), was linearized with SmaI and fused to the 540 bp fragment , from pLJ in both orientations using DNA ligase.
-~ Clones of pLPPsi with the antisense orientation were selected on the basis of their KpnI digestion patterns. Plasmid pCMVIE-BGH (30), with an 8 bp BglII
linker introduced at an XmaIII site just 3' of the CMVIE CAP, was cleaved with both BglII and SmaI and the 5.4 kb fragment containing the CMV promoter, the bGH poly-A addition site and a tetracycline resistance selectable marker isolated. The "sticky ends" of this fragment were filled in by incubation with ~' deoxynucleoside triphosphates tA,T,G and C) in the presence of the DNA polymerase I Klenow fragment and blunt-end ligated to the 540 bp SmaI fragment from pLJ
in both orientations. Clones of pCP~as with the antisense orientation were selected on the basis of their KpnI digestion patterns.
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. Transqenic Mouse Production `, The procedure for the production of transgenic
5 mice by direct microinjection of DNA into the male .~ pronucleus of fertilized mouse eggs has been described (24). DNA extraction from mouse tails and slot blot hybridization analysis of the progeny resulting from ~ these microinjected eggs were performed as previously ;; ~0 described (25).

. EXAMPLE III
Cell Culture ~,~ Mouse NIH 3T3 cells were maintained in Dulbecco's `, 15 Modified Eagle's medium (DMEM) containing 10% Nu-serum. Stable cell lines expressing antisense Psi sequences were established by co-transformation with :~ pCPPsias (36 ~g) and pSV40neo (0.36 ~g) (ATCC 33964) ~ using a modified DEAE dextran-dimethyl sulfoxide shock ,, 20 procedure (26).

. EXAMPLE IV
~ DNA and RNA Analysis and Enzyme Assays J The integrity of DNA sequences in transgenic mice 1 25 was analyzed by the method of Southern (27). RNA was ', prepared from lymphocytes by a single step isolation method (28) using Ficoll-Hypaque gradient isolation :1 procedure (29). RNA was then subjected to i electrophoresis at 70V for 4.5h in a 1.2%
30 agarose/formaldehyde gel and transferred onto nitrocellulose paper for hybridization and radioautography as described (30). Reverse - Transcriptase assays were performed as described previously (31). In order to distinguish the 35 antisense RNA strand, a strand specific RNA probe was ~ used for Northern hybridization assays of lymphocyte .
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~, ~ RNA. This probe was synthesized from a pSP65 vector ''~ containing the 540 bp SmaI Psi sequence containing ~, fragment inserted in the sense orientation using SP6 ~ 5 RNA polymerase and labeled guanosine 5'-triphosphates s~ (Promega Riboprobe System, Promega Corp., Madison ~ Wisconsin).

``2 EXAMPLE V
LPsias and CPsias Transgenic Mice Linear DNA fragments containing the M-MuLV
packaging sequences in inverted orientation under the regulation of the M-MuLV LTR or the Cytomegalovirus immediate-early promoter were respectively prepared from pLPPsias as a 2.2kb HindIII fragment or from pCPPsias as a 2.3 k~ EcoRI-Cla~I fragment.
To introduce these sequences into mouse germ lines, the fragments were separately microinjected into the pronuclei of fertilized mouse eggs (200 to 500 copies per egg). These eggs were subsequently ~-~ transplanted into the oviducts of pseudopregnant foster recipient mice. When the resulting offspring were about one month old, a segment of the tail of each animal was removed and DNA was prepared from it.
~; 25 Transgenic mice were detected by DNA slot blot and Southern transfer hybridizations. Two lines of transgenic mice (LPPsias and CPPsias) were analyzed and used in this study. LPPsias transgenics trace to a single founder mouse resulting from the microinjection of the 2.2 kb HindIII fragment from pLPPsias and CPPsias transgenics trace to a single founder from the microinjection of the 2.3 kb EcoRI-ClaI fragment from pCPPsias.
Tail DNA (10 ~g) from the LP~as founder and his ,~ 35 offspring was digested with SacI and EcoRI restriction endonucleases and CPPsias founder and offspring DNA
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was digested with Kpn I and then subjected to electrophoresis at 50V for 9h in a 0.8% agarose gel.
The DNAs were transferred onto nitrocellulose paper by the technique of Southern (27). Following incubation, the nitrocellulose paper was hybridized to a nick translated DNA probe (5 x lO8 to lO X lO8 cpm/~g) prepared from pBR322 for LPPsias DNA (this probe hybridizes to the LPPsias sequences because of the presence of pBR322 sequences in this construction but -~ does not crosshybridize with endogenous retroviral ....~
sequences present in the animal) or a l.3 kb Sac I~
EcoRI fragment from pCPPsi for CPPsias DNA. After washing and drying, the nitrocellulose was autoradiographed by exposure to Kodak film at -70QC
for 16 h. From the Southern blots it was clear that both LPPsias and CPPsias mice contain complete transcription units for the production of the 540 b antisense sequences integrated into their chromosomal components. For LPPsias DNA, the 1470 bp hybridizing SacI-EcoRI fragment shown by each LPPsias mouse ,;., confirmed the presence of the appropriate sequences ~^ and CPPsias mouse DNA from each animal showed the characteristic 400 bp KpnI hybridizing fragment.

;, ~
:~ EXAMPLE VI
Expression of Antisense Psi RNA in LPPsias and CPPs~as Transqenic Mice The transcriptional units introduced into these two lines of mice were constructed to provide the ~ 30 appropriate tissue tropism for the transcription of : the antisense RNA within the lymphoid target tissue ~ for M-MuLV. Within the LPPsias mice the r'' transcriptional unit, including the U3R
promoter/enhancer elements and the RU5 polyadenylation signals from the M-MuLV LTR, is essentially identical to the transcriptional control sequences of the intact .,, . ~

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M-MuLV and would be expected to produce the antisense RNA at the site of viral replication.
The transcriptional control unit within the CPPsias mice, including the cytomegalovirus immediately-early promoter (32) and the bovine growth hormone polyadenylation signals (33), would also be ~, expected to have lymphoid tissue tropism (34). In order to confirm production of antisense M-MuLV RNA
within the lymphoid tissue of these mice, Northern hybridization analysis of RNA from lymphocytes .` isolated from LPPsias and CPPsias mice was performed.
RNA from both CPPsias and LPPsias mice was hybridized ' 15 to a strand specific ~NA probe complementary to the M-MuLV antisense Psi sequence with a specific activity of 1-5 x lO8 cpm/~g. The distinct 600 b hybridizing RNA fragments found in all LPPsias lymphocyte RNA and the 750b hybridizing RNA in CPPsias mice confirmed the production of the antisense Psi sequences of M-MùLV in the white blood cells of LPPsias and CPPsias transgenic mice.
The different lengths of the antisense RNA in ; LPPsias and CPPsias mice is the result of the `:
different gene constructions introduced into these two lines of mice. The CPPsias mice contain a gene construction including a small portion of exon 5 from the bovine growth hormone gene and the bovine growth hormone poly A addition signals resulting in a longer antisense RNA product.
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Inhibition of M-MuLV Re~lication in Cells Expressina Antisense Psi RNA
A stable cell line expressing antisense Psi sequences was established by co-transformation of mouse NIH 3T3 cells with linearized pCPPsias (36 ~g) , and pSV40neo (0.36 ~g) (ATCC 33694) using a modified DEAE-dextran dimethyl-sulfoxide-shock procedure (26).
Stable transformants were selected in G4l8DMEM medium, Southern analysis performed to establish the presence '~ of presence of integrated CPPsias sequences, and Northern analysis carried out to confirm the presence of antisense M-MuLV RNA production by cloned cell q lines. Both control mouse NIH 3T3 cells and the CPPsias transformed cell line were challenged with M-MuLV (4XlO6 PFU/75 cm plate) for 24 hrs., washed free of virus, cultured for an additional 48 hrs, filtered through 0.45 um filters to remove cells and cellular material from the medium and virus particles concentrated from the medium by sucrose gradient centrifugation (35). RNA was prepared from the viral pellet by a phenol-chloroform extraction procedure (35) and the presence of M- MuLV genomic RNA detected by Northern Analysis using a random primer labeled 540 bp Sma I DNA fragment from pCPPsias tl x lO8 - 5 x lO4 cpm/~g) as a M-MuLV virus specific prob~. No viral RNA was produced from antisense Psi RNA expressing NIH
3T3 cells after challenge with M-MuLV, while a substantial amount of viral genomic RNA (8. 3 Rb) was produced from control NIH 3T3 cells challenged with M-MuLV.
Reverse transcriptase activity in the supernatant from the stable cell line expressing antisense Psi RNA
after infection with M-MuLV was measured and compared to normal mouse NIH 3T3 cells infected with M-MuLV
(3l) . Substantial reverse transcriptase activity was ., .
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observed (see Table II) even though this same supernatant was shown to be devoid of viral gen~mic RNA.
:. S
EXAMPLE VII
Inhibition of Leukemia In Antisense Psi RNA
Expressing Transgenlc Mice In order to test the level of inhibition of M-MuLV induced leukemia in antisense Psi transgenic ~A mice, littermate control and tranegenic mice were challenged with M-MuLV at birth. Transgenic male ~; LPsias and CPsias mice (Fl hybrids of C57B6 and SJL) , ,, were mated to non-transgenic (C57B6/SJL) females.
Within several hours after birth each offspring from these matings were injected intraperitoneally with 0.l ml containing l X 105 M-MuLV infectious virions. At 4 weeks of age, a DNA sample from the tail of each mouse pup was analyzed by slot blot hybridization and each mouse ear notched to code transgenic and non-transgenic offspring. At 12 to 14 weeks of age both transgenic and control mice with sacrificed and assayed for the presence of leukemia symptoms. Mice were judged to be leukemic if three criterion were met; spleen weight in excess of 0.5g, a hematocrit value lower than 35% and typical leukemia morphology in Giemsa-stain lymphocytes (36). In a typical leukemia morphology the number of red blood cells (RBC) was dramatically decreased, the shape of RBC was abnormal, the lymphocyte cell number and size remarkably increased, and the lymphocyte shape changed into a malignant appearance. In a normal blood cell morphology, the majority of the cells were red blood cells and the RBCs were round and smooth looking.
Only one or two lymphocytes could be seen in a typical slide. In Table III these data for each transgenic and control mouse are shown. While significant ,, i SUBSTITUTE SHEET
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~ 7 g q percentage (33%) of the control mouse showed the symptoms of leukemia, none of the LPPsi as or CPPsi as -~ mice were judged to be leukemic. Numerous of the 5 control mice were obviously severely impaired, showing typical leukemia lymphocyte morphology, containing spleens with weights in excess of 0.5g and several had spleens larger than l.Og (Fig. 6) while no antisense ^ 2si transgenic mice appeared abnormal prior to ; lO sacrifice or showed enlarged spleens, low abnormal prior to sacrifice or showed enlarged spleens, low hematocrit values or leukemic lymphocyte morphology.
The difference in spleen weight between control and s transgenic mice could be as large as 22 times (#13/#17).
; Thus, inhibition of packaging resulted in retention of normal blood cell morphology and norma' spleen weight and normal hematocrit values after viral challenge, in dramatic contrast with the control mice.
' 20 The data presented in Table III clearly suggests a strong inhibition of leukemia initiation which is M-MuLV replication dependent in mice producing the antisense RNA. Since the antisense RNA produced in - these mice only contains sequences complementary to M-i 25 MuLV packaging sequences and not to coding sequences i (21), the site of interference in the viral ~? replication cycle is concluded to be the packaging step. RNA complementary to Psi sequences appears to be highly effective at competing with the interactions 30 between Psi and capsid protein.

EXAMPLE VIII
Inhibition of Productive Virus I fection in Vitro in Cells Ex~ressinq Psi Antisense RNA
In order to directly study the effects of antisense Psi RNA on viral replication in the M-MuLV
system, stable cell lines expressing CPPsias sequènces .
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EXAMPLE IX
HYpothetical Use of Antisense Sequences Com~lementary Packaging Sequences of HIV-l in the Treatment AIDS
~ 20 The packaging sequence of HIV-l, isolate ELI is r,~ located within bases 200-340. There is a significant `j3 homology between bases 280-330 of this virus and the ~1 packaging sequence of the M-MULV virus.
Oligonucleotides complementary to the packaging ; 25 sequence of the HIV-l region between pase pairs 280--330 (in the proviral genome of HIV-l, isolate ELI) are ~ synthesized using standard techniques. The largest of ; such oligonucleotides is 50 bases in length and is complementary to the entire 280-330 sequence.
Alternatively, a 50 base oligonucleotide containing methylphosphonate analogues of the natural mononucleosides is synthesized according to known methods.
~ Doses ranging between about 500 mg and 10 grams :~, ` 35 of these antisense oligonucleotides, having either the .. l ; ... .

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HvDothetical Use of Antisense Sequences Complementary to the Packaainq Sequences of~ovine LeuXosis Virus i in the Treatment of Bovine Leukemia Q Oligonucleotides complementary to the packaging sequence of the bovine leukosis virus (BLV) between ; base pairs 341 and 417 are synthesized using standard techniques. The largest of such oligonucleotides is 50 bases in length and is complementary to the 355-405 sequence. Alternatively, a 2-base oligonucleotide , containing methylphosphonate analogues of the natural mononucleosides is synthesi~ed according to known methods.
Doses ranging between 500 mg and 100 grams of these antisense oligonucleotides, having either the natural or analogue nucleotides, are injected IV into cows infected with the bovine leukosis virus.
:j EXAMPLE XI
Plaaue Assay Comparing Acute Inhibitory Effect of Various Antisense Molecules Against Mo-MLV
`I A plaque assay was conducted to determine the relative inhibitory effect of various antisense molecules (30 mer, 38 mer, 40 mer, 50 mer and 60 mer) directed against the packaging sequences ~+ (69-106) and ~ (216-570) of MO-MLV. The target sequences of j these molecules are described below:
38mer:
from base 69 to base 106, located at 5' 3I half of U5 region.
60mer:

from base 216 to base 275 50mer:
from base 301 to base 350 40mer:

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~ from base 541 to base 570 (The base :~ locations described above are based on the virus genomic RNA sequence of Shinnick et al., Nature 293:543-548 (1981).) , The plaque assay generally followed the method of Klement, et al., P.N.A.S. 13:753-58 (1969) and Rowe, ; et al., Virology, 42:1136-39 (1970). NIH 3T3 cells ~, 10 were inoculated into a six-well plate. The next day, they were infected with Mo-MLV, lml of lX106 PFU/well.
After 5-6 days, the cells were irradiated with W for 30-45 secs. to limit their growth, and then Xc cells were laid atop the NIH 3T3 cells, 0.5 x 105 cells/well.
After 3-4 days, the cells were fixed with 2ml/well of , methanol (lmin.) and stained with lml/well hematoxylin (304 mins). Plaques were counted under low magnification with a dissecting microscope. Results are shown in Figure 4.
The result of the viral plaque assay with ~ different oligonucleotides has shown that the 50mer .' oligonucleotide has the best inhibition effect. This means that in comparison with the control, the 50mer oligo reduced the plaque number by 6-7 times. The ;~ 25 50mer sequence (RNA 300b-350b, 3'GACAT AGACC GCCTG
GGCAC CACCT TGACT GCTCA AGCCT TGTGG GCCGG 5') `~ (SEQIDNO:5) comprises a sequence complementary to the core of the Mo-MuLV packaging sequence.
Significantly, it also comprises a sequence complementary to the core portion (19bp) of the HIV-1 packaging sequence.
Use of sense strand in a parallel procedure had no effect, thus clearly showing that the antisense sequence is responsible.
It is noted that at day 3 in Figure 4, there were about 7 times the number of plaques in control cells ; ~, ..
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Table I
Animal Retroviruses Avian Erthyroblastosis Virus ~vian Leukosis Virus (or Lymphoid Leukosis virus) , 5 Avian Myeloblastosis Virus ~; Baboon Endogenous Virus ,", Bovine Leukemia Virus ''~.' Bovine Syncytial Virus ~, Caprine Encephalitis-Arthritis Virus (or Goat ~ Leukoencephalitis Virus) .. s Avian Myelocytomatosis virus ~ lO Corn Snake Retrovirus Chicken Syncytial virus .~ Duck Infectious Anemia Virus .~ Deer Kidney Virus :~. Equine Dermal Fibrosarcoma Virus :j~ Equine Infectious Anemia Virus Esh Sarcoma Virus ~: Feline Leukemia Virus .~ Feline Sarcoma Virus . 15 Feline Syncytium-forming virus Fujinami Sarcoma Virus Gibbon Ape Leukemia Virus (or Simian Lymphoma Virus or ~:. Simian Myelogenous Leukemia Virus) , Golden Pheasant Virus . Lymphoproliferative Disease Virus J Myeloblastosis-associated Virus 20 Myelocytomatosis Virus -. Mink Cell Focus-Inducing Virus ' Myelocytomatosis Virus 13 Mink Leukemia Virus i Murine Leukemia Virus ;.~. Mouse Mammary Tumor Virus ; Mason-Pfizer Monkey Virus ~, Murine Sarcoma Virus j~ 25 Myeloid LeUkemia Virus :1 Myelocytomatosis Virus 1, . Progressive Pneumonia virus Rat Leukemia Virus Rat~Sarcoma Virus :~ Rous-Associated Virus 0 :i Rous-Associated Virus 60 3 Rous-Associated Virus 61 ,' 30 Reticuloendotheliosis-Associated Virus Reticuloendotheliosis Virus . Reticuloendotheliosis Virus-Transforming .~ Ring-Necked Pheasant Virus ~ Rous Sarcoma Virus :~ Simian Foamy Virus ;? Spleen Focus-Forming Virus Squirrel Monkey Retrovirus ;~ 35 Spleen Necrosis Virus ~ Sheep Pulmonary Adenomatosis/Carcinoma Virus f ,, .f SUBSTITUTE SHEET

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. 5TABLE II
~;~ REvERSE TRANSCRIPTASE (RT) ASSAY

. Infecting RT Activity in , Cell Line Cell/ml Viru~ Titer ~PFU) Supernatant 0 NIH 3T3 lo6 5 X 105 100.0 ~'~ Clone 9-5 lo6 5 X 105 39,7 ,~ Clone 11-2 105 5 X 105 32.5 ,~ Clone 12-6 106 5 X 105 40.1 ,.,f~ *In arbitrary units (assay value of normal mouse NIH
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~Incidence of leukemia in control and anti-sense w transgenic mice challenged with Maloney murine ,leukemia virus Leukemia ~ou~e Tran3genic Hematocrit Weiyht(g) LYmhcyte Leukemla . 1 NON 49% 0.19 ~ 2 NON 33% 0.25 3 NON 49% 0.15 4 NON 46% 0.16 -. 5 NON 43% 0.13
6 NON 35% 0.63
7 NON 35% 0.18
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NON 48% 0.11 ~ 14 NON 45% 0.lS
:.',. 10 15 CWa~ 46% 0.13 '~ 16 CWas 54% 0.09 17 CWas 45% 0.08 .~
CWas 48% 0.11 21 CWas 47% 0.11 CWas 55% 0.15 26 CWas 46% 0.12 27 CWas 49% 0.10 28 CWas 44% 0.10 CWas 43~ 0.14 34 CWas 48% 0.12 42 CWas 49% 0.14 46 LWas 42% 0.17 '~1 47 LWa~ 46% 0.25 LWas 49% 0.19 51 LWas 48% 0.18 53 LWas 48% 0.17 54 LWas 50% 0.12 LWas ~49% 0.19 56 LWas 49% 0.16 57 LWas 40% 0.09 59 LWa~ 49% 0.13 ~; 61 LWas 46% 0.09 62 LWas 44% 0.12 63 . LWas 51% 0.14 64 LWas 46~ 0.13 * died a few hours prior to evaluation ~ definitive leukemia ' y .~. ` SUBSTITUTE SHEET
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~able IV: -Table of Packaging Sequences:
~ Each entry includes both a reference for the ,`~!,~ 5 published genomic sequence generally and a reference for the location of the packaging sequence within the -~ genome.
1. Reticuloendotlieliosis ~irus (Rev) Genome: Wilhelmsen, et al. J. Virol. 52:172-182 -~i (1984). bases 1-3149; Shimotohno, et al. Nature 285:550-554 (1980). bases 3150-3607. Packaginq ; Sequence (~):144-base between the Kpn I site at 0.676 kbp and 0.820 kbp relative to the 51 end of the i' provirus.
~ J. Embretson and H. Temin J. Virol. 61(9):2675-`: 2683 (1987).
. , 2. Human immunodeficiency virus type 1 (IIIV-l) Genome Gallo et al. Science 224:500-503 (1984) .
Packa~inq sequence (~):19 base pairs between the 5' ~ LTR and the gag gene inîtiation codon. A. Lever, J.
:~ Virol. 63(9):4085-4087 (1989).
s 3. Moloney murine leukemia virus (Mo-MuLV) ; Genome: Shinnick, et al. Nature 293:543-548 (1981).
~ 20 Packaainq sequence (~):350 nucleotides between the ; splice site and the AUG site for coding sequence of ~; gag protein. R. Mann, R. Mulligan and D. Baltimore, -~ Cell 33:153-159 (1983). Second ~ackaqina sequence ):Only in the 5' half of the U5 region.
J. Murphy and S. Goff, J. Virol. 63(1):319-327 (1989).
.4 ~, 4. Avian sarcoma virus (ASV) ;~ 25 Genome: Neckameyer and - Wang J. Virol. 53:879-884 (1985). Packaging sequence (~):150 base pairs between 300 and 600 bases from the left (gag-pol) end of the provirus. P. Shank and M. Linial, J. Virol.
36(2):450-456 (1980).
5. Rous sarcoma virus (RSV) Genome: Schwartz, et al. Cell 32:853-869 (1983) .
Packaaing sequence (~):230 base pairs from 120-base tPB site beginning) to 22-base before gag start codon.
S. Kawai and T. Koyama (1984), J. Virol. 51:147-153.
6. Bovine leukosis virus (BLV) Genome: Couez, et al. J. Vlrol. 49:615-620, 1984, `~ bases 1-341; Rice, et al. Virology 142:357-377, 1985, bases 1-4680; Sagata, et al. Proc. Natl. Acad.
Sci. 82:677-681, 1985, complete BLV provirus.
Packaging sequence: the present inventors predict hat , i ;-.
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REFERENCES
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"Molecular Biology of Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., l9B4.
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3. Barklis, E., R.C. Mulligan, and R. Jaenisch.
~x 1986. Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells.
Cell 47:391-399.
4. ~ann, R. and D. Baltimore. 1985. Varying the .'5 position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAS.
J. Virol. 54:401-407.
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6. Cone, R.D., A. Weber-Benarous, D. Baorto and R.C. Mulligan. 1987. Regulated expression of the 3 complete human ~-globin gene encoded by a transmissible retrovirus vector. Mol. Cell. Biol.
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7. Freifelder, D. 1987. Molecular BioloqY:
EukaEY~_ Viruses, Jones and Bartlett Publishers, Inc., Boston Portola Valley, 2nd ed.
8. Temin, H.M. 1972. RNA-directed DNA
30 synthesis. Sci. Amer.
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Baltimore. 1979. A detailed model of reverse transcription and tests of crucial aspects. Cell 8:93-100.
10. Conie, R., and R. Mulligan. 1984. High- -efficiency gene transfer into mammalian cells:
.~

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Generation of helper-free recombinant retrovirus with ` broad mammalian host range. Proc. Natl. Acad. Sci.
USA 8I:6349-6353.
~ 5 11. Mann, R., R.C. Mulligan, and D. Baltimore.
-~ 1983. construction of a retrovirus packaging mutant and its use to produce helper-free, defective retrovirus. Cell 33:153-159.
12. Shank, P.R., and M. Linial. 1980. Avian ~- 10 oncovirus mutant (SE21Qlb) deficient in genomic RNA:
characterization of a deletion in the provirus. J.
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13. Bender, M.A., T.D. Palmer, R.E. Gelinas, and A.D. MilIer. 1987. Evidence that the packaging signal f m Moloney murine leukemia virus extends into the ~; gag region. J. Virol. 61: 639-1646.
14. Weintraub, H., J.G. Izant, and R.M. Harland.
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19. Mizumo, T., M.Y. Chou, and M. Inouye. 1984.
;cl A unique mechanism regulating gene expression:
~ 35 Translational inhibition by a complementary RNA

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`.~ WO92~17211 ~ 78q PCl/U~i92/02911 . ` i . -- 5 3 --.` ` .
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. WO 92/1721 I PCr/USg2/0291 1 5 4 ~
,'''' ~ 2~7gq ~' ' ;
~, ` 36. Ruscetti, s., L. Davis, J. Feild, and A.
Oliff. 1981. Friend murine leukemia virus-induced leukemia is associated witli the formation of mink ~, 5 cell focus-inducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing ,~ xenotropic viral envelope genes. J. Exp. Med.
54:907-920.
37. Smith, C.C., O.P. Tslo Paul and P.S. Miller Proc. Natl. Acad. Sci. USA 83:2787-2791 (1986) (complementary oligonucleotide methods used in ;~ antiviral research) ~' 38. Ruden, T. and E. Gilboa J. Virol 63:677-682 ~ (1989) ; 15 The references cited in this specification are all incorporated by reference~herein, whether specifically incorporated or not.
While this invention has been described in - connection with specific embodiments thereof, it will be understood that it is capable of further `j modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention x and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

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Claims (17)

1. An antisense nucleic acid molecule which is substantially complementary to the packaging sequence or a portion thereof of a lymphotropic retrovirus, which nucleic acid molecule is capable of hybridizing to the packaging sequence of the retrovirus and inhibiting retrovirus-mediated disease.
2. The antisense nucleic acid molecule of claim 1 wherein the nucleic acid is an RNA.
3. The antisense nucleic acid molecule of claim 1 wherein the nucleic acid is a DNA.
4. The antisense nucleic acid molecule of claim 1 wherein the nucleic acid is a non-naturally occurring analog of DNA or RNA characterized by the presence of one or more non-naturally occurring nucleosides and/or internucleoside linkages.
5. The antisense nucleic acid molecule of claim 1 wherein the nucleic acid comprises one or more methylphosphonates.
6. The antisense nucleic acid molecule of claim 1, having a length of less than 100 bases.
7. The antisense nucleic acid molecule of Claim 1 which is substantially complementary to at least a portion of the packaging sequence of a lymphotropic retrovirus selected from the group consisting of human immunodeficiency virus type 1, Moloney murine leukemia virus, and bovine leukosis virus.
8. A recombinant DNA molecule comprising a DNA sequence encoding the antisense RNA molecule of Claim 2, which is operably linked to a promoter capable of directing its transcription in a host cell.
9. The recombinant DNA molecule of Claim 8 wherein said promoter is the murine Moloney leukemia virus LTR promoter.
10. The recombinant DNA molecule of Claim 8 wherein said promoter is the cytomegalovirus immediate early promoter.
11. A host cell transformed or transfected with the recombinant DNA molecule of claim 8.
12. The host cell of claim 11 wherein the antisense RNA molecule does not hybridize to any host cell nucleotide sequence that is necessary for host cell survival.
13. A method for rendering a lymphocyte resistant to productive infection by a lymphotropic retrovirus comprising inserting into the genome of said lymphocyte a DNA sequence encoding the antisense RNA molecule of claim 2, which DNA sequence is operably linked to a promoter capable of directing its transcription in said cells, and which transcribed antisense RNA molecule hybridizes to the packaging sequence of said lymphotropic retrovirus and thereby inhibits the packaging of viral RNA and rendering said lymphocyte resistant to said infection.
14. A method for rendering a mammal resistant to productive infection by a lymphotropic ratrovirus comprising introducing into the genome of at least some of the cells of said mammal a DNA
sequence encoding the antisense RNA molecule of claim 2, which DNA sequence is operably linked to a promoter, capable of directing its transcription in said cells and which transcribed antisense RNA
molecule hybridizes to the packaging sequence of said lymphotropic retrovirus and thereby inhibits the packaging of viral RNA and rendering said mammal resistant to said infection.
15. A method according to claim 10 wherein said DNA sequence has been introduced into said mammal or an ancestor of said mammal at an embryonic stage.
16. A transgenic or chimeric non-human mammal prepared by the method of claim 14, and progeny thereof which express said antisense RNA.
17. A method for rendering a mammal resistant to productive infection by a lymphotropic retrovirus comprising administering to said mammal the antisense nucleic acid molecule of claim 1 which hybridizes to the packaging sequence of said lymphotropic retrovirus, thereby inhibiting the packaging of viral RNA and rendering said mammal resistant to said infection.
CA002107789A 1991-04-05 1992-04-03 Retrovirus inhibition with antisense nucleic acids complementary to packaging sequences Abandoned CA2107789A1 (en)

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DE4126484A1 (en) * 1991-08-10 1993-02-11 Bayer Ag Anti-sense-RNA expression vectors - contain hybrid promoter sequences and viral DNA sequences i.e. HIV, in anti-sense direction, useful in prodn. of HIV-resistant cells
DE4225094A1 (en) * 1992-04-28 1993-11-04 Frank Andreas Harald Meyer Compsn. for gene therapy, esp. of viral infections and tumours - comprises anti-sense DNA or RNA inserted into long terminal repeat or transposable element and e.g. virus envelope as carrier
DE69334238D1 (en) 1992-09-25 2008-09-25 Aventis Pharma Sa ADENOVIRUS VECTORS FOR THE TRANSMISSION OF FOREIGN GENES IN CELLS OF THE CENTRAL NERVOUS SYSTEM, ESPECIALLY IN THE BRAIN
EP0598935A1 (en) * 1992-11-24 1994-06-01 Bayer Ag Expression vectors and their use to produce HIV-resistant human cells for therapeutic use
US5583035A (en) * 1992-12-07 1996-12-10 Bayer Aktiengesellschaft HIV antisense expression vectors
FR2702152B1 (en) * 1993-03-03 1995-05-24 Inst Nat Sante Rech Med Recombinant viruses and their use in gene therapy.
US5712384A (en) * 1994-01-05 1998-01-27 Gene Shears Pty Ltd. Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs
KR100696410B1 (en) * 1994-01-05 2008-11-07 진 쉬어즈 피티와이., 엘티디. Ribozymes and Retrocombination Retroviruses Having Retroviral Packaging Sequence Expression Constructs
CA2180358A1 (en) * 1994-01-05 1995-07-13 Geoffrey P. Symonds Ribozymes targeting the retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs
AU2133695A (en) * 1994-04-06 1995-10-30 Sadna Joshi-Sukhwal Inhibition of hiv-1 multiplication in mammalian cells
US20020058636A1 (en) * 1994-09-21 2002-05-16 Geoffrey P. Symonds Ribozymes targeting the retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs
DE60238486D1 (en) 2001-07-10 2011-01-13 Johnson & Johnson Res Pty Ltd METHOD FOR THE GENETIC MODIFICATION OF HEMATOPOETIC TEMPORARY CELLS AND USES OF MODIFIED CELLS
US7994144B2 (en) 2001-07-10 2011-08-09 Johnson & Johnson Research Pty, Limited Process for the preparation of a composition of genetically modified hematopoietic progenitor cells

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