WO 1992017211 A1
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.
RETROVIRUS INHIBITION WITH ANTISENSE NUCLEIC ACIDS COMPLEMENTARY TO PACKAGING SEQUENCES
BACKGROUND OF THE INVENTION
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.
Description of the Background 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
SUBSTITUTESHEET 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 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 retroviral integration resembles that of various eucaryotic mobile genetic elements such as Copia and 412 of Drosophila or Ty-1 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 DNA. There is a characteristic duplication of short stretches of the hosts DNA at the site of integration.
SUBSTITUTESHEET The retroviral protein directly involved in inserting the viral DNA into the host DNA is called the integrase protein (IN) . The sequence of the IN 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- MLV; in avian viruses and the human immunodeficiency virus it is 32kd) . During integration the IN protein removes 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 protein of all retroviruses is thought to have an endonuclease activity which results in the production 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 to the host DNA results in the duplication of the short sequences of the host DNA.
Progeny viral genomes and RNAs 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. 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 tumorogenesis, retroviruses are the only taxonomic group of RNA viruses that are oncogenic. Various
SUBSTITUTESHEET retroviruses such as the Human Immunodeficiency Virus (HIV) , which is the etiological agent responsible for acquired immune deficiency syndrome in humans, are also responsible for several very unusual diseases of 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 from the cytoplasmic membrane of the host cell as the maturing virus buds from that cell. It is covered by glycoprotein spikes, coded for by the viral genome. The cytoplasmic membranes of infected cells actively transcribing the proviral sequences and processing 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. The capsid contains a ribonucleoprotein complex that includes the genomic RNA, reverse transcriptase, the 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 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.
There are several classification schemes applicable to retroviruses. What is, perhaps, the simplest classification scheme is based on morphology.
SUBSTITUTESHEET 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 found only within cells. They range in size from 60- 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 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 herein, it is necessary to understand the organization of a typical retrovirus. The Moloney murine leukemia
SUBSTITUTESHEET 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 Ver a, 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 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 RNA molecule. The 5' end of the molecule carries a typical in RNA cap structure (m7 G5 'ppp5 'Gm) . A poly- A tail of about 200 residues is attached to the 3' end, and several internal adenosine residues are methylate .
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 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- 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. Next are the three major coding regions: sag, or the group-specific antigen gene, which code for the
SUBSTITUTESHEET viral core proteins; pol. which encodes the viral polymerase (or reverse transcriptase) ; and env, which encodes the envelope proteins and glycoproteins. 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. 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 approximate molecular weight of the protein in kDa as determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) (23) . Four internal structural proteins are products of the viral gag 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 the env gene. They are the glycoprotein, gp70, and the nonglycosylated pl5 protein. The pl5 protein is a transmembrane protein which attaches to 9P70 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.
SUBSTITUTESHEET 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 pol 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. r 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-l retrovirus also possesses genes modulating viral replication, including vif, vpr, tat, rev, vpu. and nef (Haseltine, W.A. , J. Acguired Immune Deficiency 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
SUBSTITUTESHEET packaging sequences were deleted from 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 sequences. The "helper" cell provided: (a) the gag- 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 a DNA copy upon arrival into the infected cell. The sequences required for RNA packaging into virions have been defined and Shown to reside between the 5• LTR and the beginning of the early portion of the gag gene.
J . 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 promoter 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
SUBSTITUTESHEET 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 plasmid rizant et al. , Cell 36:1007 (1984).
Antisense oligonucleotides have been shown to be inhibitory in various viral systems. Zamecnik and Stephensen, Biochemistry, 75:280-84 (1978) inhibited Rous sarcoma virus (a retrovirus) production in 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 blocking circulatizatoin, 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 5' or 3' flanking regions of the env gene.
Gupta, J. Biol. Chem., 262:7492-96 (1987) inhibited translation of the Sendai virus nucleocapsid protein (NP) and phosphoprotein (P.C) mRNAs by means of antisense DNAs complementary to the 5' flanking region. Oligonucleotides complementary to the coding 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 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.
SUBSTITUTESHEET (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 this emphasis, the assays used to test antisense sequence for antiviral activity measure the expression of a HIV-1 gene product. See S. Gott, J. Virol (1981) 38(1) :239 (RT Test); J. McDougal, J. Imniunol. Meth. (1985) 76:171 (antigen detection). RNA northern blot analysis, the expression assay that is most typically used to test antisense nucleic acid inhibition of HIV- 1 (See Thomas, PNAS, 77:3201, 1980), cannot 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 Hindlll-Pst I fragment from the 5' end of the proviral DNA. The latter target is said to include the 5' splice site, the tRNA primer 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.
SUBSTITUTESHEET 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, they are more prone to hybridize to cellular DNA, 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 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. , Biochemistry 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 oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5 was shown to
SUBSTITUTESHEET selectively inhibit viral infection (Smith, C.C. et al.. Proc. Natl. Acad. Sci. USA 11:2787-2791 (1986)) .
SUMMARY OF THE INVENTION
An object of the present invention is to overcome the deficiencies noted above.
More particularly, the present inventors have 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 cell lines isolated, the cells were resistant to M- MuLV infection, and produced only virus devoid of packaged 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
St -JSTITUTESHEET 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 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 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 a retrovirus comprising inserting into the genome of the cell a DNA sequence, operably linked to a promoter, wherein the DNA sequence is transcribable 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
SUBSTITUTESHEET a DNA sequence containing the packaging sequence of 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 sequence is 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 sequence, a transgenic in which said DNA sequence 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.
SUBSTITUTESHEET BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing the construction of plasmids pLPPsias (A) and pCPPsias (B)-
Figure 2 is a partial sequence (SEQIDN0:1) 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 gig 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 of MoMLV, did not show significant inhibition of the virus infection while the best inhibition observed was with anti-sense oligos complementary to the open bold sequence, i.e., the core (bases 301-350) of packaging site. Figure 3 is a partial sequence (SEQIDNO:2) 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.
SUBSTITUTESHEET DESCRIPTION OF THE PREFERRED EMBODIMENTS
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 antisense nucleic acids (DNA or RNA) complementary to a part of the packaging sequences, which hybridizes o 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 5 gene competes with normal messenger RNA for binding to ribosomal RNA, while one directed against the packaging sequence competes with a gag-encoded core protein for binding to genomic RNA. RNA-RNA interactions are stronger than RNA-protein o 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 to the Psi sequences of Moloney murine leukemia virus (M-MuLV) . It was discovered that such animals completely resisted challenge with this leukemia virus.
The present invention is therefore directed to the use of antisense RNA and DNA molecules 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 intended to encompass sequences capable of hybridizing to the packaging sequence of a retrovirus. The
SUBSTITUTESHEET 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 Ly phadenopathy Associated Virus) , Human Immunodeficiency Virus-2 (HIV-2) , Human T-Cell Lymphotropic Virus-I (HTLV-l) , and Human 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 gag protein coding sequences) , sequences within this region (such as, for example bases 200-340 in the proviral genome of HIV-1, 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.
SUBSTITUTESHEET 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-1. 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 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-l, which are complementary to viral genomic RNA, viral mRNA, as well as the viral DNA sequences. For the packaging sequence of HIV-l, see (Lever. J. Virol.. 63(9) :4085-4097 (1989); Korman, et al., (Proc. Natl. Acad. Sci. USA 84:2150-2154 (1987).
SEQ ID NO: 4 is a 44mer sequence shown below, which is complementary to the HIV-l packaging 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
SUBSTITUTESHEET 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 seguence 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.
SUBSTITUTESHEET It is desirable to avoid sequences of antisense RNA which would form secondary structure due to intramolecular hybridization, since this would render 5 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 o 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, 5 alternatively, from mononucleosides having substitutions at the non-bridging phosphorous bound oxygens. A preferred analogue is a methylphosphonate analogue of the naturally occurring mononucleosides. More generally, the mononucleoside is any analogue o 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 5 (1981)). 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.. supra) .
Basic procedures for constructing recombinant DNA and RNA molecules in accordance with the present invention are disclosed by Sambrook, J. et al.. In: Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Press, Cold Spring Harbor,
SUBSTITUTESHEET NY (1989) , which reference is herein incorporated by reference.
Oligonucleotide molecules having a strand which 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: 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 be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra) r and by Haymes, B.D., et al. (In: Nucleic Acid Hybridization. A Practical Approach. IRL Press, Washington, DC (1985)), which 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 sequences occurs when the expression vector is introduced into an appropriate host cell. If a 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.
SUBSTITUTESHEET A DNA sequence encoding the antisense RNA of the present invention may be recombined with vector DNA in accordance with conventional techniques, including 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 joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by
Sambrook et al., supra. 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 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 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 sequence coding for the desired RNA product may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as those which provide for termination and polyadenylation. Thus, by retaining 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 the host cell may be substituted.
SUBSTITUTESHEET 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. 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 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. Molec. Biol. 195:471-480 (1987) Hu, M. et al.. Gene 42:21-30 (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.) .81:2035-2039 (1984)); the PR and PL promoters of bacteriophage lambda (The Bacteriophage Lambda. 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 (1980)); the trp, recA. heat shock, and lacZ promoters of E. coli. ; the int promoter of bacteriophage lambda;
SUBSTITUTESHEET the bla promoter of the /S-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. (Biochimie 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. if}: 15-442 (1984)).
Eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D. , et al.. J. Mol. APPI. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. , et al.. Nature (London) 290: 304-310 (1981) and the yeast σal4 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)). F°r 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
SUBSTITUTESHEET early promoter (32) , optionally used in conjunction with the bovine growth hormone polyadenylation signals (33) , and the promoter of the Moloney-MuLV LTR, for 5 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 o advantage of inducibility. The SV40 early promoter 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 5 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 o appropriate. Alternatively an antisense DNA molecule may be provided. DNA is more readily synthesized in vitro than RNA.
The antisense molecule may be an analogue of DNA or RHA. The present invention is not limited to use of 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 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 l'-alpha-anomers) . The naturally occurring linkage is
SUBSTITUTESHEET 3 » 0
O" - P = 0.
Alternative linkages include the following:
3'0 s- - P = 0
3»0 CH3 - P = O
NR2 - P = O
(where R is hydrogen and/or alkyl)
3'0 RO - P = O
(where R is hydrogen or alkyl)
S- - P = S. 05«
It is also possible to replace the 3'0-P-05• with other linkages such as 3•0-CH2C(O)-05• , 3•0-C(O)-NH5• , and 3•C-CH2 CH2 S-C5• .
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.
Antisense molecules suitable for use in the present invention include but are not limited to dideoxyribonucleoside methylphosphonates, see Mill, et al.. Biochemistry, 18:5134-43 (1979),
SUBSTITUTESHEET oligodeoxynucleotide phosphorothioates, see Matsukura, et al., Proc. Nat. Acad. Sci., 84:7706-10 (1987), oligodeoxynucleotides covalently linked to an 5 intercalating agent, see Zerial, et al. , Nucleic Acids Res., 15:9909-19 (1987), oligodeoxynucleotide conjugated with poly(L-lysine) , see Leonetti, et al.. Gene, 72:32-33 (1988), and carbamatelinked oligomers assembled from ribose-derived subunits, see Summerton, o J- , Antisense Nucleic Acids Conference, 37:44 (New York 1989) .
While direct administration of antisense nucleic acid drugs provides acute protection, the protective period is dependent on the half-life of the molecule. 5 Moreover, the supply of antisense nucleic acid can be increased only 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 o RNA, into one or more cells of the human or animal subject, thus creating a transgenic or chimeric animal having enhanced resistance to retroviral infection. The recombinant DNA may be delivered to the animal by, e.g., microinjection of the expression cassette into the animal at the oocyte stage, retroviral vector transfection of the embryo, or intravenous injection of the retroviral vector into the fetal or postnatal animal.
Thus, the present invention is also directed to 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 retroviral packaging sequence. "Antisense" DNA is introduced into the animal to be made transgenic, or
SUBSTITUTESHEET 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. 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 of the transgene in the transgenic animal. There are several means by which such a gene can be introduced into the genome of the animal embryo so as to be 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 introduced into the fetal or postnatal animal.
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 expressed in all such cells. The presence of the transgene in the germ cells of the transgenic "founder" animal in turn means that all its progeny will carry the transgene in all of their germ cells and somatic cells. Introduction of the transgene at a 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 the progeny of this founder animal that inherit the transgene conventionally, from the founder's germ cells, will carry the transgene in all of their germ cells and somatic cells.
SUBSTITUTESHEET 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. 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, is achieved by DNA extraction and slot blot hybridization analysis of the DNA for the presence of the transgene (McGrane, M. et al.. J. Biol. Chem. 261:11443-11451 (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, replicatipn deficient, retroviral vector containing DNA sequences encoding and expressing anti¬ sense RNA complementary to the packaging sequences of HIV. In this procedure bone marrow cells would be removed from the patient, treated with the recombinant retroviral vector and cells in which the DNA genome of the vector had integrated in their chromosomes selected using FACS cell sorting based upon a light visualization marker also incorporated into the retorviral vector (i.e., the β-galactosidase gene).
SUBSTITUTESHEET 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. 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 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 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.
SUBSTITUTESHEET 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 illustration, and are not intended to be limiting of the present invention, unless so specified.
EXAMPLE I Plasmids Recombinant plasmids pLPPsias and pCPPsias were constructed as shown in Fig. 1. Plasmids pLJ(21) was cleaved with Smal and the 540 bp Smal fragment containing the M-MuLV sequences isolated. Plasmid p3'-LTR-2, containing the 3'M-MuLV LTR (22), was linearized with Smal 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 Kpnl digestion patterns. Plasmid pCMVIE-BGH (30), with an 8 bp Bglll linker introduced at an Xmalll site just 3' of the CMVIE CAP, was cleaved with both Bglll and Smal 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 (A,T,G and C) in the presence of the DNA polymerase I Klenow fragment and blunt-end ligated to the 540 bp Smal fragment from pLJ in both orientations. Clones of pCPPsias with the antisense orientation were selected on the basis of their Kpnl digestion patterns.
SUBSTITUTESHEET EXAMPLE II Transgenic Mouse Production The procedure for the production of transgenic 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 described (25) .
EXAMPLE III Cell Culture Mouse NIH 3T3 cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 10% Nu- serum. Stable cell lines expressing antisense Psi sequences were established by co-transformation with pCPPsias (36 μg) and pSV40neo (0.36 μg) (ATCC 33964) using a modified DEAE dextran-dimethyl sulfoxide shock procedure (26) .
EXAMPLE IV DNA and RNA Analysis and Enzyme Assays The integrity of DNA sequences in transgenic mice 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 procedure (29) . RNA was then subjected to electrophoresis at 70V for 4.5h in a 1.2% 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 antisense RNA strand, a strand specific RNA probe was used for Northern hybridization assays of lymphocyte
SUBSTITUTESHEET RNA. This probe was synthesized from a pSP65 vector containing the 540 bp Smal Psi sequence containing fragment inserted in the sense orientation using SP6 RNA polymerase and labeled guanosine 5*-triphosphates (Promega Riboprobe System, Promega Corp., Madison Wisconsin) .
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 Hindlll fragment or from pCPPsias as a 2.3 kb EcoRI-Clal 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. 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 Hindlll fragment from pLPPsias and CPPsias transgenics trace to a single founder from the microinjection of the 2.3 kb EcoRI- Clal fragment from pCPPsias.
Tail DNA (10 μg) from the LPPsias founder and his offspring was digested with Sad and EcoRI restriction endonucleases and CPPsias founder and offspring DNA
SUBSTITUTESHEET 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 108 to 10 X 108 cpm/μg) 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 1.3 kb Sac I- EcoRI fragment from pCPPsi for CPPsias DNA. After washing and drying, the nitrocellulose was autoradiographed by exposure to Kodak film at -70@C 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 Kpnl hybridizing fragment.
Expression of Antisense Psi RNA in LPPsias and CPPsias Transgenic Mice
The transcriptional units introduced into these two lines of mice were constructed to provide the appropriate tissue tropism for the transcription of the antisense RNA within the lymphoid target tissue for M-MuLV. Within the LPPsias mice the 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
SUBSTITUTESHEET 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 to a strand specific RNA probe complementary to the M-
MuLV antisense Psi sequence with a specific activity of 1-5 x 108 cpm/μg. 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-MuLV 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.
SUBSTITUTESHEET EXAMPLE VI
Inhibition of M-MuLV Replication in Cells Expressing 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 μg) and pSV40neo (0.36 μg) (ATCC 33694) using a modified DEAE-dextran dimethyl-sulfoxide-shock procedure (26) . Stable transformants were selected in G418DMEM 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 lines. Both control mouse NIH 3T3 cells and the CPPsias transformed cell line were challenged with M- MuLV (4X106 PFU/75 cm plate) for 24 hrs., washed free of virus, cultured for an additional 48 hrs, filtered through 0.45 urn 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 (1 x 108 - 5 x 108 cpm/μg) as a M-MuLV virus specific probe. 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 Kb) 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 (31) . Substantial reverse transcriptase activity was
SUBSTITUTE SHEET observed (see Table II) even though this same supernatant was shown to be devoid of viral genomic RNA.
Inhibition of Leukemia In Antisense Psi RNA Expressing Transgenic Mice
In order to test the level of inhibition of M- MuLV induced leukemia in antisense Psi transgenic 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 0.1 ml containing 1 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
SUBSTITUTESHEET 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 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 Psi transgenic mice appeared abnormal prior to 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 transgenic mice could be as large as 22 times (#13/#17) .
Thus, inhibition of packaging resulted in retention of normal blood cell morphology and normal spleen weight and normal hematocrit values after viral challenge, in dramatic contrast with the control mice. 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- MuLV packaging sequences and not to coding sequences (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 between Psi and capsid protein.
Inhibition of Productive Virus Infection in vitro in Cells Expressing 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 sequences
SUBSTITUTESHEET were produced and challenged with M-MuLV. While control cells showed substantial quantities of M-MuLV viral RNA in RNA preparations from the cell supernatant, supernatants from cell lines expressing antisense Psi RNA were devoid of M-MuLV genomic viral RNA suggesting the inability of these cells to produce functional virus containing packaged viral RNA. In spite of the lack of viral RNA in the cell supernatant, significant reverse transcriptase activity was measured by a sensitive reverse transcriptase assay (31) (Table II) suggesting that the antisense Psi RNA expressing cells are producing empty viral particles because of antisense blockage of the packaging process.
Hypothetical Use of Antisense Sequences Complementary to the Packaging Seguences of HIV-l in the Treatment AIDS The packaging sequence of HIV-l, isolate ELI is located within bases 200-340. There is a significant homology between bases 280-330 of this virus and the packaging sequence of the M-MuLV virus. Oligonucleotides complementary to the packaging 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 of these antisense oligonucleotides, having either the
SUBSTITUTESHEET natural or analogue nucleotides, are injected IV into AIDS patients each day for several weeks.
Hypothetical Use of Antisense Sequences Complementary to the
Packaging Sequences of Bovine Leukosis Virus in the Treatment of Bovine Leukemia
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 synthesized 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.
Plaque Assay Comparing Acute Inhibitory Effect of Various Antisense Molecules Against Mo-MLV
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 ψ+ (69-106) and *• (216-570) of MO-MLV. The target sequences of these molecules are described below:
38mer: from base 69 to base 106, located at 5* half of U5 region.
60mer: from base 216 to base 275 50mer: from base 301 to base 350 40mer:
SUBSTITUTESHEET from base 429 to base 468 30mer: 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 were inoculated into a six-well plate. The next day, they were infected with Mo-MLV, 1ml of 1X106 PFU/well. After 5-6 days, the cells were irradiated with UV for 30-45 sees, 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 50mer sequence (RNA 3OOb-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-l packaging sequence.
Use of sense strand in a parallel procedure had no effect, thus clearly showing that the antisense seguence is responsible. It is noted that at day 3 in Figure 4, there were about 7 times the number of plaques in control cells
SUBSTITUTESHEET as for cells treated with the 50 mer, clearly demonstrating viral inhibition. The later convergence of plaque contents was expected, as the cells were treated only once with the antisense molecules, which were gradually degraded by cell nucleases. Use of resistant analogues, or replenishing the supply of antisense molecules by subsequent administrations or by providing for intracellular expression of the antisense molecule would overcome this problem.
SUBSTITUTESHEET Table I Animal Retroviruses
Avian Erthyroblastosis Virus
Avian Leukosis Virus (or Lymphoid Leukosis virus) Avian Myeloblastosis Virus
Baboon Endogenous Virus
Bovine Leukemia Virus
Bovine Syncytial Virus
Caprine Encephalitis-Arthritis Virus (or Goat
Avian Myelocytomatosis virus Corn Snake Retrovirus Chicken Syncytial virus
Duck Infectious Anemia Virus
Deer Kidney Virus
Equine Dermal Fibrosarcoma Virus
Equine Infectious Anemia Virus
Esh Sarcoma Virus
Feline Leukemia Virus
Feline Sarcoma Virus 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 Myeloblastosis-associated Virus Myelocytomatosis Virus
Mink Cell Focus-Inducing Virus Myelocytomatosis Virus 13 Mink Leukemia Virus Murine Leukemia Virus Mouse Mammary Tumor Virus Mason-Pfizer Monkey Virus Murine Sarcoma Virus Myeloid Leukemia Virus Myelocytomatosis Virus Progressive Pneumonia virus Rat Leukemia Virus Rat Sarcoma Virus Rous-Associated Virus 0 Rous-Associated Virus 60 Rous-Associated Virus 61 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 Spleen Necrosis Virus
Sheep Pulmonary Adenomatosis/Carcinoma Virus
SUBSTITUTESHEET Simian Sarcoma-Associated Virus (or Wooly Monkey
Simian Sarcoma Virus (or Wooly Monkey Virus) .
TABLE II REVERSE TRANSCRIPTASE (RT) ASSAY
*In arbitrary units (assay value of normal mouse NIH 3T3 cells set to 100)
SUBSTITUTESHEET TABLE III
Incidence of leukemia in control and anti-sense w transgenic mice challenged with Maloney murine leukemia virus
SUBSTITUTE SHEET Table IV:
Table of Packaging Sequences:
Each entry includes both a reference for the published genomic sequence generally and a reference for the location of the packaging sequence within the genome.
1. Reticuloendotlieliosis virus (Rev)
Genome: Wilhelmsen, et al. J. Virol. 52:172-182 (1984). bases 1-3149; Shimotohno, et al. Nature 285:550-554 (1980). bases 3150-3607. Packaging
Sequence (^):144-base between the Kpn I site at 0.676 kbp and 0.820 kbp relative to the 51 end of the provirus.
J. Embretson and H. Temin J. Virol. 61(9):2675- 2683 (1987) .
2. Human immunodeficiency virus type 1 (IIIV-1) Genome: Gallo et al. Science 224:500-503 (1984) .
Packaging seguence (^):19 base pairs between the 5' LTR and the gag gene initiation codon. A. Lever, J. Virol. 63(9) :4085-4087 (1989).
3. Moloney murine leukemia virus (Mo-MuLV) Genome: Shinnick, et al. Nature 293:543-548 (1981). Packaging 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 packaging sequence (φ+, :θnly in the 5' half of the U5 region. J. Murphy and S. Goff, J. Virol. 63(1) :319-327 (1989).
4. Avian sarcoma virus (ASV) 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) . Packaging seguence (^):230 base pairs from 120-base (PB site beginning) to 22-base before gag start codon.
5. Kawai and T. Koya a (1984), J. Virol. 51:147-153.
6. Bovine leukosis virus (BLV)
Genome: Couez, et al. J. Virol. 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 that
SUBSTITUTESHEET it lies between the end of the primer binding site at about base 340 and the initiation codon for gag at about base 41-8.
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(complementary oligonucleotide methods used in antiviral research)
38. Ruden, T. and E. Gilboa J. Virol 63:677-682 (1989) 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 βe understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention 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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