INHIBITION OF HERPESVIRIDAE INFECTION BY ANTISENSE OLIGONUCLEOTIDES.

Background Of The Invention

The present invention is directed to antisense oligomers which are complementary to a vital region of a viral genome which are active as antiviral agents. The present invention is also directed to methods of interfering with replication of a virus after infection of host cells by the virus and to antisense oligomers which are useful in interfering with viral replication.

In viral replication, viral genes are typically activated and expressed in phases. In the replication of a virus such as a virus of the Herpes family, genes are expressed in three phases. Phase I involves the expression of about five genes. These genes are mostly regulatory in nature and are termed "alpha genes." The second set of genes expressed are called "beta genes;" their function is to make proteins that affect nucleic acid metabolism and synthesis or replication of viral DNA. The third set of genes, the gamma genes, comprise about 30 to 40 genes that control the synthesis of structural proteins of the virus. The gamma genes are induced after the onset of viral DNA synthesis. In the usual course of viral infection and replication, the infected cell is "killed", it may be killed outright, but alternatively may be constructively "killed" by being unable to divide or express its own genes. In order to prevent production of viral progeny and/or minimize the number of viral progeny produced, the viral replication process should be interrupted at a stage prior to where viral progeny are produced. At a time soon after infection, viral components reprogram the infected cell's metabolism for the production of viral progeny, and at this time, the cell is slated for eventual death.

SUBSTITUTE

The use of antisense oligonucleotides that are complementary to and bind to specific target nucleic sequences, particularly specific messenger RNA's, has been suggested as a means to deactivate specific genes. (See Weintraub, "Antisense RNA and DNA, Scientific American, pages 40 to 46 (January 1990) ) .

The use of antisense oligomers which are complementary to certain splice junction mRNA's of herpes simplex virus type 1 ("HSV-1") to specifically inhibit virus replication has been reported (See Kulka et al., Proc. Nat. Acad. Sci. (USA) 6:6868-6872 (September, 1989)) .

Summary Of The Invention

According to the present invention, antisense oligomers are provided that are complementary to a vital region of a viral genome which act as antiviral agents. Such vital regions comprise nucleic acid sequences necessary for viral replication and are included in one or more essential genes. Thus, in one aspect, the present invention is directed to an oligomer complementary to such a vital region or mRNA transcript thereof, which when hybridized to said target sequence, inhibits or interferes with viral DNA synthesis or replication. In one preferred aspect the target sequence comprises a portion of a mRNA transcript of a gene essential for viral DNA synthesis or replication. Suitable target sequences include sequences at or proximate to a 5*-terminal translational start or a 3'-terminal polyadenylation signal of the gene.

The present invention is also directed to methods of interfering with replication of a virus after it has infected host cells. The present invention is also directed to oligomers which are useful in interfering with and/or inhibiting such viral replication. According to methods of the present invention, said virus or viral DNA or their environment is contacted with an oligomer which is complementary to a target sequence which comprises a

SUBSTITUTE SHEET

vital region of the viral genome or mRNA transcript thereof. Optionally, two or more oligomers may be used wherein each oligomer is complementary to a different target sequence. Target sequences may be portions of the same gene or of different genes.

In one aspect, the present invention is directed to a method of interfering with replication of a virus after infection of host cells by the virus wherein the cells or their growth environment is contacted with an amount of an oligomer which is complementary to and which hybridizes with a messenger RNA sequence for a gene essential for viral DNA synthesis and/or replication, that is effective to interfere with expression or function of said gene.

In one preferred aspect of the present invention, the methods of the present invention are especially useful in interfering with viral replication in infections resulting from viruses of the family Herpesviridae, particularly human herpes viruses, especially Herpes Simplex viruses. Thus, the present invention is also directed to methods of inhibiting or interfering with replication of a human herpes virus, especially a Herpes Simplex virus, by contacting the virus viral DNA or cells infected therewith with an oligomer complementary to a nucleic acid target sequence essential for viral DNA synthesis or replication and wherein the oligomer can selectively hybridize with said target sequenced. For Herpes Simplex viruses, preferably the target sequence comprises a mRNA transcript of an essential ,3-gene. Suitable ,3 genes include UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52. Suitable regions of these genes for selection of a target sequence include a sequence at or proximate to a 5'-translational start or a 3'-polyadenylation signal.

Among other factors, in one preferred aspect, the present invention is based on our finding that oligomers complementary to mRNA transcripts of genes that code for the β group of polypeptides that are essential for viral

SUBSTITUTE SHEET

replication are especially effective in decreasing and/or inhibiting viral replication in Herpes Simplex viruses.

According to an additional aspect of the present Invention, methods of treating an organism infected with a Herpes-viradae virus are provided using these antisense oligomers and methods.

Definitions

As used herein, the following terms have the following meanings, unless expressly stated to the contrary:

The term "nucleoside" includes a nucleosidyl unit and is used interchangeably therewith.

The term "nucleotide" refers to a subunit of a nucleic acid consisting of a phosphate group, a 5 carbon sugar and a nitrogen containing base. In RNA the 5 carbon sugar is ribose. In DNA, it is a 2-deoxyribose. The term also includes analogs of such subunits.

The term "nucleotide ultimer" refers to a chain of nucleotides linked by phosphodiester bonds, or analogs thereof.

An "oligonucleotide" is a nucleotide multimer generally about 3 to about 100 nucleotides in length, but which may be greater than 100 nucleotides in length. They are usually considered to be synthesized from nucleotide monomers.

A "deoxyribooligonucleotide" is an oligonucleotide consisting of deoxyribonucleotide monomers.

A "polynucleotide" refers to a nucleotide multimer generally about 100 nucleotides or more in length. These are usually of biological origin or are obtained by enzymatic means.

A "nucleotide multimer probe" is a nucleotide multimer having a nucleotide sequence complementary with a target nucleotide sequence contained within a second nucleotide multimer, usually a polynucleotide. Usually the probe is selected to be perfectly complementary to the

SUBSTITUTE SHEET

corresponding base in the target sequence. However, in some cases it may be adequate or even desirable that one or more nucleotides in the probe not be complementary to the corresponding base in the target sequence. A "non-nucleotide monomeric unit" refers to a monomeric unit which does not significantly participate in hybridization of an oligomer. Such monomeric units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and optionally include groupings capable of interacting after hybridization of oligomer to the target sequence, such as crosslinking alkylation, intercalating and chelating agents.

A nucleotide/non-nucleotide polymer" refers to a polymer comprised of nucleotide and non-nucleotide monomeric units.

An "oligonucleotide/non-nucleotide multimer" is a multimer generally of synthetic origin having less than

100 nucleotides, but which may contain in excess of 200 nucleotides and which contains one or more non-nucleotide monomeric units.

A "monomeric unit" refers to a unit of either a nucleotide reagent or a non-nucleotide reagent of the present invention, which the reagent contributes to a polymer. A "hybrid" is the complex formed between two nucleotide multimers by Watson-Crick base pairing s between the complementary bases.

The term "oligomer" refers to oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothioate analogs of oligonucleotides, phosphoamidate analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, such as phosphotriesters and other oligonucleotide analogs and modified oligonucleotides, and also includes nucleotide/non-nucleotide polymers. The term also includes nucleotide/non-nucleotide polymers wherein one or more of the phosphorous group linkages between monomeric

units has been replaced by a non-phosphorous linkage such as a formacetal linkage or a carbamate linkage.

The term "alkyl- or aryl-phosphonate oligomer" refers to nucleotide oligomers (or nucleotide/non-nucleotide polymers) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one alkyl- or aryl- phosphonate linkage replaces a phosphodiester linkage.

The term "methylphosphonate oligomer" (or "MP- oligomer") refers to nucleotide oligomers (or nucleotide/non-nucleotide polymer) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one methylphosphonate internucleoside linkage replaces a phosphodiester internucleoside linkage. In some of the various oligomer sequences listed herein "p" in, e.g., as in ApA represents a phosphate diester linkage, and "p." in, e.g., as in CpG represents a methylphosphonate linkage. Certain other sequences are depicted without the use of p or p. to indicate the type of phosphorus diester linkage. In such occurrences, A as in ATC indicates a phosphate diester linkage between the 3'- carbon of A and the 5' carbon of T, whereas A, as in ATC or ATC indicates a methylphosphonate linkage between the 3'-carbon of A and the 5'-carbon of T or T. The term "antisense oligomer" refers to an oligomer which is complementary to the "sense" strand of a DNA duplex and to the mRNA transcript synthesized from that sequence. A DNA duplex is comprised of two complementary DNA strands, one termed the "sense" strand and one termed the "antisense" strand. Messenger RNA transcripts are synthesized using the antisense DNA strand as a template and hence are homologous (with the replacement of T with A) to the sense strand.

The term "vital region" of a viral genome or viral DNA refers to a nucleic acid sequence which is necessary for viral replication such that if the sequence is deleted

or rendered nonfunctional, the virus is incapable of replication.

The term "deblocking conditions" describes the conditions used to remove the blocking (or protecting) group from the 5 ^f -OH group on a ribose or deoxyribose group.

The term "deprotecting conditions" describes the conditions used to remove the protecting groups from the nucleoside bases. The term "tandem oligonucleotide" or "tandem oligomer" refers to an oligonucleotide or oligomer which is complementary to a sequence 5• or 3' to a target nucleic acid sequence and which is co-hybridized with the oligomer complementary to the target sequence. Tandem oligomers may improve hybridization of these oligomers to the target by helping to make the target sequence more accessible to such oligomers, such as by decreasing the secondary structure of the target nucleic acid sequence.

The melting temperature or "Tm" of a duplex (such as a double stranded nucleic acid DNA:DNA or RNA:DNA) is defined a the temperature at which half the helical structure is lost.

Detailed Description Of The Invention

The present invention is directed to antisense oligomers useful as antiviral agents and to methods of interfering with viral replication in a host cell after its infection using such antisense oligomers, wherein said oligomers are complementary to (and which hybridize with) a target nucleic acid sequence of a gene essential for viral replication or a viral messenger RNA transcript of said gene.

Preferred Target Sequences

In general, preferred are target nucleic acid sequences which comprise a vital region of the viral genome. These target sequences may comprise portions of

SUBSTITUTE SHEET

an essential gene for viral DNA replication or a mRNA transcript thereof which are "available", i.e. are in a state where the complementary oligomer is able to hybridize with the target sequence. Thus, these target sequences are preferably single stranded and relatively free of secondary structure and bound protein.

Preferred target sequences include mRNA transcripts of genes which are "essential" for DNA replication. Moreover, mRNA transcripts which are present in low numbers comprise particularly advantageous target sequences for this antisense therapy. With fewer mRNA transcripts, a lower concentration of oligomer can hybridize with and interfere with the function of a larger percentage of the mRNA from a particular gene. Certain genes which code for mRNA's which are present in large amounts comprise less preferred target sequences, since if the function of these mRNA's is only partially blocked, the unhybridized mRNA's may proceed with the normal replicative cycle of the virus. Moreover, a proportionally larger amount of oligomer would be required to block an equivalent fraction of the mRNA.

Preferred are essential genes which are expressed during the earlier stages of DNA replication, but after the cell is "committed" to death due to infection by the virus. By blocking DNA replication at such an earlier stage, few if any functional virus particles are made. Since the cell has already been "committed" to death it will die whether or not functional virus particles are made and after death, will be dealt with by the host organism's immune system. These cells include cells which, due to the viral infection, are unable to divide and/or express their own genes. If viral DNA replication is blocked before the cell has been committed to death, the viral DNA in the cell will not be destroyed and viral DNA replication may recommence later on.

SUBSTITUTESHEET

Suitable target sequences include sequences which are at or proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal.

Preferred target sequences include those which have a relatively high local G-C base content. Sequences having a relatively high local G-C content are preferred in part because they tend to hybridize more tightly to the complementary oligomer and exhibit a correspondingly higher Tm. Especially preferred target sequences have a high G-C base content on both ends of the target sequence that is complementary to and hybridizes with the complementary oligomer, which enables the ends of the oligomer to hybridize more tightly to the target sequence.

Preferred Oligomers These oligomers may comprise either ribonucleoside or deoxyribonucleoside monomeric units; however, deoxyribonucleoside monomeric units are preferred.

Preferred are oligomers which comprise from about 6 to about 40 nucleotides, more preferably from about 12 to about 20 nucleotides. Although oligomers which comprise more than about 20 nucleotides may be used, where complementarity to a longer sequence is desired, it may be advantageous to employ shorter tandem oligomers to maximize solubility and transport across cell membranes while competing for the development of a secondary structure of the target nucleic acid, such as a mRNA. Alternatively, it may be advantageous to use more than one oligomer, each oligomer complementary to a distinct target sequence which may be part of the same gene or a different gene.

Although nucleotide oligomers (i.e., having the phosphodiester internucleoside linkages present in natural nucleotide oligomers, as well as other oligonucleotide analogs) may be used according to the present invention, preferred oligomers comprise oligonucleoside alkyl and aryl-phosphonate analogs, phosphorothioate oligonucleoside

SUBSTITUTE SHEET

analogs, phosphoro-amidate analogs and phosphotriester oligonucleotide analogs. However, especially preferred are oligonucleoside alkyl- and aryl-analogs which contain phosphonate linkages replacing the phosphodiester linkages which connect two nucleosides. The preparation of such oligonucleoside alkyl and aryl-phosphonate analogs and their use to inhibit expression of preselected nucleic acid sequences is disclosed in U.S. Patent Nos. 4,469,863; 4,511,713; 4,757,055; 4,507,433; and 4,591,614, the disclosures of which are incorporated herein by reference. A particularly preferred class of those phosphonate analogs are methylphosphonate oligomers.

Such alkyl- and aryl-phosphonate oligomers advantageously have a nonionic phosphorus backbone which allows better uptake of oligomers by cells. Also, the alkyl- and aryl-phosphonate intermonomeric linkages of such alkyl- and aryl-phosphonate oligomers are advantageously resistant to nucleases.

Where the oligomers comprise alkyl- or aryl- phosphonate oligomers, it may be advantageous to incorporate nucleoside monomeric units having modified ribosyl moieties. The use of nucleotide units having 2'- 0-alkyl- and in particular 2'-0-methyl-, ribosyl moieties, in these alkyl or aryl phosphonate oligomers may advantageously improve hybridization of the oligomer to its complementary target nucleic acid sequence.

Synthetic methods for preparing methylphosphate oligomers ("MP-oligomers") are described in Lee, BL. efc al. , Biochemistry 27:3197-3203 (1988) and Miller, P.W. , et al. , Biochemistry 2j5:5092-5097 (1986), the disclosure of which are incorporated herein by reference.

Preferred are oligonucleoside alkyl- and aryl- phosphonate analogs wherein at least one of the phosphodiester internucleoside linkages is replaced by a 3' - 5' linked internucleoside methylphosphonyl (MP) group (or "methyl-phosphonate") . The methylphosphonate linkage is isosteric with respect to the phosphate groups of

SUBSTITUTESHEET

oligonucleotides. Thus, these methylphosphonate oligomers

("MP-oligomers") should present minimal steric restrictions to interaction with complementary polynucleotides or single-stranded regions of nucleic acid molecules. These MP-oligomers should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules. It has been found that certain MP-oligomers are more resistant to nuclease hydrolysis, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent

No. 4,469,863).

If desired, labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalating agents such as acridine, alkylating agents or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o- phenanthroline-copper or EDTA-iron may be incorporated in the MP-oligomers.

Preferred are MP-oligomers having at least about 6 nucleosides which is usually sufficient to allow for specific binding to the desired nucleic acid sequence. More preferred are MP-oligomers having from about 6 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides. Due to a combination of ease of preparation, with specificity for a selected sequence and minimization of intra-oligomer- internucleoside interactions such as folding and coiling, particularly preferred are MP-oligomers of from about 12 to 20 nucleosides.

One group of preferred MP-oligomers includes MP- oligomers where the 5*-internucleoside linkage is a phosphodiester linkage and the remainder of the internucleoside linkages are methylphosphonyl (or methylphosphonate) linkages. Having a phosphodiester

SUBSTITUTESHEET

linkage on the 5'-end of the MP-oligomer permits kinase labelling and electrophoresis of the oligomer.

Preferred Embodiment of the Invention

Infections due to viruses of the family Herpesvirdae comprise particularly suitable targets for therapy using the antisense oligomers and methods of the present invention. Herpes viruses vary greatly in their biological properties. Some have a wide host cell range, multiply efficiently and rapidly destroy the cells which they infect (HSV-1, HSV-2) . Others have a narrow host cell range. A ubiquitous property of these herpes viruses is their capacity to remain latent in the host in which they multiply. The mechanism by which the virus perpetuates itself appears to reflect a function of dedicated viral genes as well as association with appropriate cells. In general, infections caused by herpes viruses have been found to be persistent. Herpes viruses for which therapy using these antisense oligomers appears promising include human herpes viruses 1 to 7 which include Herpes simplex Virus Type 1, Herpes Simplex Virus Type 2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus and human herpes viruses 6 and 7.

In one preferred embodiment, the present invention is directed to antisense oligomers which are useful as antiviral agents against herpes simplex virus ("HSV") , particularly type 1 ("HSV-1"), and to methods of controlling HSV-1 infections by inhibiting and/or interfering with replication of HSV-l.

According to the present invention, antisense oligomers are provided that are complementary to "essential" genes.

In Herpes viruses, as much as about one half of viral genes are non essential; that is, they may be deleted or at least reduced in expression or treated with antisense oligomers and not effect viral replication.

UBSTITUTESHEET

Preferred target sequences for these antisense oligomers comprise essential genes, that is genes which when deleted or their function is compromised, significantly affect viral replication, particularly the synthesis and/or replication of DNA. Also, as noted, preferred target sequences include mRNA transcripts of such essential genes, wherein copies are present only in low numbers. For this reason, we have found that essential β genes of HSV-1 to comprise particularly suitable target sequences for these antisense oligomers. HSV-1 has about 15 β genes, of which at least about 8 have been reported to be essential. These essential β genes include the genes termed UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52. These genes have been reported to code for proteins which are necessary for viral DNA synthesis and/or replication. Seven of these genes have been reported to be required for viral-origin-dependent DNA synthesis and to map in the L component of the viral DNA. These seven genes have been reported to specify the following: a DNA polymerase (UL30) with an apparent molecular weight of 140,000; a single-strand specific- DNA-binding protein designated as ICP8 (UL29) with an apparent molecular weight of 124,000; a protein binding to the origin of viral DNA synthesis (UL9) with a translated molecular weight of 94,000; a protein that binds to double-stranded DNA (UL42) with a molecular weight of 62,000; and three additional proteins (UL5, predicted molecular weight of 99,000; UL8, predicted molecular weight of 80,000; and UL52, predicted molecular weight of 114,000). These three proteins form a complex in which each protein is present in equimolar ratios and which functions as a primase and helicase. The protein specified by UL5 has independently been shown to act as a DNA dependent ATPase. The above noted seven proteins appear to be all that is necessary for ori _s -dependent amplification of DNA transfected into cells. For this reason, oligomers which

are complementary to the mRNA of one of these seven genes are particularly preferred and comprise especially suitable antiviral agents against HSV-1.

Of the above noted seven essential genes, preferred are the genes denoted UL5, UL8 and UL52. It is believed that the mRNA transcripts of these genes comprise target sequences which are particularly susceptible to inhibition using these antisense oligomers.

Portions of these essential genes which may be relatively more available to these antisense oligomers comprise especially suitable target sequences. It is believed that sequences that are proximate to the 5'- terminal translational start of these mRNA transcripts or to the 3'-terminal polyadenylation signal comprise especially suitable target sequences in view of their demonstrated susceptibility to inhibition of viral function due to hybridization of an antisense oligomer. Preferred target sequences include portions of these mRNA transcripts in which it appears that secondary structure of the mRNA does not interfere with its ability to hybridize to a complementary oligomer.

Antisense oligomers complementary to selected regions of mRNA transcripts of these seven genes have been assayed for antiviral activity using a Virus Titer Reduction Assay (see Example A) and a Direct Plaque Assay (Example B) and have been found to demonstrate antiviral activity (see Tables II, III and IV) .

To assist in understanding the present invention, the following examples are included which described the results of a series of experiments. The following examples relating to this invention should not, of course, be construed in specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the present invention as hereinafter claimed.

SUBSTITUTE SHEET

Example 1

Preparation Of Phosphate Diester Oligomers

Phosphate diester oligomers are prepared using a Biosearch model 8750 DNA synthesizer using standard phosphoro-amidite chemistry (M.H. Caruthers, et al. , Methods of Enzymol. 154:287-313 (1985)) according to the manufacturers recommendations. The 5'-dimethoxytrityl protecting group is left on at the end of the synthesis to permit purification on a Sep-Pak™ C18 cartridge (Millipore/Waters, Bedford, MA) as described by K.M. Lo et al. (Proc. Natl. Acad. Sci. (USA) .81:2285-2289 (1984)). During this procedure, the dimethoxytrityl protecting group was removed.

Example 2 Preparation Of Methylphosphonate Oligomers

Methylphosphonate oligomers are synthesized using methylphosphonamidite monomers, according to the chemical methods described by P.S. Miller et al. (Nucleic Acids Res. 11:6225-6242 (1983)), A. Jager and J. Engels (Tetrahedron Letters 25_:1437-1440 (1984)) and M.A. Dorman et al. (Tetrahedron Letters 40:95-102 (1984)). Solid phase synthesis is performed on a Biosearch Model 8750 DNA synthesizer according to the manufacturer's recommenda¬ tions with the following modifications: "G" and "C" monomers are dissolved in 1:1 acetonitrile/dichloromethane at a concentration of 100 mM. "A" and "T" monomers are dissolved in acetonitrile at a concentration of 100 mM. DEBLOCK reagent = 2.5% dichloroacetic acid in dichloromethane. OXIDIZER reagent = 25 g/L iodine in 2.5% water, 25% 2,6-lutidine, 72.5% tetrohydrofuran. CAP A= 10% acetic anhydride in acetonitrile. CAP B = 0.625% N,N- dimethylaminopyridine in pyridine. The 5'-dimethoxytrityl protecting group is left on at the end of the synthesis to facilitate purification of the oligomers, as described below.

SUBSTITUTESHEET

The crude, protected methylphosphonate oligomers are removed from the solid support by mixing with concentrated ammonium hydroxide for two hours at room temperature. The solution is drained from the support using an Econo- Column™ (Bio-Rad, Richmond, CA) and the support is washed five times with 1:1 acetonitrile/water. The eluted oligomer is evaporated to dryness under vacuum at room temperature. Next, the protecting groups are removed from the bases with a solution of ethylenediamine/ethanol/ acetonitrile/water (50:23.5:23.5:2.5) for 6 hours at room temperature. The resulting solutions are then evaporated to dryness under a vacuum.

The 5'-dimethoxytrityl ("trityl") containing oligomers are purified from non-tritylated failure sequences using a Sep-Pak™ C18 cartridge (Millipore/Waters Bedford, MA) as follows: The cartridge is washed with acetonitrile, 50% acetonitrile in 100 mM, triethylammonium bicarbonate (TEAB, pH 7.5) and 25 mM TEAB. Next, the crude methylphosphonate oligomer is dissolved in a small volume of 1:1 acetonitrile/water and then diluted with 25 mM TEAB to a final concentration of 5% acetonitrile. This solution is then passed through the cartridge. Next, the cartridge is washed with 15-20% acetonitrile in 25 mM TEAB to elute failure sequences from the cartridge. The trityl-on oligomer remaining bound to the cartridge is then detritylated by washing with 25 mM TEAB, 2% trifluoroacetic acid, and 25 mM TEAB, in that order. Finally, the trityl-selected oligomer is eluted* from the cartridge with 50% acetonitrile/water and evaporated to dryness under vacuum at room temperature.

The methylphosphonate oligomers are further purified by reverse-phase HPLC chromatography as follows: A Beckman System Gold HPLC is used with a Hamilton PRP-1 column (Reno, NV, 10 μ, 7 mm i.d. x 305 mm long) . Buffer A = 50 mM triethylammonium acetate (pH 7) ; Buffer B = 50% acetonitrile in 50 mM triethylammonium acetate (pH 7) . The sample, dissolved in a small volume of 10-50%

5.7 CONFIRMATION OF TRANSFORMATION

Transformation may be confirmed using procedures known in the art. For instance, confirmation may be accomplished by putting the shoot in a rooting medium comprising a selection agent, as in Section 5.6, supra . In another embodiment, leaf pieces removed from transformed shoots are placed on a callus medium which may comprise effective amounts of a nutrient medium, one or more growth regulator(s) , i.e., phytohormones, an energy source, and a selection agent, specific examples of which are described in Section 5.4, supra , and subsequently determining the callus response of the leaf pieces to these growth conditions.

The presence of a reporter gene may also be determined to confirm transformation, e.g., by GUS (0-glucuronidase) or luciferase assays, if the Agrobacteri urn comprises a DNA fragment encoding a β- glucuronidase or luciferase gene, by using procedures known in the art. See, e.g., Jefferson et al. (1986) Proc. Natl. Acad. Sci. USA 83:8447-8451 (GUS) and Ow et al. (9186) Science 234:856-859 (luciferase) .

The exogenous DNA fragment may be detected by DNA detection means using procedures known in the art. These include but are not limited to Polymerase Chain Reaction (PCR) technology, restriction enzyme digestion, Southern blot hybridization, and Northern blot hybridization (see, e.g., Maniatis et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) . 5.8 APPLICATIONS AND USES

The method of the invention may be used to introduce a wide variety of gene-encoded traits in the chrysanthemum plants using genes from any source (e.g., bacterial, fungal, plant, mammalian) . Examples of such traits included new colors or color patterns (e.g., encoded by genes for indigo, delphinidin, anthocyanin, carotenoids, betalains) ; herbicide resistance (e.g..

chlorosulfuron, glyphosphate, sulfonylurea) ; pesticide or disease resistance (e.g., known fungal, bacterial, viral, insect, or nematode diseases) ; resistance to environmental extremes (e.g., drought, temperature salinity); and horticultural characteristics (e.g., habit, flowering time, etc.). The method may also be used to alter or modulate the effect of genes present in plants, e.g., by transformation with antisense constructs, or with "sense constructs" (see Napoli et al. (1990) The Plant Cell 2:279-289 and van der Krol et al. (1990) The Plant Cell 2:291-299.

The method may also be used as a means to generate somaclonal variation in chrysanthemums, as an alternative to known methods, e.g., treatment with radiation or chemical mutagens. While not being bound by any theory or mechanism, it is believed that interaction of introduced Agrobacterium DNA or pieces of the same with endogenous plant components, e.g., plant nucleases may lead to the creation of somaclonal variants. This may provide a milder and more controllable means of generation of somaclonal variation than existing methods. This approach provides a means to produce a variety of new, readily selectable traits in host chrysanthemums, unrelated to the particular DNA actually introduced by the Agrobacterium.

The following examples illustrated the practice of the present method.

6. EXAMPLE 1: TRANSFORMATION OF DENDRANTHEMA GRANDIFLOR VARIETY TOON HERMANS

6.1 GROWTH OF SOURCE PLANTS

Rooted, in vitro shoots of Dendranthema grandiflora variety Toon Hermans were obtained from Fides (Holland) . They were maintained and propagated by putting nodal segments into Magenta G-7 (Magenta Corp.) boxes with 80 ml of an MS-based medium (MS major salts, minor salts and iron (Murashige et al. (1962) Phys.

IAZ E-I

A. COMPLEMENTARY TO AREA AROUND TRANSIATIONAL START

OLIGOMER NO. SEQUENCE

0015 5'-CAT-GAC-COC-ACC-ACG-CTC-3 '

0013 5 ⁱ -CTg-COq-TQT-CCA-TCO-CAC-3 ^■

0021 5•-GAT-ATC-TGC-GOT-GTC-CAT-3 •

0028 5i-CTC-TCC-TCC-ACOCAC-AC-3 '

0046 5'-CCT-CCA-CCC-ACA-GCG-TAT-3• 0047 5•-GGC-CCC-TAA-GOA-TCA-CGG-3 • 0007 5•-CCA-CGA-AAG-GCA-TGA-CCG-3• 0056 5 ^» -CTq-ACC-AAA-CAT-CGC-QCA-3• 0054 5 ^« -GCT-TTG-TCT-CCA-TGT-CCT-3 ^» 0039 5•-GCC-ACC-GGA-AAA-CAT-CGC-3 • 0016 5'-GAA-TCC-GTC-ATC-CCA-ACG-3' 0052 5'-GTC-CQC-GCQ-CCC-AAG-QGC-3' 0019 5•-GTC-T C-CTQ-CCC-CAT-TQC-3 ^» 0053 5 ^t -CCT-CTC-CCC-GCQ-GTT-CCC-3 '

A, C, G or T ^*■*■ Phosphate diester linkage ht C, g or T ^* = Methylphosphonate linkage

SEQUENCE T — GTT**GTC— TT* AAT-GGA-3 CTG-GTT'-G'yT-TGT* TGT-CTT-3 AAT- O-qC -GGT' TGT-TTG-3 AAT-AAC-ACA-TAA- A T-TGO-3 -GCO-TOT-TTQ-ATC- ^■ TTA-ATA-3 -ACC-CTG-ATC-GQC- CGT-CGC-3 -AAC-AGA-AAOGAC- ATC-TTG-3 -ATT-GGT-CAA-ACT- ^■ GAG-GCA-3 -TCT-CAG-GGC-AAT- •GTT-TTT-3 -CAC-qqq-qGA-qco- ■CTC-T G-3 -T A-T G-GGC-GC - ^■ CAC-GAG-3 -ATG-TCG-TAG-CAA- ^■ TAA-TTT-3 -GTC-GGC-CGC-AGA- •CAT-TTA-3 -ACC-AAG- T -ATT- ■TAC-AT -3 -TTG-GOC-AAT-ACC' ■AAG-TT -3 -GCG-ACA-CGC-GGG- AAA-GTG-3 -CAC-ACA-CAT-GAA- CCA-CAC-3 -GAG-GGT-GGO-GGC- GCC-AGG-3 -GTA-CGT-GGC-ATG- ^■ TAT-TTA-3 -ACC-AAT-CAO-ACA- ■CCA-TAA-3 -CCT-CCG-GCA-CAG' ACA-AGG-3

A, c, G or T - Phosphate diester linkage i» S» S or I « Methylphosphonate linkage

ESHEET

21

TABLE II

ANTI-HERPES SIMPLEX VIRUS TYPE 1 ACTIVITY (TRANSIATIONAL START)

Q IgQMSR PQ _T GENE OCATION INHIBITION (Gene Product) fftTG START +1) (MAXIMUM) +1

0015 DNA DNP. ATPase -15 -+3 63%

HELICASE/PRIMASE

0013 -5- -+13 78% 0021 +1- -+18 45% 0028 +19- -+35 62%

UL9 ORIGIN BINDING PROTEIN

0007 -5 -+13 49%

HL1£ SPLICE JUNCTION

0056 -6- -+12 7%

IZ 2 DNA BINDING PROTEIN

0054 _5_. -+13 68%

UL30 DNA POLYMERASE

0039 -3- -+15 50%

UL42 DNA SYNTHESIS PROTEIN

0016 -7 -+11 26%

UL52 HELICASE/PRIMASE

0052 -21 4 63% 0019 -3- -+15 79% 0053 +16- -+33 74%

τπ-uTΞ SHEET

TABLE III

ANTI-HERPES SIMPLEX VIRUS TYPE 1 ACTIVITY (POLY A SIGNAL)

OLIGOWRP jm, SE E LOCATTQW (Gene Product) INHIBTTTQW +1 (MAXIMUM) A(A) TAAA

IΪ S DNA DNP. ATPase

0005 -3 +15 0006 +5 +22 73% 0020 0008 +12 +29 69% 0023 +23 -+40 18% 17% 0059 +41- —+58 96% +59- -+76 18%

UL8 HELICASE/PRIMASE

0051 -15 +3 0024 +4 +21 86% 0014 71% 0022 +22- -+39 10% +40- -+57 58%

21≥ SPLICE JUNCTION

0057 -13 H5

32%

UL29 DNA BINDING PROTEIN

0055 +4 +21

10%

UL30 DNA POLYMERASE

0040 _ +3 ₁ -20

55%

UL42 DNA SYNTHESIS PROTEIN

0025 -6 (-12 0017 +4 +21 17% 0018 0026 +22- 33%

-+39 54% 0151 +40- — ⁽ -58 93% +59- -+76

44%

ULS2 HELICASE/PRIMASE

0027 +3 +20 0038 0035 +21- 87%

—1-38 98% +39- --+56 66%

SUBSTITUTESHEET

TAB E IV

SEQUENCE

5 ' -CTO-CGG-TGT-CCA-TCO-CAC-3 • 5 • -CTQ-CQQ-TGT-CCA-TCG-CAC-3 « 5 ^> -CTG-CGG-TGT-CCA-TCG-CAC-3 ' 5 ' -CTG-CGG-TGT-CCA-TCG-CAC-3 * 5 ' -CTQ-CGG-TGT-CCA-TCG-CAC-3 • 5 • -CTG-CGG-TGT-CCA-TCG-CAC-3 '

0004 UL8 5 • -GAT-ATC-TGC-GGT-GTC-CAT-3 • 200μM 32%

A, C, G or T = Phosphate diester linkage J, S» S or ϊ = Methylphosphonate linkage

IΔB E_

A. MULTIPLE OLIGOMERS COMPLEMENTARY TO ONE GENE AlSDS Together

(32%)

82% (62%)

99%

B. TWO OLISOMERS COMPLEMENTARY TO TWO SENSE Oligoaer Npt QSΏΛ Together

0013 UL8

67%

0019 UL52