JP2001114798A - Oligonucleotides with high chiral purity phosphorothioate linkages - Google Patents

Abstract

(57) [Summary] (Modifications) [Problem] To provide an oligonucleotide having a phosphorothioate bond of high chiral purity. A phosphorothioate oligonucleotide, wherein all nucleoside units are linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages, preferably the target RNA or Provided are phosphorothioate oligonucleotides that are complementary to at least a portion of the sequence of DNA, as well as chemical and enzymatic methods for synthesizing sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] A related cross-reference the application of the application, filed on August 29, 1994, is a US continuation-in-part application of patent application Ser. No. 08 / 297,703, which is filed on October 16, 1991 No. 07 / 777,007, which is a continuation-in-part of U.S. patent application Ser. This application is also a continuation-in-part of U.S. Patent Application No. 08 / 058,023, filed May 5, 1993, which is incorporated by reference in U.S. Patent Application No. 07/777, filed October 15, 1991. , 670 (U.S. Pat. No. 5,212,
No. 295, issued May 18, 1993), which is a continuation-in-part of US patent application Ser. No. 07 / 777,007. Each of the above-mentioned patent applications is assigned to the assignee of the present invention, and the entire disclosure of each application is incorporated herein by reference.

[0002] The present invention relates to sequence-specific phosphorothioate oligonucleotides containing nucleosides linked by intersugar linkages, and their synthesis and use. More specifically,
The intersugar linkages connecting the nucleosides of the oligonucleotides of the invention may be substantially pure all Sp or all R
p is a chiral phosphorothioate linkage. Such oligonucleotides are produced by chemical or enzymatic synthesis. They are particularly well suited as diagnostic, therapeutic and research reagents.

[0003] oligonucleotides of the invention are single-stranded RNA or single-stranded DNA
Is known to hybridize to Hybridization is based on the target RN of the base of the oligonucleotide.
A sequence-specific base pair hydrogen bond to the base of A or DNA. Such base pairs are said to be complementary to each other.

In determining the degree of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of the oligonucleotide to bind to the complementary nucleic acid is compared by measuring the melting temperature of a particular hybridization complex. be able to. Melting temperature (Tm)
Is the physical property characteristic of a double helix, the temperature (° C.) at which 50% of the helical (hybridized) to coiled (unhybridized) is present
Represents Tm is measured by using the UV spectrum to determine the formation and disintegration (melting) of the hybridization complex. Base stacking that occurs during hybridization is accompanied by a decrease in UV absorption (lightening). Therefore, the reduction in UV absorption is higher T
m. The higher the Tm, the higher the bond strength between the chains.

[0005] Using oligonucleotides, enzymatic cleavage of target RNA using the intracellular enzyme RNase H can be performed. The mechanism of such RNase H cleavage requires that the 2'-deoxyribofuranosyl oligonucleotide hybridize to the target RNA. The resulting DNA-RNA duplex is RNa
Activates the seH enzyme, and the activated enzyme cleaves the RNA strand. Breakage of the RNA strand disrupts the normal function of the target RNA. Phosphorothioate oligonucleotides work by this type of mechanism. But D
For the NA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be
To survive in cells for a sufficient time for ase H activation,
It must be reasonably stable to nucleases.
In non-cellular applications, such as the use of oligonucleotides as research reagents, such nuclease stability may not be necessary.

[0006] Some publications by Walder et al.
The interaction between Nase H and the oligonucleotide is described. Of particular interest are: (1) D
agle et al. , Nucleic Acids
Research 1990, 18, 4751;
(2) Dagle et al. , Antisense
Research And Development
1991, 1, 11; (3) Eder et al. ,
J. Biol. Chem. 1991, 266, 647
2; and (4) Dagle et al. , Nucl
eic Acids Research 1991,1
9,1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since these are substrates, the target RN by RNase H
Activate cleavage of A. However, the authors further note that in Xenopus embryos, both phosphodiester and phosphorothioate linkages are also subject to exonuclease degradation.
Such nuclease degradation is detrimental because it rapidly depletes oligonucleotides available for RNase H activation.

As described in references (1), (2) and (4), phosphoramidates are used to stabilize oligonucleotides against nuclease degradation while still providing RNase H activation. 2'-deoxyoligonucleotides were constructed with short sections of phosphodiester linked nucleotides located between areas of alkyl phosphonate or phosphotriester linkages. Since the phosphoramidate-containing oligonucleotide was stabilized against exonuclease, it was described in Reference (4).
In, the authors note that each phosphoramidate linkage resulted in a 1.6 ° C decrease in the measured Tm value of the phosphoramidate-containing oligonucleotide. Such a decrease in Tm indicates a decrease in hybridization between the oligonucleotide and its target nucleic acid strand.

[0008] The application of oligonucleotides as diagnostic, research reagents, and therapeutic agents includes transporting or being taken up by cell membranes of the oligonucleotides, properly hybridizing to target RNA or DNA, And subsequently it is necessary to stop or destroy nucleic acid function. These essential functions depend, in part, on the initial stability of the oligonucleotide to nuclease degradation. In addition, their function depends on the specificity of the oligonucleotide for the target RNA or DNA molecule.

A serious deficiency of oligonucleotides for these purposes is that they are susceptible to enzymatic degradation by various ubiquitous nucleases that may be intracellular and extracellular. Unmodified, "wild-type" oligonucleotides are not useful as therapeutics because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides to confer nuclease resistance has been a major focus of research aimed at developing oligonucleotide therapy and diagnostics.

[0010] Modification of oligonucleotides to enhance nuclease resistance has generally been performed on the sugar-phosphate backbone, especially on the phosphorus atom. Phosphorothioates have been reported to be resistant to nucleases. In addition, phosphorothioate oligonucleotides are generally more chemically stable than natural phosphodiester oligonucleotides. Phosphorothioate oligonucleotides also exhibit solubility in aqueous media. Furthermore, phosphorothioate oligonucleotide-RNA
Heteroduplexes can serve as substrates for exogenous RNase H. Furthermore, phosphorothioate oligonucleotides exhibit high thermodynamic stability. However, if the oligonucleotide is the target RNA or D
Although the ability to bind NA reliably is important for hybridization to target RNA or DNA, modifications at the phosphorus atom of the oligonucleotide
Although they exhibit varying degrees of nuclease resistance, they have the disadvantage of generally poor hybridization properties [Cohen, J. et al. S. , Ed. , Oligonucl
eotides: Antisense Inhibit
ors of Gene Expression (CR
C Press, Inc. , Boca Raton, F
L, 1989].

[0011] The reason for this poor hybridization may be the prochiral nature of the phosphorus atom. Modification of the modified phosphorus at the internal phosphorus atom of the oligonucleotide results in the Rp and Sp stereoisomers.

The modified phosphorus oligonucleotides obtained to date, wherein the resulting molecule has an asymmetric substituent, are
ⁿ (n is equal to the number of phosphorothioate sugar linkages in the oligonucleotide) it was racemic mixtures with isomers. Thus, 15 containing 14 asymmetric centers
The -mer phosphorothioate oligonucleotide has 2
^It contains ¹⁴ or 16,384 diastereomers.
In this regard, in the racemic mixture, only a small percentage of the oligonucleotides appear to specifically hybridize to the target mRNA or DNA with sufficient affinity.

[0013] Chemically synthesized phosphorothioate oligonucleotides having chirally pure intersugar linkages have so far been limited to molecules having only one or two diastereomeric intersugar linkages. . Since the synthesis of oligonucleotides with chirally pure intersugar linkages has not yet been achieved by automated synthesis, the effect of chirality introduced into a chemically synthesized racemic mixture of sequence-specific phosphorothioate oligonucleotides Was not evaluated until recently. This is because sulfur is incorporated non-stereospecifically during automated synthesis. See, for example, Stec et al. , J. et al. Chrom
matography, 326: 263 (1985).
Have synthesized certain oligonucleotide phosphorothioates with racemic intersugar linkages, but can elucidate the diastereomers of certain small oligomers with one or at most two diastereomeric intersugar linkages. It was just done.

However, Stec et al. [Nuc
leic Acids Res. , 19: 5883 (1
991)] subsequently reported automated stereocontrolled synthesis of oligonucleotides. The method described in the above-mentioned literature is 5
It utilizes base-catalyzed nucleophilic substitution at the valency phosphorothioyl center.

The synthesis of phosphorothioates with all Rp intersugar linkages using enzymatic methods has been studied by several authors [Burgers and Ec].
kstein, J .; Biological Chemi
try, 254: 6889 (1979); Gupta
et al. , J. et al. Biol. Chem. , 256:
7689 (1982); Brody and Frey,
Biochemistry, 20: 1245 (198
1); and Eckstein and Jobin, B
iochemistry, 2: 4546 (198
3)]. Brody et al. (Biochemistry, 2).
1: 2570 (1982)], and Romaniuk
And Eckstein [J. Biol. Chem. ,
257: 7684 (1982)] synthesized enzymatically poly TpA and poly ApT phosphorothioate. B
urgers and Eckstein [Proc. Na
tl. Acad. Sci. U. S. A. , 75: 479
8 (1978)] enzymatically synthesized polyUpA phosphorothioate. Cruse et al. [J. Mol. Bio
l. , 192: 891 (1986)] produced a hexamer of three diastereomeric Rp GpC phosphorothioate dimers linked by a natural phosphodiester bond.

[0016] The relative ability of an oligonucleotide to bind to a complementary nucleic acid can be compared by measuring the melting temperature of a particular hybridization complex. Melting temperature (Tm) is a physical property characteristic of a double helix and represents the temperature (° C.) at which 50% of the helical versus coiled (unhybridized) is present. Tm
Is measured by using the UV spectrum to determine the formation and collapse (melting) of hybridization. Base stacking that occurs during hybridization is accompanied by a decrease in UV absorption (lightening). Thus, a decrease in UV absorption indicates a higher Tm. The higher the Tm, the higher the bond strength between the chains. Non-Watson-Crick base pairs have a strong destabilizing effect on Tm.

Preliminary report [Stec, J. et al. W. , Oli
goucleotides asAntisense
Inhibitors of Gene Express
session: Therapeutic Implica
tions, Meeting abstracts, J
Une 18-21, 1989], from two thymidine methylphosphonate tetramer diastereomers linked by natural phosphodiester bonds, thymidine homopolymers having all but one modified phosphate bond at the intersugar bond An octamer ("other than one") Rp configuration or a "other than one" Sp configuration was formed. Rp “other than one” methylphosphonate sequence nonspecific thymidine homooctamer, ie, (dT) ₈
Is a 15-mer deoxyadenosine homopolymer, ie, (dA) _15, and a similar thymidine homopolymer with “except one” Sp configuration methylphosphonate linkages, ie, all but one except the intersugar linkages of Sp Note that a more thermodynamically stable hybrid (Tm 38 ° C.) was formed than the hybrid formed by d (T) ₁₅ and d (A) ₁₅ (Tm <O ° C.). Having a natural phosphodiester bond (d
T) ₈ , a hybrid between octatimidylic acid and d (A) ₁₅ , is reported to have a Tm of 14 ° C.

More recently, Ueda et al. [Nucleley.
c Acids Research, 19: 547 (1
991)] enzymatically synthesized mRNA for intermittently incorporating Rp diastereomeric phosphorothioate linkages for use in translation systems. Ueda et al.
T7 coliphan DNA with 17 promoters for 7 RNA polymerase and one stop site was used. In vitro synthesis with T7 RNA polymerase
MRNA with hundreds to tens of thousands of nucleotides was generated.

Backbone chirality will also affect the sensitivity of phosphorothioate oligonucleotide-RNA heteroduplexes to RNase H activity. The ability to serve as a template for RNase H has significant therapeutic implications, as it has been suggested that RNase H causes cleavage of the RNA component in the RNA-DNA oligonucleotide heteroduplex. For oligonucleotides containing a racemic mixture of Rp and Sp intersugar linkages, it is not known whether all phosphorothioate oligonucleotides can function equally as substrates for RNase H. For various catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (in-line mechanism) and a reversal of configuration. Thus, only a small percentage of oligonucleotides containing the correct chirality for maximum hybridization efficiency and translation termination will be present in the racemic mixture. That is, increasing the percentage of phosphorothioate oligonucleotides that can serve as a substrate for RNase H in a heteroduplex will lead to more effective compounds for antisense therapy.

[0020] To enhance the reliability of hybridization, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are highly desirable. Moreover, such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages will lead to more effective therapeutic compounds. However, so far, synthesis of such molecules has been largely unsuccessful. Therefore, a simple method for synthesizing phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages is highly desirable.

It has been recognized that the nuclease resistance of oligonucleotides and the reliability of hybridization are very important in the development of oligonucleotide therapy. Oligonucleotides having nuclease resistance are also desired as research reagents and diagnostics.

SUMMARY OF THE INVENTION In accordance with the present invention, phosphorothioates in which all nucleoside units are linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages An oligonucleotide is provided. Preferably, the phosphorothioate oligonucleotide of the invention comprises a target R
Complementary to at least a portion of the sequence of NA or DNA.

Further, according to the present invention, there are provided chemical and enzymatic methods for synthesizing sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. Here, the phosphorothioate oligonucleotide consists of at least 10 nucleoside units linked together by either substantially all Rp or substantially all Sp intersugar linkages. Preferably, the phosphorothioate oligonucleotide consists of about 10 to about 50 nucleoside units linked by a substantially chirally pure intersugar linkage. More preferably, the phosphorothioate oligonucleotide consists of about 15 to about 30 nucleoside units linked by a substantially chirally pure intersugar linkage. Most preferably, said phosphorothioate oligonucleotide consists of about 17 to 21 nucleoside units linked by a substantially chirally pure intersugar linkage. The method involves combining a sequence primer, a template, and an excess of all four chirally pure nucleosides 5'-O- (1-thiotriphosphate). The method further comprises synthesizing the complementary oligonucleotide by addition of a polymerase followed by cleavage of the primer from the complementary oligonucleotide. Further, the method includes dissociating the complementary oligonucleotide from the template.

In another embodiment of the method of the present invention for synthesizing a sequence-specific phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages, a sequence primer, template and nucleoside 5'-O-
Combine the racemic mixture of (1-thiotriphosphate). Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages and complementary to the template are synthesized by adding a polymerase and a selected metal ion. The oligonucleotide thus synthesized is dissociated from the template and the primer.

Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can be used as target RNs.
It is useful for increasing the thermodynamic stability of heteroduplexes formed with A and DNA and eliciting RNase H activity. Further, the oligonucleotides of the present invention are useful for modulating the activity of RNA.

The phosphorus atom in a phosphodiester bond detailed description oligonucleotides of the invention can be said to be "prochiral". Substitution or modification of a non-bonded oxygen atom of a phosphodiester bond results in the formation of a chiral sugar-phosphate bond. The resulting intersugar linkage is either an Sp intersugar linkage or an Rp intersugar linkage. Displacement of the non-bonded oxygen atom of the natural phosphodiester bond with sulfur to give a phosphorothioate bond results in the formation of a chiral center and Sp and Rp diastereomers. Molecules in which substantially all of the phosphorus atoms in the sugar backbone are Sp or Rp are referred to herein as chirally pure.

Ribonucleoside-5'-O- (1-thiotriphosphate) (NTPαS) and 2'-deoxyribonucleoside-5'-O- (1-thiotriphosphate) (dNTPαS) were obtained from Ludwig and Ec
kstein [J. Org. Chem. , 631 (19
89)] as a racemic mixture of Sp and Rp. In this exemplary synthetic scheme, the unprotected nucleoside is converted to 2-chloro-4H-1,
React with 3,2-benzodioxaphosphorin-4-one, which phosphitylates the 5'-hydroxyl group. Subsequent reaction with pyrophosphate produces a cyclic triphosphate derivative that is reactive toward sulfur, with a mixture of Rp and Sp nucleosides 5'-O- (1-thiotriphosphate), ie, α-thiotriphosphate. Is obtained. The product is, for example, DEAE
-Purified by Sephadex chromatography,
It can be identified (by characteristic Rp or Sp chemical shifts) using MR spectroscopy.

As shown in the examples below, pure R
The p and Sp nucleoside-5′-O- (1-thiotriphosphate) diastereomers can be used, for example, in reverse phase HP
It can be easily isolated on a production scale using LC chromatography. The nucleotide diastereomers isolated by such HPLC are further characterized by analytical HPLC comparison of such Rp and Sp nucleoside 5'-O- (1-thiotriphosphate) diastereomers with commercial samples. can do.

A sequence-specific natural oligonucleotide,
That is, enzymatic synthesis of a natural phosphodiester oligonucleotide can be performed by using an appropriate nuclease in the presence of a template and a primer. Similarly, racemic mixtures of phosphorothioate oligonucleotides with chirally mixed intersugar linkages can be synthesized. According to the present invention, such an enzymatic synthesis can be carried out in the presence of a sequence-specific template and primers, with all enantiomerically pure S
p or all Rp nucleosides 5'-O- (1-
By using thiotriphosphate) as a substrate for a suitable nuclease, it can be further developed to include the synthesis of sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For example, a commercially available DNA polymerase, Sequenase ^™ (US Biochemical, Ind.)
c. , Cleveland, OH), a phosphorothioate oligonucleotide can be synthesized using a phosphodiester oligonucleotide template and a racemic phosphorothioate oligonucleotide primer. By using this polymerase, both the phosphodiester and phosphorothioate primers can be extended.

The yield of enzymatically synthesized phosphorothioate oligonucleotides can be determined by repeatedly adding template and primers, by repeatedly adding polymerase, by repeatedly adding nucleoside triphosphates, or by several of these. Or it can be optimized by combining all. For example, repeated additions of template and primer maximize yields from the enzymatic cascade. Further optimization can be performed by prehybridizing the template and primer together in the system buffer, followed by cooling, and adding nucleoside triphosphate and polymerase.

An appropriate polymerase can be selected to produce a DNA or RNA phosphorothioate oligonucleotide. Such polymerases include T7 DNA polymerase, modified T7 DNA polymerase, such as Sequenase ^™ , E. et al. c
oli DNA polymerase, DNA polyclenow fragment polymerase, Luteus
Polymerase, T4 bacteriophage polymerase,
Modified T4 DNA polymerase, T7 RNA polymerase and E. coli. coli RNA polymerase, but is not necessarily limited to these.

Enzymatic synthesis proceeds with a reversal of configuration around the chiral center of the phosphorus atom. That is, use of all Sp α-thiotriphosphates results in substantially all Rp phosphorothioate oligonucleotides, and use of all Rp α-thiotriphosphates results in substantially all Sp phosphorothioate oligonucleotides. In another aspect of the invention, the phosphorothioate oligonucleotide is a nucleoside-5 ′ by using a metal ion in the reaction solution to facilitate preferential incorporation of one or the other chiral α-thiotriphosphate.
It can be synthesized from a racemic mixture of -O- (1-thiotriphosphate). As mentioned above, polymerase synthesis of phosphorothioate oligonucleotides is achieved with a reversal of configuration around the chiral center of the precursor nucleoside-α-thiotriphosphate. Without wishing to be bound by theory, all Rp configuration optimizations are described, for example, by E.W. It is believed that this can be achieved by adding high concentrations of magnesium ions in the reaction buffer using E. coli polymerase. Similarly, without wishing to be bound by theory again, all Sp
Is believed to be obtained by using a high manganese ion concentration in the reaction buffer.

According to the present invention, "substantially all" means that at least 75% of the intersugar linkages include all oligonucleotides that are chirally pure. More preferably, the oligonucleotide having about 85% to about 100% chirally pure intersugar linkages is
Substantially chirally pure. Most preferably, about 9
Oligonucleotides having 5% to about 100% chirally pure intersugar linkages are substantially chirally pure.

In the context of the present invention, the term “phosphorothioate oligonucleotide” refers to a naturally occurring base,
Includes phosphorothioate oligonucleotides formed from sugars and phosphorothioate linkages. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Natural sugars include β-D-ribofuranosyl and β-D-2′-deoxy-erythro-pentofuranosyl. "Phosphorothioate oligonucleotides" also refer to nucleoside-5'-O-
The (1-thiotriphosphate) analog is a substrate for a suitable polymerase and, to some extent, includes a modified base or modified sugar incorporated into the phosphorothioate nucleotide unit of the oligonucleotide. Modified bases of the oligonucleotides of the invention include adenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl-adenines, 5-
Halouracil, 5-halocytosine, 6-azauracil,
6-azacytosine and 6-azathymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenine, 8-hydroxyl adenine and other 8
-Substituted adenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thiolalkylguanine, 8-hydroxylguanine and other 8-substituted guanines, other aza and deazauracil, other aza and deazathymidine, other Including aza and deazacytosine, other aza and deazaadenine, other aza and deazaguanine, 5-trifluoromethyluracil and 5-trifluorocytosine. The sugar component may be deoxyribose or ribose. The oligonucleotides of the present invention may also include modified nucleobases or other modifications consistent with the spirit of the invention, particularly those that increase their nuclease resistance to facilitate their use as therapeutic, diagnostic or research reagents. May be included. The oligonucleotides of the invention can be used as diagnostic, therapeutic and research reagents. They are,
An effective amount of an oligonucleotide of the invention can be used in pharmaceutical compositions by adding it to a suitable pharmaceutically acceptable diluent or carrier. They can further be used to treat organisms having a disease characterized by the undesired production of proteins. An organism can be contacted with an oligonucleotide of the invention having a sequence that is capable of specifically hybridizing to a strand of a target nucleic acid encoding an undesired protein.

The formulation of the therapeutic composition and its subsequent administration is considered to be within the skill of those in the art. Generally, for therapy, the oligonucleotides according to the invention will be administered to a patient in need of such treatment, usually in a pharmaceutically acceptable carrier, of the age and seriousness of the disease state of the patient to be treated. 0.0 per kg of body weight, depending on the degree
Administer doses ranging from 1 μg to 100 g. further,
The treatment regimen lasts for a period that will vary depending on the characteristics of the particular disease, its severity, and the overall condition of the patient,
It can be extended from once a day to once every few years.
Following treatment, patients are monitored for changes in their condition and for relief of symptoms of the disease condition. If the patient does not respond significantly to the current dose level, the dose of the oligonucleotide can be increased, or if relief of the symptoms of the disease state is observed, or if the disease state is eliminated, The dose can be reduced.

In some cases, it may be more effective to treat patients with the oligonucleotides of the present invention in combination with other traditional therapeutic modalities. Dosage may be adjusted until healing occurs or until a reduction in disease state is achieved.
The course of treatment, from days to months, depends on the severity and responsiveness of the condition to be treated. Optimal dose schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. The optimal dose will vary depending on the relative potency of the individual oligonucleotides, and can generally be estimated based on the EC ₅₀ found to be effective in vitro and in vivo in animal models.
Generally, doses will be from 0.01 μg / kg to 10 μg / kg body weight.
0 g, once or more daily, 1 weekly
Once a month or once a year or once every few years
Times, given.

Following a successful treatment, the oligonucleotide was added at 0.01 mg / kg body weight to prevent recurrence of the disease state.
It would be desirable to have maintenance therapy in which the patient is administered a maintenance dose ranging from μg to 100 g, once a day or more to once every several years.

The pharmaceutical compositions of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, nasal),
It can be done orally or parenterally. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

Formulations for topical administration include transdermal patches,
Ointments, lotions, creams, gels, drops, suppositories, sprays, solutions and powders may be included.
Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may be necessary or desirable. Coated condoms, gloves, etc. may also be useful.

Compositions for oral administration include powders, granules, suspending or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickening agents, fragrances, diluents, emulsifiers, dispersing aids or binders may be desired.

Compositions for intrathecal or intraventricular administration include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Formulations for parenteral administration include sterile aqueous solutions which may contain buffers, diluents and other suitable additives.

The phosphorothioate oligonucleotides of the present invention can be compared to both natural phosphodiester oligonucleotides and racemic phosphorothioate nucleotides with respect to hybridization, nuclease resistance and their effect on RNase H activity. Similarly, pure phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can be evaluated for their ability to increase the efficacy of treatment in an in vivo test system. Increasing the efficacy of such treatments includes such properties as pharmacokinetics or metabolism, toxicology, delivery (ie, absorption and distribution), and species comparison.

Homopolymers with all Rp or all Sp intersugar linkages were useful for initial studies of stability and other properties. However, these oligonucleotides have little therapeutic use because they are not specific to the target molecule. In order to specifically hybridize to a target nucleic acid, a phosphorothioate oligonucleotide having a specific sequence is required.

Sequence-specific phosphorothioate oligonucleotides with substantially chirally pure intersugar linkages can increase the thermodynamic stability of heteroduplexes with target RNA and DNA and confer RNase H activity. Useful.

A radiolabel can be used to help identify phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For a racemic phosphorothioate oligonucleotide synthesized by an automated synthesizer, [ ³⁵ S] (radiolabeled elemental sulfur) can be used for the oxidation reaction of the hydrogen phosphonate oligomer obtained from the synthesizer. Labeling of enzymatically synthesized phosphorothioate oligonucleotides can be performed with [α- ³² P] ATP and ligase or in a polymerase reaction with [α- ³⁵ S] ATP. Also, radiolabeled nucleoside triphosphates can be used in probes and sequencing analyses. Autoradiograms are created in a standard way.

[0046] The template of the present invention is most preferably a region of a nucleic acid sequence that directs the synthesis of a disease enhancing protein. A short oligonucleotide that base pairs with the region of the template oligonucleotide acts as a primer that forms the starting point for oligonucleotide synthesis by the polymerase.

[0047] Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are synthesized using primers selected to have sites thereon that are susceptible to nuclease cleavage, eg, restriction endonuclease cleavage. can do. The cleavage site,
It may be located at the 3 'end of the primer. Cleavage at the site with an appropriate restriction endonuclease yields an oligonucleotide that obtains the first 5 'terminal nucleoside from the primer. Another nucleoside of the phosphorothioate oligonucleotide of the present invention is a nucleoside chiral thiotriphosphate added by enzymatic means.

By selecting appropriate restriction nucleases with the selected primers, the various 5'-terminal nucleosides of the desired phosphorothioate oligonucleotide can be appropriately located at the 5 'end of the phosphorothioate nucleotide. That is, any endonuclease recognition site can be designed so long as one nucleoside from the primer is the first 5 'nucleoside of the newly synthesized sequence-specific phosphorothioate oligonucleotide of the present invention by staggered cleavage. This produces various nucleosides at the 5 'end of the enzymatically synthesized phosphorothioate oligonucleotide of the invention.

Upon completion of the enzymatic extension of the primer on a suitable template of the desired sequence, the phosphorothioate oligonucleotide of the invention can be released from the primer by using a suitable nuclease. For example, a primer having the sequence CTGCAG at the 3 ′ end can be used to incorporate a guanosine nucleoside at the 5 ′ end of the desired phosphorothioate oligonucleotide. Next, Pst1
By using a restriction nuclease, the AG bond can be cleaved. Thus, the guanosine nucleoside component of the AG bond can be incorporated as the 5 'terminal nucleoside of the desired phosphorothioate oligonucleotide. Other restriction endonucleases include, but are not limited to, BamH1, Smal, and HinDIII restriction endonucleases.

The oligonucleotide still associated with the template is dissociated from the template,
For example, it can be purified by gel electrophoresis and / or chromatography. For example, a suitable purification is SepPac (Millipore, Milfor
d, MA) can be performed using standard polyacrylamide / urea gel electrophoresis in combination with chromatography. Another useful chromatography technique that can be used is HPLC chromatography.

The chiral phosphorothioate oligonucleotides of the present invention can also be prepared as described in Sec et al. [Nuc
leic Acids Res. , 19: 5883 (1
991)] and Stec and Lesnikow
ski (Methods in Molecular)
Biology, S.M. Agrawal, Ed. , Vol
ume 20, p. 285, 1993], and can be chemically synthesized via a 1,3,2-oxathiaphospholane intermediate.

The phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages synthesized according to the method of the present invention can be analyzed by a number of methods. For example, a configuration analysis of the resulting substantially chirally pure sequence-specific phosphorothioate oligonucleotide having all Sp or all Rp intersugar linkages can be determined using [ ³¹ P] NMR chemical shifts. it can. Such chemical shifts have been used to identify Rp epimers of phosphorothioate dinucleotides (Ludwig a).
nd Eckstein, J. et al. Org. Chem. , 6
31-635 (1989)].

[0053] The sequence accuracy of the phosphorothioate oligonucleotides of the present invention can be determined using the sensitivity of the heteroduplex to S1 nuclease.

The sequence of the phosphorothioate oligonucleotide is 3 'of the phosphorothioate oligonucleotide.
The hydroxyl group is [α- ³² P] cordycepin triphosphate (ie 3′-deoxyadenosine-5 ′)
Labeling with (triphosphate) can further increase the validation. The resulting oligonucleotide can be subjected to enzymatic degradation.

[0055] The relative ability of a phosphorothioate oligonucleotide having a substantially chirally pure intersugar linkage to bind to a complementary strand is determined by the phosphorothioate oligonucleotide having a substantially chirally pure intersugar bond and its complement. The comparison can be made by measuring the melting temperature of the hybridization complex with the target strand. Melting temperature (Tm) is a physical property characteristic of a double helix, meaning the temperature in degrees Celsius at which a 50% helical to coiled (unhybridized) ratio is present. Tm is measured by measuring the formation and collapse (melting) of hybridization using the UV spectrum. Base stacking that occurs during hybridization is accompanied by a decrease in UV absorption (lightening). Consequently, a decrease in UV absorption indicates a higher Tm.
The higher the Tm, the greater the binding strength of the chains. Non-Watson-Crick base pairs have a strong destabilizing effect on Tm. Thus, to obtain optimal binding of an oligonucleotide to its target RNA,
It is desirable that the base pairs have as close to optimal accuracy as possible.

The phosphorothioate oligonucleotides of the present invention can be further evaluated for resistance to the ability of various exonucleases and endonucleases to degrade. The phosphorothioate oligonucleotide is treated with nuclease, followed by electrophoresis (PAGE), for example, on a polyacrylamide gel, and a suitable stain [eg, Stains All ^™ (Si
gma Chem. Co. , St. Lois, MO)]
Can be analyzed by staining using Degradation products can be quantified using laser densitometry.

Using fetal calf serum and human serum,
Nucleolysis (nuc) on phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages
oleolytic activity can be assessed. For example, phosphorothioate oligonucleotides having substantially all Rp intersugar linkages can be evaluated in this manner. Tests on combinations of molecules capped at the 3 'or 5' end (with one or several phosphorothioate linkages per cap) can be used to determine the combination that produces the greatest nuclease stability. Capping can be performed by chemically synthesizing the cap portion of the sequence using purified Rp monomers and then incorporating the cap into an oligonucleotide on a DNA synthesizer. Analyzes on capping can determine the importance of chirality on nucleolytic stability and the number of linkages required for maximal stability.

The sensitivity of the phosphorothioate oligonucleotide-RNA heteroduplex to the catalytic activity of RNase H can also be evaluated. Phosphorothioate oligonucleotides can be incubated with radiolabeled target mRNA (eg, synthesized via T7 RNA polymerase) at various hybridization temperatures. Next, Minshul
and Hunt [Nuc. Acid Res. , 64
33 (1986)). RNase H from E. coli
And 37 ° C. The product was then analyzed by Northern blot analysis for RNase.
It can be evaluated for H activity. In this method, the product is electrophoresed on a 1.2% agarose / formaldehyde gel and transferred to nitrocellulose. Next, a filter is searched using a random primer [ ³² P] -labeled cDNA complementary to the target mRNA, and quantified by autoradiography. By comparing various phosphorothioate analogs, one can examine the effect of chirality on the ability to act as a substrate for RNase H when complexed with RNA.

E. A comparison of the heteroduplex sensitivity to the catalysis of E. coli RNase H and mammalian RNAse H can be made. The heteroduplex is incubated in rabbit reticulocyte lysate under translational conditions and the endogenous RNase H
Can be assayed by Northern blot analysis for catalytic cleavage of mRNA. This allows
The effect of chirality on mammalian RNAseH activity can be examined.

[0060] Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can also be evaluated for inhibition of gene expression in a model system of cell culture. Designed to target reporter genes to determine whether phosphorothioate oligonucleotides with substantially pure chirally pure intersugar linkages are more potent or more specific inhibitors of gene expression The resulting phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can be synthesized and tested in cell culture models of gene expression. Examining the effect of antisense on gene expression using the vector pSV2CAT has been described previously [Henthorn et al. ,
Proc. Natl. Acad. Sci. U. S.
A. , 85: 6342 (1988)]. This vector contains the bacterial chloramphenicol acetyltransferase gene under the regulatory control of the SV40 promoter. A sequence complementary to the translation initiation of CAT mRNA,
Using a 15-mer phosphorothioate oligonucleotide with all Rp intersugar linkages, pSV2CAT
To HeLa cells and then all Rp
The cells can be treated with a phosphorothioate oligonucleotide having an intersugar linkage for 48 hours to assay intracellular CAT activity. Next, the activity in inhibiting gene expression of phosphorothioates with substantially chirally pure intersugar linkages was demonstrated by using chemically synthesized random phosphorothioates with diastereomeric intersugar linkages and natural phosphodiesters of the same sequence. It can be compared directly with oligonucleotides.

The vector pSV2APAP [Marcus
-Sekura et al. , Nucleic Ac
ids Research, 15: 5749 (198
7)] contains the mammalian placental alkaline phosphatase gene (PAP). It can also be used as a reporter to determine the effect of antisense on gene expression. PAP has advantages over CAT as a reporter gene in that it is a mammalian gene rather than a bacterial gene containing introns and other RNA processing signals. At this time, PA
Expression of P is believed to more closely mimic events in the expression of natural mammalian genes. CAT m
A 15-mer phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages, as described above for RNA, is used in parallel with chemically synthesized racemic phosphorothioate and natural phosphodiester oligonucleotides having similar sequences. You can find out. PAP and CAT reporter constructs are used as controls in replicate experiments to determine non-specific effects on gene expression.

Further, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are:
Its ability to act as an inhibitor of RNA translation in vivo can be assessed. A variety of therapeutic areas can be targeted for such manipulation by the oligonucleotides of the present invention. One therapeutic area is hepatitis caused by hepatitis C virus (HCV). The following phosphorothioate oligonucleotides have application in the treatment of HCV hepatitis: oligo # 259, CCTTTCGCGA CCC
AACACTA (SEQ ID NO: 1), oligo # 260, GCCTTTCGCG
ACCCAACACT (SEQ ID NO: 2), oligo 270, GTACCACAAG
GCCTTTCGCG (SEQ ID NO: 3), oligo # 330, GTGCTCA
TGG TGCACGGTCT (SEQ ID NO: 4) and oligo # 340,
TTTAGGATTC GTGCTCATGG (SEQ ID NO: 5). Another area of treatment is inflammatory diseases mediated by intercellular adhesion molecules (ICAM-1). Oligonucleotides that have application in the treatment of inflammatory diseases include ISIS-2302, GCCCAAGC
TG GCATCCGTCA (SEQ ID NO: 6) is included. Another therapeutic area includes infections caused by cytomegalovirus (CMV). ISIS-2922 is a commercial
V is a phosphorothioate oligonucleotide having application in the treatment of retinitis, the sequence GCGTTTGCTC TTCTTCTTGC G
(SEQ ID NO: 7). Another therapeutic area includes cancers mediated by protein kinase C-α (PKC-α). ISIS-3521 is a phosphorothioate oligonucleotide having application in the treatment of such cancers and has the sequence GTTCTCGCTG GTGAGTTTCA (SEQ ID NO: 8). Yet another therapeutic area includes C-raf kinase-mediated cancer. ISIS-5132 is a phosphorothioate oligonucleotide having application in the treatment of such cancers and has the sequence TCCCGCCTGT GACATGCATT (SEQ ID NO: 9). Yet another therapeutic area includes cancer mediated by Ha-ras or Ki-ras. The following phosphorothioate oligonucleotides have applications in the treatment of such cancers: ISIS-2503, TCCGTCAT
CG CTCCTCAGGG (SEQ ID NO: 10), ISIS-257
0, CCACACCGAC GGCGCCC (SEQ ID NO: 11), and IS
IS-6957, CAGTGCCTGC GCCGCGCTCG (SEQ ID NO: 1
2). Another therapeutic area is HIV-mediated AID
S and other related infections. ISIS-53
20 is a phosphorothioate oligonucleotide having application in the treatment of AIDS and has the sequence TTGGGGTT (SEQ ID NO: 17). In the above sequence, the individual nucleotide units of the oligonucleotide are 5 'from left to right.
From the 3 'direction.

In the context of the present invention, “hybridization” means a hydrogen bond between complementary nucleotide units, which may be a Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bond. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds. As used herein, "complementary" also refers to sequence complementarity between two nucleotide units. For example, an oligonucleotide and DNA or RNA are considered to be complementary to each other at that position if the nucleotide unit at one position of the oligonucleotide can hydrogen bond with the nucleotide unit at the same position on the DNA or RNA molecule. Can be Oligonucleotides and their DNA
Alternatively, RNA is complementary to each other if a sufficient number of corresponding positions in each molecule are occupied by nucleotide units that can hydrogen bond with each other. That is, "specifically hybridizable" and "complementary" refer to oligonucleotide and target RNA or DN.
A term used to indicate a sufficient degree of complementarity such that stable and specific binding to A occurs. It will be appreciated that an oligonucleotide need not be 100% complementary to its target DNA sequence in order to be able to specifically hybridize. Under conditions in which binding of the oligonucleotide to the target RNA or DNA molecule interferes with the normal function of the target RNA or DNA, and specific binding is desired, for example, under physiological conditions in the case of in vivo assays or therapeutic treatments, Or, in the case of an in vitro assay, the oligonucleotide will specifically hybridize under the conditions under which the assay is performed, provided that the oligonucleotide has sufficient complementarity to avoid non-specific binding to the non-target sequence. Can be.

In particular, the oligonucleotides of the present invention are disclosed in the following US patent application (assigned to the assignee of the present invention):
As specified therein, it can be used in therapy, as a diagnosis, and for research. These applications have the following title: Compositions
and Methods for Modulatin
gRNA Activity, No. 463,358, filed Jan. 11, 1990; Antisense Ol
igonucleotides Inhibitors
of Papilloma Virus, No. 445,196, filed Dec. 4, 1989; Oligonu.
cleide Therapiesfor Mod
ulrating the Effects of He
rpesvirus, filed February 26, 1990;
No. 85,297; Reagents and Meth
ods for Modulating Gene E
xpression Through RNA Mim
icry, No. 497,09, filed March 21, 1990.
No. 0; Oligonucleotide Modula
Tion of Lipid Metabolis
m, No. 516,969, filed Apr. 30, 1990.
No. Oligonucleotides for Mod
ulrating the Effects of Cy
tomegavirus Infections,
No. 568,366 filed on Aug. 16, 1990; An
Tisense Inhibitors of the
Human Immunodeficiency V
irus, No. 521,90, filed May 11, 1990.
No. 7: Nuclease Resistant Pyr
imidized modified oligogonuc
leoteides for Modulation of
Gene Expression, July 2, 1990
No. 558,806 filed on Nov. 7; Novel Poly
Amine Conjugated Oligonuc
leoteides, filed on July 27, 1990, No. 55
8,663; Modulation of Gene
Expression Through Inter
reference with RNASecondary
Structure No. 5 filed May 4, 1990
18,929; Oligonucleotide M
odulation ofCell Adhesio
n, No. 567,286, filed August 14, 1990;
Inhibition of Influenza V
No. 567, filed Aug. 14, 1990.
No. 287; Inhibition of Candid
a, No. 568,672, filed Aug. 16, 1990;
And Antisense Oligocleot
ids Inhibitors oPapillom
Avirus, PCT / U filed December 3, 1990
S90 / 07067. These patent applications disclose a number of means by which oligonucleotide interactions can result in improved regulation of RNA and DNA activity. The disclosure of the aforementioned U.S. patent application is incorporated herein by reference, in that the particular sequences disclosed in the above patent application can be used with the present invention.

[0065]

The following examples are illustrative and do not limit the invention. Example 1 All Sp or all Rp 5'-O-
Isolation of (1-thiotriphosphate) nucleosides 5'-O- (1-thiotriphosphate) deoxynucleoside and ribonucleoside were obtained from ODS Hyper
rsil (Shandon Southern, Run
con, UK) using a solvent A (5 m
C-18 reverse phase high performance liquid chromatography (HPLC) eluting with an isocratic mixture of 30 mM potassium phosphate, pH 7.0 containing M tetrabutylammonium ion, and solvent B (5 mM tetrabutylammonium hydroxide in methanol). ). Alternatively, effective separation is achieved using 100 mM triethylammonium bicarbonate, pH 7.5, containing a linear gradient from 0% to 15% acetonitrile over 20 minutes.

In order to confirm the purity of the enantiomers separated by such HPLC, Sp
And the Rp deoxynucleotide enantiomer,
E. FIG. I. Deoxynucleoside 5'- commercially available from Dupont, Wilmington, DE
Compare with O- (1-thiotriphosphate) s.

Example 2 Racemic phosphorothioate
Substantially all of the oligonucleotides have an Rp intersugar linkage.
Enzymatic synthesis of phosphorothioate extension of all Rp synthetic racemic phosphorothioate oligonucleotide primer phosphorothioate extension product which is modified T7DNA polymerase I, Sequenase (Sequenase ^TM) (U.S.Biochem
icals Corp. Cleveland, OH). Using this T7 DNA polymerase, 2
The 18-mer phosphorothioate oligonucleotide primer hybridized to the 1-mer natural phosphodiester oligonucleotide is extended. 1X Sequenase ^™ reaction buffer (US Bioch) containing 30 picomoles (pmol) of primer and template
Chemicals Corp. , Cleveland, O
H) Heat (final volume 10 μl) at 95 ° C. for 5 minutes and slowly cool to room temperature. 180 pmol of deoxy 5 '-[α- ³⁵ S] cytidine triphosphate and Sequenase ^™ enzyme (U.S. Biochemical)
s Corp. , Cleveland, OH)
Incubate at 37 ° C for 20 minutes. The product, 20
Analyze by polyacrylamide gel electrophoresis (PAGE) using a% polyacrylamide / 7M urea denaturing gel. The product autoradiograph is compared to a control reaction without primer / template. The final product is further characterized, for example, by enzymatic degradation. One such degradation is snake venom phosphatase degradation. E. FIG.
Snake venom phosphatase degradation of dinucleoside monophosphorothioate synthesized using E. coli DNA polymerase I indicates that the dinucleoside is in the Rp configuration.

Example 3 Substantially Pure Rp Intersugar Linkage
Phosphorothioate oligonucleotide CCGACTATG having
Synthesis of CA AGTAC (SEQ ID NO: 13) Large-scale enzymatic synthesis of sequence-specific all-Rp phosphorothioate oligonucleotides was performed using a 55-mer natural phosphodiester template and a 41-mer natural phosphodiester primer. The template sequence is: GTACTTGCAT AGTCGATCGG AAAATAGGGT TCTCATCTCC CGGGAT
TTGG TTTGAG (SEQ ID NO: 14). The primer sequence was CTCAACCAAA TCCCGGGAGA TGAGAACCCT ATTTTCCGAT C (SEQ ID NO: 15). The template was chosen to have a sequence complementary to the desired specific sequence CGACATAGCA AGTAC (SEQ ID NO: 13). Sequenase ^™ buffer diluted 5 × to 1 × (US Biochemi)
cals Corp. , Cleveland, OH) was used. 2 for both template and primer
A concentration of 0 nM was added to 40 μL of this buffer. The template and primer were hybridized at 95 ° C. for 5 minutes and cooled to room temperature. After cooling the buffer, the buffer was adjusted to 7 mM DTT. Next, 20 μL of 1: 8 diluted Sequenase ^™ enzyme and 320 μM Sp G
TPαS, CTPαS, ATPαS and TTPαS were added. The reaction solution was adjusted to 140 μL with H ₂ O. The reaction was incubated at 37 ° C. for 18 hours. The reaction solution is extracted twice with an equal volume of phenol as usual, precipitated by treatment with 2.5 volumes of 100% ethanol at -20 ° C as usual, pelleted, washed with 500 μL of 70% ethanol. Again, pelletized and dried. The precipitate is suspended in 20 μL H ₂ O for 30 minutes, then
1 mM CaCl ₂ , 25 mM Tris H in 0 μL H ₂ O
Cl, pH was adjusted to 8.0. The solution was maintained at 95 ° C. for 5 minutes and quenched (ie, cooled very quickly with ice). The template and primers were removed from the synthesized oligonucleotide by adding 4.6 μM DNase I and incubating at 37 ° C. for 10 minutes. The reaction mixture was extracted twice with phenol and precipitated with ethanol as described above. The precipitate was resuspended in H ₂ O and separated by SepPak ^™ chromatography (Milli
20 in combination with Pore, Milford, MA)
Purified using% polyacrylamide / 7M urea gel electrophoresis.

In another synthetic method, PstI restriction nuclease (Life Technologies, Ind.)
c. , Gaithersburg, MD) was used to cleave the primer-bound phosphorothioate oligonucleotide at the restriction site. The desired phosphorothioate oligonucleotide CCGACTATGCA AGTAC (SEQ ID NO: 13) is
epPak ^™ chromatography (Millipor
e, Milford, Mass.) using polyacrylamide / 7 M urea gel electrophoresis. The yield was optimized using an enzymatic cascade influenced by repeated template-primer additions throughout the reaction. 7 ml from 20 ml reaction by cascade incremental synthesis
5 A ₂₆₀ units of all Rp configuration phosphorothioate oligonucleotide CCGACTATGCA AGT
AC (SEQ ID NO: 13) was obtained.

Example 4 Using Automated DNA Synthesis
Phosphorothioates with racemic mixtures of intersugar linkages
Oligonucleotide Synthesis Oligonucleotides can be prepared using an automated DNA synthesizer (Applied Bis) using hydrogen phosphonate chemistry in a conventional manner.
[Ag] synthesized by the ossystems model 380B).
rawaletal. Proc. Natl. Aca
d. Sci. U. S. A. , 85: 7079 (198
8)]. After the final coupling step, the phosphorothioate linkage was generated by oxidizing the bound oligomer with sulfur in carbon disulfide / triethylamine / pyridine.
After sulfur oxidation, the oligonucleotide is dissociated from the support using standard ammonium hydroxide deprotection methods,
The base protecting group was removed. Phosphorothioate oligonucleotides are available on oligonucleotide purification columns (OPC;
ABI, Foster City, CA) chromatography and HPLC (Beckman System).
Gold HPLC). HPL
The C-purified oligonucleotide was subsequently precipitated with ethanol and evaluated for final purity by gel electrophoresis on 20% acrylamide / 7 M urea or analytical HPLC. Oligonucleotide sequence authenticity was assessed by iodine oxidation in pyridine / water and standard sequencing. These oligonucleotides contain a mixture of combinations of all possible Rp and Sp isomers at each phosphorus bond.

Example 5 Thermodynamic and kinetic hives
T7 RNA polymerase for redidation analysis
Synthesis of Complementary DNA or RNA Sequences Using A. Synthesis of short complementary DNA oligonucleotides with natural phosphodiester linkages was performed utilizing standard automated synthesis on an ABI model 380B DNA synthesizer. The correct length oligonucleotide was purified by HPLC and sequenced by standard techniques.

Short complementary RNA oligonucleotides were synthesized for hybridization analysis using T7 RNA polymerase. High concentrations and large amounts of T7 RNA polymerase were required to initiate the many cycles required to synthesize short RNA. Because of this need, T7 RNA polymerase has
A polymerase expression vector, BL21 / pAR121
E. coli strain containing 9 (Brookhaven Natio)
nal Laboratory, Upton, NY). Isolation from 2 L of cells yielded about 300,000 to 500,000 units of T7 RNA polymerase. Absorbance value = 1.2A _600. This was concentrated enough for the synthesis of short (10-30 nucleotide) RNA species. For the synthesis, an ABI synthesizer (AB
(I, Foster City, CA) using T7 promoter and a template containing a complementary target sequence and a T7 promoter hybridization sequence. The template and promoter were purified by HPLC to ensure that the correct species was present for enzymatic synthesis. The synthesized product was purified on a 20% polyacrylamide / 8M urea gel and sequenced in a conventional manner.

Example 6 A heat-denatured oligonucleotide (a phosphorothioate oligonucleotide of the invention or other) was prepared using complementary DNA
Alternatively, together with the RNA oligonucleotide, a 100 mM ionic strength buffer (89.8 mM NaCl, 10 mM
Sodium phosphate, pH 7.0, 0.2 mM EDT
Incubated in A). Heat the sample to 90 ° C,
Initial absorbance was measured using a Guilford Response II spectrophotometer (Corning).
The sample is then gradually cooled to 15 ° C.
The change in absorbance was monitored at 60 nm. The temperature was increased by 1 degree, the absorbance was read, and the denaturation profile was analyzed by taking the first derivative of the melting curve. The data was also analyzed using a two-phase linear regression analysis to determine Tm and Delta G. The results of these tests are shown in Table 1.

[0074]

[Table 1]

Example 7 Radiolabeled oligonucleotide
To quantitate the binding stringency of the various synthesis of phosphorothioate oligonucleotides, utilizing filter binding assay. That is, these are DNA or RN
The tendency to hybridize with A to form a heteroduplex was quantified. These assays require a radiolabeled oligonucleotide.

Phosphorothioate oligonucleotides with all Rp intersugar linkages were synthesized by enzymatic methods from [ ³⁵ S] -monomers purified from Sp monomers. For automated synthesis of phosphorothioate oligonucleotides containing chiral mixed sugar linkages, oligonucleotides containing hydrogen phosphonates were synthesized and then sulfurized in a pyridine / carbon disulfide mixture in the presence of elemental [ ³⁵ S].
The resulting radiolabeled phosphorothioate oligonucleotide can be purified by OPC chromatography and HPLC. The target mRNA was applied to a nitrocellulose filter, baked at 80 ° C. for 2 hours, blocked, and subsequently hybridized with a radiolabeled phosphorothioate oligonucleotide. Binding stringency was assessed by quantifying the radiolabeled oligonucleotide eluted from the filter after increasing the temperature or increasing the ionic strength of the elution buffer (eg, Tris NaCl buffer). The eluted oligonucleotides were also evaluated by their mobility in an anion exchange HPLC protocol utilizing an isocratic phosphate buffer. The results were compared to the mobility of a standard oligonucleotide prepared to have a racemic mixture of intersugar linkages.

Example 8 Determination of the nuclease degradation rate of phosphorothioate oligonucleotides in a medium containing 10% nuclease-digested fetal calf serum (FCS) was determined in Dulbecco's Modified Essential Medium (DMEM) containing 10% heat-inactivated FCS. It was carried out in. Heat inactivation of FCS was performed at 55 ° C. for 1 hour before adding to the medium.
Racemates and oligonucleotides with chirally pure intersugar linkages were tested separately for resistance to nuclease digestion. Each 66 μg / ml oligonucleotide was separately added to the medium and incubated at 37 ° C. at the time intervals shown in Table 2. Take a 15 μl aliquot and add 15 μl 9 M urea in 0.1 M Tris-H
Cl (pH 8.3), 0.1 M boric acid and 2 mM ED
Added to TA. Aliquots were mixed by vortexing and stored at -20 <0> C. 20% polyacrylamide /
Polyacrylamide gel electrophoresis (PAGE) analysis was performed on a 7M urea slab gel. After electrophoresis “Stain
s All "(Sigma. Chem. Co., St.
(Louis, MO). After destaining, the gel was subjected to an UltraScan XL apparatus (Pharm
acia LKB Biotechnology, Up
psala, Sweden) and analyzed by laser densitometry. Integration was performed and data were expressed as percent reduction from full length (n) before incubation to n-1. Oligonucleotide sequence CGACTATGCA AGTAC with a chirally pure intersugar linkage of Rp
The results for (SEQ ID NO: 6) are shown in Table 2.

[0078]

[Table 2]

As is evident from Table 2, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages exhibited higher nuclease digestion resistance than phosphorothioate oligonucleotides having racemic intersugar linkages.

Example 9 Analysis of RNase H The sensitivity of racemates and phosphorothioates having substantially chirally pure intersugar linkages to RNase H was analyzed. Oligonucleotides (2-fold molar excess over RNA) and 5 μg (3.1 kb) of in vitro synthesized mRNA (using T7 RNA polymerase promoter) are incubated in 5 μl of RNase H hybridization buffer at 60 ° C. for 30 minutes. did. The sample was gradually cooled to room temperature and then 3.7 m
g / ml BSA, 20 units of E. coli. coli RN
case H (Promega), 142 mM DTT,
It was adjusted to 150 mM KCl and 3 mM MgCl _2. The samples were incubated at 37 ° C. for 30 minutes. Next, the sample was extracted with phenol and precipitated with ethanol.
Analyzed by electrophoresis on agarose gel, followed by ethidium bromide staining. Markers were run on the gel at the same time as the sample to determine the approximate length of the RNA sample.

Example 10 Patients suffering from HCV-induced hepatitis were oligo # 259 (SEQ ID NO: 1) and oligo # 26 synthesized according to the method of Example 3 or Example 19, respectively.
0 (SEQ ID NO: 2), oligo 270 (SEQ ID NO: 3), oligo # 330 (SEQ ID NO: 4) or oligo 340 (SEQ ID NO: 5). Oligonucleotides at 1-1000 μg / kg body weight are incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. The treatment can be repeated as necessary until the disease is eliminated.

Example 11 A patient suffering from an inflammatory disease mediated by ICAM-1 was treated with ISIS-2302, ie, synthesized according to Example 3 or Example 19, and the sequence GCCCAAGCTGGCATCCGT
Treat with oligonucleotides having CA (SEQ ID NO: 6). Oligonucleotides at 1-1000 μg / kg body weight are incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. The treatment can be repeated as necessary until the disease is eliminated.

Example 12 A patient suffering from retinitis caused by cytomegalovirus was treated with ISIS-2922,
Or synthesized according to Example 19, the sequence GCGTTTGC
Oligonucleotides of 1-1000 μg / kg body weight, treated with an oligonucleotide having TC TTCTTCTTGC G (SEQ ID NO: 7), are incorporated into a pharmaceutically acceptable carrier and administered intravitrally. The treatment can be repeated as necessary until the infection is eliminated.

Example 13 A patient suffering from PKC-α-mediated cancer was treated with ISIS-3
521, treated with an oligonucleotide synthesized according to Example 3 or Example 19 and having the sequence GTTCTCGCTG GTGAGTTTCA (SEQ ID NO: 8). 1-10
Oligonucleotides at 00 μg / kg body weight are incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. The treatment can be repeated as necessary until the disease is eliminated.

Example 14 Patients with C-raf kinase-mediated cancer were treated with IS
Treated with IS-5132, an oligonucleotide synthesized according to Example 3 or Example 19 and having the sequence TCCCGCCTGT GACATGCATT (SEQ ID NO: 9). 1
Oligonucleotides at -1000 μg / kg body weight are incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. The treatment can be repeated as necessary until the disease is eliminated.

Example 15 Patients suffering from C-raf kinase-mediated cancer were treated with I synthesized according to Example 3 or Example 19, respectively.
SIS-2503 (SEQ ID NO: 10), ISIS-257
0 (SEQ ID NO: 11) or an oligonucleotide having ISIS-6957 (SEQ ID NO: 12). 1-
Oligonucleotides at 1000 μg / kg body weight are incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. The treatment can be repeated as necessary until the disease is eliminated.

Compounds 1, 2 and 3 of Examples 16, 17 and 18, respectively, were prepared according to Stec et al. [Nuc
leic Acids Res. , 19: 5883 (1
991)] and Stec and Lesnikow
ski [Methodsin Molecular B
iology, S.M. Agrawal, Ed. , Volu
me20, p. 285, 1993).

Example 16 2-chloro-1,3,2-O
Synthesis of xathiaphospholane (1) pyridine (1 mol), benzene (400 mL), 2-
A mixture of mercaptoethanol (0.5 mol) and phosphorus trichloride (0.5 mol) is stirred at room temperature for 30 minutes. The pyridinium chloride is filtered off, the solvent is evaporated off under reduced pressure and the crude product is purified by distillation under reduced pressure. 70-7
The fraction boiling at 2 ° C./20 mmHg was collected and ³¹ PN
Characteristic determination by MR.

Example 17 N, N-diisopropylamido
No. 1,3,2-oxathiaphospholane (2) was synthesized with n-pentane (300 m
L) and dissolved in diisopropylamine (0.4 mol)
Is added dropwise. The reaction mixture is stirred at room temperature for 30 minutes, after which the diisopropylamine hydrochloride is filtered off, the solvent is evaporated off under reduced pressure and the crude product is purified by vacuum distillation. Product 2 is obtained as a fraction boiling at 70 ° C./0.1 mmHg, which is characterized by ³¹ P NMR and mass spectroscopy.

Example 18 5'-O-dimethoxytriti
Luthymidine-3'-O- [2-thiono-1,3,2-o
Xiathiaphospholane ] (3) Synthesis of 5'-O-dimethoxytritylthymidine (10 mmol
l) and 1H-tetrazole (11 mmol) are dried in vacuo and dissolved in dichloromethane (25 mL). Compound 2 (11 mmol) is added to the solution and the reaction mixture is stirred at room temperature for 2 hours. Dry elemental sulfur (15 mmol) is added and the reaction mixture is stirred at room temperature for 16 hours and left to stand.
Then unreacted sulfur is filtered off and the filtrate is concentrated under reduced pressure.
The residue is dissolved in chloroform (3 mL) and purified by silica gel (230-400 mesh) column chromatography, eluting first with chloroform, then with chloroform: methanol (97: 3). Separate diastereomeric species of compound 3 are obtained by column chromatography on silica gel. Compound 3 is dissolved in ethyl acetate and loaded on a silica gel 60H column. Ethyl acetate was used as the elution solvent, and elution was performed using HPTLC (silica gel 6).
0, ethyl acetate as the developing solvent).
The fraction containing the separated diastereomer of compound 3 was concentrated under reduced pressure, and the residue was subjected to ³¹ P NMR and HPLC (L
(Icrospher Si 100, 5 μM, ethyl acetate as eluent, flow rate 3 mL / min). The early eluting fraction corresponds to the Sp diastereomer and the late eluting fraction is the Rp diastereomer.

Example 19 5'-OH nucleoside and dia in solid phase automated synthesis
Stereospecificity of the reaction with stereomerically pure compound 3
Controlled Applied Biosystems (Foster
(City, CA) using a model 380B automated DNA synthesizer according to the method of Stec et al. 5'-OH
The reaction between a nucleoside and a diastereomerically pure nucleoside oxathiaphospholane, such as compound 3, is carried out using 1,8-diazabicyclo (5.4.0) as a catalyst.
Requires the use of undec-7-ene (DBU). Commercially available linkers used to attach oligonucleotides to a support matrix in automated DNA synthesis are unstable to DBU and require modifications in the linker. A suitable linker for oligonucleotide synthesis by the oxathiaphospholane method is DBU
A "succinate-sarcosinyl" linker that is resistant to succinate and can be hydrolyzed with concentrated ammonium hydroxide at room temperature within one hour.

(A) Synthesis of 5'-O-dimethoxytrityl nucleoside bound to a solid matrix using "succinic acid-sarcosinyl" linker (1) N-Fmoc-sarcosine (Bachem Bi)
science, Inc. , Philadelphi
a, PA) (1.6 mmol) with a long-chain alkylamine-
CPG (LCA-CPG, Sigma, St. Louis)
(s, MO) (2 g) and dried under vacuum. Anhydrous D
MF (5 mL), pyridine (0.5 mL) and DCC
(2.4 mmol) was added and the reaction mixture was shaken at room temperature for 12 hours. Then the solvent is filtered off and the support is treated with methanol:
Acetonitrile: pyridine (1: 1: 1, 3 × 20 m
L). The support can be made of piperidine 1 in pyridine.
By treating with 10 mL of 0% solution, N-Fmoc
The protecting group was removed. N-Sarcosinylated LCA-CPG
With methanol: acetonitrile: pyridine (1: 1:
(1, 3 x 20 mL) and dried under vacuum.

(2) 5′-O-dimethoxytrityl nucleoside was added to DMF (2 mL) and pyridine (0.2 m
L) and sarcosinylated LCA-CPG obtained as described in (1) in the presence of DCC (50 mg). The reaction mixture was shaken at room temperature for 12 hours, then methanol: acetonitrile: pyridine (1: 1: 1, 3 × 2
0 mL). After drying, the support was treated with N-methylimidazole: THF (1 mL) and acetic anhydride / lutidine (1 mL) for 15 minutes. Next, the support was treated with methanol: acetonitrile: pyridine (1: 1: 1, 3 × 1).
0 mL) followed by acetonitrile (3 × 10 mL) and then dried under vacuum.

(B) The diastereomerically pure activated nucleoside was then added on top of the oligonucleotide bound to the sarcosinyl LCA-CPG support in the presence of a 300-fold molar excess of DBU. The diastereomers of the activated nucleoside can be purified by column chromatography (silica gel 60) before use in the coupling reaction.
H, using ethyl acetate as the elution solvent, eluting with HPT
LC (silica gel 60, monitored by ethyl acetate as the developing solvent)]. Table 3 shows the synthesis protocol.
Is shown in

[0095]

[Table 3]

The diastereomeric purity of the phosphorothioate oligonucleotide was determined by ³¹ P NMR using HPL.
C (Lichropher Si100, 5AM, ethyl acetate as eluent, flow rate 3 mL / min), can be determined enzymatically or by electrophoresis.

Example 20 Disease Status in Human Patients
Therapeutic The oligonucleotides of the invention can be used to treat various disease states. Treatment of a patient diagnosed with a particular disease state involves administering to the patient an effective dose of an oligonucleotide in a pharmaceutically acceptable formulation by a suitable route. Effective oligonucleotide doses will depend on the disease state to be treated, the severity of the disease state and the age of the patient being treated. An effective dose of an oligonucleotide can be determined based on its IC ₅₀ , which is routine for one of ordinary skill in the art. Alternatively, the effective dose of the oligomer is determined by the pharmacokinetic software program TopFi.
It can be determined by using t. For example,
Oligonucleotide doses may range from 0.01 μg / kg body weight (for children) to 10 μg / kg, depending on the progression of the disease state.
It varies up to 0 g (for adults). Similarly, the frequency of administration will vary, from once or more daily, to once every 20 years, depending on the progress of the disease state.

The route of oligonucleotide administration will depend on the disease state to be treated. For example, administration of the oligonucleotide to a patient being treated for an inflammatory disease can be by the oral or rectal route. In the treatment of patients suffering from AIDS, the most effective method of oligonucleotide administration will be by the oral route or by subcutaneous injection. Cancer, such as breast cancer, can be treated by subcutaneous injection, while colon cancer can be treated by oral or rectal administration of oligonucleotides. Diseases or disorders of the central nervous system will be best treated by intrathecal or intraventricular administration to deliver the oligonucleotide to the spinal column or brain of the patient.

Following administration of the oligonucleotide, the patient can be monitored for relief of symptoms associated with the disease state. The dose can then be adjusted (increased or decreased) depending on the severity of the disease state and the responsiveness to the treatment.

It may be preferable to administer the oligonucleotides of the invention in combination with other traditional therapies.
Oligonucleotides include AZT for the treatment of patients suffering from AIDS, sulfasalazine for the treatment of inflammatory diseases such as ulcerative colitis.
e) and in combination with agents such as, but not limited to, 5-fluorouracil for the treatment of colon cancer.

It may also be desirable to administer maintenance therapy to patients who have been successfully treated for the disease state. The dose and frequency of oligonucleotide administration as part of a maintenance regimen can range from 0.01 μg / kg to 10 μg / kg body weight.
At 0 g, it varies from once a day or more to once every few years.

Example 21 Intraventricular of oligonucleotide
Intraventricular drug administration to deliver the administered drug directly to the patient's brain would be desirable for treatment of patients with brain afflicting diseases. To perform this mode of oligonucleotide administration, a silicone catheter was surgically introduced into the ventricle of the brain of a human patient, and a subcutaneous infusion pump (Medtronic Inc., Minn.) Was surgically implanted in the abdomen.
eapolis, MN) [Cancer R
essearch, 44: 1698 (1984)]. The pumps are used to inject the oligonucleotides and to perform precise dose adjustments and dose schedule changes with the aid of external programming equipment. Pump storage capacity is 18-
20mL, infusion rate from 0.1mL / h to 1mL
/ H range. Frequency of daily to monthly administration and 0.01 μg to 1 mg / kg of body weight of drug to be administered
Depending on the dose in the range of 00 g, the pump reservoir is 3-
Can be replenished at 10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump.

Example 22 Intrathecal injection of oligonucleotides
Intrathecal drug administration to introduce the drug to the spine of the administration patient,
It would be desirable to treat patients with diseases of the central nervous system. In order to carry out this route of oligonucleotide administration, a silicone catheter was surgically inserted into L3-
4 implant into the lumbar spine space and connect it to a subcutaneous infusion pump surgically implanted in the upper abdomen [The Ann
als of Pharmacotherapy, 2
7: 912 (1993) and Cancer, 41: 1.
270 (1993)]. The pumps are used to inject the oligonucleotides and to perform precise dose adjustments and dose schedule changes with the aid of external programming equipment.
The reservoir capacity of the pump will be 18-20 mL and the infusion rate will range from 0.1 mL / h to 1 mL / h. Frequency of daily and monthly administration and body weight of drug to be administered 1k
Depending on the dose ranging from 0.01 μg to 100 g per g, the pump reservoir can be refilled at 3-10 week intervals. Refilling of the pump is performed by a single percutaneous puncture of the self-sealing septum of the pump.

Example 23 A patient suffering from AIDS is treated with ISIS-5320, an oligonucleotide synthesized according to Example 3 or Example 19 and having the sequence TTGGGGTT (SEQ ID NO: 17). 1-1000 μg of oligonucleotide per kg of body weight is incorporated into a pharmaceutically acceptable carrier and administered intravitreally. The treatment can be repeated as necessary until the infection is eliminated.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 A61P 29/00 31/14 31/14 31/18 31/18 35/00 35/00 C12N 15/09 ZNA C12N 15/00 ZNAA (31) Priority claim number 08 / 468,569 (32) Priority date June 6, 1995 (1995.6.6) (33) Priority claim country United States (US (31) Priority claim number 08 / 466,692 (32) Priority date June 6, 1995 (June 6, 1995) (33) Priority claim country United States (US) (31) Priority claim number 08 / 471,966 (32) Priority date June 6, 1995 (June 6, 1995) (33) Priority claiming country United States (US) (31) Priority claim number 08 / 469,851 (32) Priority date June 6, 1995 (June 6, 1995) (33) Priority country United States (US) ( 31) Priority claim number 08 / 470,129 (32) Priority date June 6, 1995 (June 6, 1995) (33) Priority claim country United States (US) (72) Inventor Hawk, Glen United States of America 21771, Maryland, Mount Airy, Leafy Hollow Circle 919

Claims (6)

[Claims] 1. An oligonucleotide represented by SEQ ID NO: 6, wherein at least 75% of the nucleoside units are linked together by Sp phosphorothioate 3′-5 ′ linkages. 2. The oligonucleotide of claim 1, wherein all of the nucleoside units are linked together by Sp phosphorothioate 3'-5 'linkages. 3. A composition comprising the oligonucleotide of claim 1 and an acceptable carrier. 4. An oligonucleotide represented by SEQ ID NO: 6, wherein at least 75% of the nucleoside units are linked together by Rp phosphorothioate 3′-5 ′ linkages. 5. The oligonucleotide according to claim 4, wherein all of the nucleoside units are linked together by Rp phosphorothioate 3′-5 ′ linkages. 6. A composition comprising the oligonucleotide of claim 4 and an acceptable carrier.