WO1995033759A2 - Antisense oligonucleotides and methods for the treatment of schistosomiasis - Google Patents
Antisense oligonucleotides and methods for the treatment of schistosomiasis Download PDFInfo
- Publication number
- WO1995033759A2 WO1995033759A2 PCT/US1995/009598 US9509598W WO9533759A2 WO 1995033759 A2 WO1995033759 A2 WO 1995033759A2 US 9509598 W US9509598 W US 9509598W WO 9533759 A2 WO9533759 A2 WO 9533759A2
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- administering
- oligonucleotide
- oligonucleotide according
- schistosome
- effective amount
- Prior art date
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- C12N2310/53—Physical structure partially self-complementary or closed
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates to the field of antisense oligonucleotides and their use in the study and treatment of schistosomiasis.
- Schistosomiasis is a debilitating or fatal tropical disease that afflicts in excess of 250 million people in developing countries worldwide.
- the disease a blood fluke infection, is caused by a parasite of the genus Schistosoma.
- the species responsible for the disease are S. haematobium, S. japonicum, S. mansoni, S. mekongi, and S. intercalation.
- Adult schistosome worms live in the human mesenteri venules of the bloodstream where they have been estimated to consume their dry weight in glucose every 5 hours.
- Antisense oligonucleotide technology may provide a novel approach to the treatment of schistosomiasis. See generally Agrawal, Trends in Biotech. 10, 152 (1992). By binding to the complementary nucleic acid sequence (the sense strand), antisense oligonucleotides are able to inhibit splicing and translation of RNA. In this way, antisense oligonucleotides are able to inhibit protein expression. Antisense oligonucleotides have also been shown to bind to genomic DNA, forming a triplex, and inhibit transcription. Furthermore, a 17-mer base sequence statistically occurs only once in the human genome, and thus extremely precise targeting of specific sequences is possible with such antisense oligonucleotides.
- Zamecnik and Stephenson were the first to propose the use of synthetic antisense oligonucleotides for therapeutic purposes. Stephenson and Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 75, 285 (1978); Zamecnik and Stephenson, Proc. Natl Acad. Set U.S.A. 75, 280 (1978). They reported that the use of a oligonucleotide 13-mer complementary to the RNA of Rous sarcoma virus inhibited the growth of the virus in cell culture.
- Antisense oligonucleotides have also been shown to inhibit protein expression in mammalian systems.
- Burch and Mahan, J. Clin. Invest. 88, 1190 (1991) disclosed antisense oligonucleotides targeted to murine and human IL-1 receptors that inhibited IL-1-stimulated PGE 2 synthesis in murine and human fibroblasts, respectively;
- Colige et al., Biochemistry 32, 7 (1993) disclosed antisense oligonucleotides that specifically inhibited expression of a mutated human procollagen gene in transfected mouse 3T3 cells without inhibiting expression of an endogenous gene for the same protein;
- Monia et al., J. Biol Chem. 267, 19954 (1992) disclosed selective inhibition of mutant Ha-ras mRNA expression with phosphorothioate antisense oligonucleotide.
- Temsamani et al. Ann. N.Y. Acad. Sci 660, 318 (1992) disclosed certain 3' end- capped oligodeoxynucleotide phosphorothioates; and Tang et al., Nucleic Acids Res. 21, 2729 (1993) disclosed self-stabilized antisense oligodeoxynucleotide phosphorothioates having a hair-pin loop structure at their 3' ends.
- modified antisense oligonucleotides are capable of withstanding nucleolytic degradation, yet are still capable of hybridizing to target sequences and, thus, inhibiting protein expression. These modified oligonucleotides are better suited for in vivo applications. Tang et al., supra, showed that self-stabilized antisense oligonucleotides showed greater in vivo stability than their linear counterparts in mice. Simons et al. Nature 359, 67 (1992) reported the use of two antisense c-myb phosphorothioate oligonucleotides that suppressed intimal accumulation of rat carotid arterial smooth muscle cells in vivo.
- oligonucleotide structure disclosed by Pederson et al. in U.S. Patent No. 5,220,007 ('007) is another modified antisense oligonucleotide that may be particularly well-suited for both in vitro and in vivo inhibition of protein expression.
- That molecule comprises an internal sequence having two or more consecutive, modified or unmodified, phosphodiester linkages.
- the internal sequence is flanked on both sides by modified nucleic acid sequences.
- the internal sequence activates RNase H, while the flanking sequences are unable to activate RNase H.
- RNase H will excise the region of the target sequence complementary to the internal sequence of the antisense oligonucleotide.
- the target mRNA is thereby inactivated and protein expression inhibited.
- 3' end-capped (Temsamani et al., supra) and self-stabilized 3' hair- pin loop (Tang et al., supra) antisense oligonucleotides have been shown to have increased stability to nucleolytic attack and therefore may be well suited for inhibition of protein expression.
- the 3' hair-pin loop structure of Tang et al. is characterized as having a 3'-terminal sequence that is substantially complimentary to and anneals to an internal sequence.
- antisense oligos can be taken up by shistosoma worms, and thus can be effective as therapeutic agents for treating schistosomiasis. There is therefore a need for antisense oligonucleotides and methods effective for the treatment of schistosomiasis.
- the present invention comprises antisense oligonucleotides and methods effective for the treatment of schistosomiasis.
- the antisense oligonucleotides of the present invention are targeted to nucleic acids that code proteins essential to the metabolism, and/or reproduction of the schistosome worm.
- Administration of the oligonucleotides of the present invention will reduce or eliminate infection by parasites of the Schistosoma genus. Accordingly, the present invention also comprises methods for using these antisense oligonucleotides to treat schistosomiasis.
- Antisense oligonucleotides of the present invention are also useful for modulating gene expression in schistosome worms. Because they are targeted to a specific nucleic acid sequence, antisense oligonucleotides are able to selectively inhibit synthesis of the protein for which the targeted nucleic acid codes. Thus, antisense oligonucleotides of the present invention are useful for in vitro probing of the metabolic and reproductive processes of the schistosome parasite, leading to development of other compounds and methods for treating schistosomiasis. Antisense oligonucleotides of the present invention may also be useful in the reversal of possible drug resistance to PZQ through specific protein inhibition.
- Figure 1 displays the mass uptake of 35 S-labeled oligonucleotides by schistosome worms in vitro.
- Figure 2 displays the time course of 35 S-labeled oligonucleotide by schistosome worms in vitro.
- Figure 3 displays the efflux of 35 S-labeled oligonucleotide by schistosome worms in vitro.
- Figure 4 displays the efflux of 35 S-labeled oligonucleotide by schistosome worms into the culture medium.
- Figure 5 displays the effect of PZQ treatment on 35 S-labeled oligonucleotide uptake by cultured schistosome worms.
- Figure 6 displays the uptake 35 S-labeled oligonucleotides by schistosome worm cell nucleus, cytoplasm and tegument membrane.
- the present invention is ultimately directed to the treatment of schistosomiasis, a disease caused by a parasitic infection by a worm of the genus Schistosoma.
- One aspect of the present invention comprises antisense oligonucleotides targeted against the nucleic acids of schistosome worms.
- the target nucleic acid codes for a protein essential for a metabolic process and/or reproductive function of the parasite.
- the oligonucleotides of the present invention inhibit production of this protein by binding to the nucleic acid, thereby inhibiting transcription or translation.
- antisense oligonucleotide and “oligonucleotide” are interchangeable and mean a deoxyribonucleotide polymer having from 2 to about 100 nucleotide monomers.
- the nucleotide bases can be any base, modified or unmodified, that is able to form Watson-Crick or Hoogsteen base pairs. Oligonucleotides according to the present invention are non-naturally occurring.
- modified means any chemical modification of the nucleotide base or the sugar phosphate backbone.
- a modification may be of from one to all moieties. The only limitation on the type and number of modifications is that they do not render the oligonucleotide unable to bind to the target nucleic acid and inhibit protein synthesis.
- co-administered means administered either before, with, or following administration of specific oligonucleotides.
- the antisense oligonucleotides of the present invention are from about 5 to about 100 nucleotides long. Preferably, they are about 16 to 50 nucleotides in length.
- the oligonucleotides are completely unmodified. In the preferred embodiment, however, the oligonucleotides of the present invention will have anywhere from one to all internucleotide linkages modified to enhance resistance to nuclease attack and/or enhance schistosome uptake.
- any modification that does not prevent or substantially diminish the ability of the oligonucleotide to inhibit protein production can be used in the present invention.
- modifications contemplated include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidate, methylphosphonate, phosphomorpholidate, carbonate, and carbamate intemucleoside linkages.
- the olgionucleotides of the present invention may also have one or both ends capped to impart nuclease resistance.
- One or more types of modifications may be present in any particular oligonucleotide, as in, for instance, oligonucleotides having structural features as described by Pederson et al. in the '007 patent, supra.
- the oligonucleotides of the present invention have one or more modifications that render them resistant to nuclease attack and enhance their uptake by schistosome worms.
- oligonucleotide having a 3' end sequence substantially complementary to an internal sequence of the oligonucleotide will form a structure in which the two regions hybridize, enhancing the oligonucleotide's resistance to nucleolytic degradation yet leaving the oligonucleotide with the ability to bind to its target nucleic acid sequence and inhibit protein synthesis.
- Such an oligonucleotide is a "self-stabilized" oligonucleotide.
- Another example is foldback triplex-forming oligonucleotides, as described by
- oligonucleotides have a first region with a sequence substantially complementary to a target nucleic acid that binds to the target via Watson-Crick interactions, forming a duplex.
- a second region of the oligonucleotide has a sequence that folds back upon the duplex, forming a triplex via Hoogsteen base pairing.
- the oligonucleotides of the present are self-stabilized or triplex-forming oligonucleotides.
- the oligonucleotides of the present invention may be obtained by any suitable method. In most instances the oligonucleotides are conveniently chemically synthesized. A variety of methods are well known to those skilled in the art and have been reviewed. E.g., Agrawal and Zamecnik, U.S. Patent No. 5,149,798; Goodchild et al., Bioconjugate Chem. 2, 165 (1990); Agrawal and Sarin, Adv. Drug Delivery Rev. 6, 251 (1991); Oligonucleotides and Analogues: A Practical Approach (F. Ekstein, Ed., IRL Press, 1991); and Uhlmann and Peyman, supra. 3'- and/or 5'-capped modifications may be prepared, for example, by the methods of Temsamani et al.,
- oligonculeotides according to the invention are efficiently taken up by schistosome worms. Furthermore, the tegument (the outer coat of schistosome worms) interferes little, if at all, with oligonucleotide uptake. Accordingly, oligonucleotides according to the present invention may be useful for both in vitro gene modulation of parasites of the genus Schistosoma as well as for in vivo treatment of mammals infected with a Schistosoma parasite. The oligonucleotides find in vitro application in regulating schistosome genes essential for metabolism and reproduction. As used herein, the term "metabolism" means any process involved in the growth, development, and/or maintenance of the parasite.
- oligonucleotides according to the invention act as probes for the physiological function of schistosoma genes.
- the use of oligonucleotides of the present invention as regulators of gene expression is also a powerful method of studying genetic mutations without going through the laborious task of actually creating the mutant.
- Antisense oligonucleotides that inhibit protein production thereby create the functional equivalent of a mutation that results in the same effect.
- Such uses will aid in the development of still other compounds/compositions and methods for combating schistosomiasis, as well as in understanding the biology of schistosoma.
- the oligonucleotides of the present invention may also be useful as an anti- schistosomiasis agent in vivo.
- Administration to an infected mammal of an antisense oligonucleotide targeted against a nucleic acid encoding a protein involved in some essential metabolic or reproductive process results in the decrease or elimination of the number of live, reproducing parasites within the infected mammal.
- the oligonucleotide 5'- GCCATAGGGGGCAGGGAAGGC-3' significantly reduced the worm burden in S. mansoni-mfected mice and reduced the number of eggs per gram of liver.
- oligonucleotides according to the present invention can be synthesized using standard techniques to have a sequence (comprising either the whole oligonucleotide or only a region of it) complementary to that gene. Then, using the techniques disclosed herein, in particular in Example 6, one may determine in a routine manner the efficacy of the anti-worm agent.
- the protein is involved in a process essential for the metabolism, reproduction, or both, of the schistosome worm.
- the method comprises contacting the worm with an effective amount of the antisense oligonucleotide. Generally, in in vitro applications, this will be in an amount of from about 1 mg/ml to about 8 mg/ml. For in vivo applications, administration of from about 1 to about
- oligonucleotide per kg body weight 30 mg oligonucleotide per kg body weight is appropriate.
- In vivo treatment of a mammal infected with a Schistosoma parasite is preferably accomplished by parental administration, most preferably by intravenous administration.
- a method of inhibiting protein synthesis in a schistosome worm comprising co-administering an oligonucleotide of the invention and praziquantel. It has unexpectedly been found that co-administration of a single 250 mg/kg body weight dose of praziquantel increases schistosome uptake of oligonucleotide in vitro by as much as 60%.
- the preferred amount of prazinquantel is any non-toxic amount that enhances the schistosome uptake of oligonucleotide. Accordingly, in a preferred embodiment, praziquantel may be optionally co-administered with an oligonucleotide of the invention, either in the same pharmaceutical composition or via separate administration.
- the time dependent oligonucleotide efflux from Schistosome Worms at two temperatures can also be determined by measuring the amount of oligonucleotide in the culture medium.
- Schistosome worms are incubated with 35 S-labeled oligonucleotides (1 ⁇ Ci/ml). After 16 hrs. the worms are washed (3X) with cold PBS and incubated in oligonucleotide-free medium at 4°C and 37°C for different times.
- the results are presented in Figure 4.
- the efflux of 35 S-labeled oligonucleotides per worm pair into the medium is represented by the percentage of cpm recorded from 1 ml of culture medium. Efflux levels measured from radiolabel in the culture medium is consistent with those levels measured from radiolabel remaining in worms.
- toxicity is not evidenced until very high levels of the oligo (around 16.0 mg/ml) are reached. At lower levels of oligo (4.0 mg/ml and below) worm survival approximates that of control worms in culture. At 8.0 mg/ml, a low level of toxicity is evident as early as 48-60 hr. in culture, with toxicity being greater for the smaller mass female worms.
- Schistosome Worms are incubated with 35 S-labeled oligonucleotides (0.6 ⁇ Ci/ml) for 20 hrs, then washed 3X with cold PBS. These worms (either male/female separated or mixed) are transferred into PBS buffer containing 2.7 mM
- the uptake and amount of 35 S-labeled oligonucleotides is represented by cpm remaining within worm body tissues and the buffer.
- the amount of 35 S-labeled oligonucleotides in tegument membrane was calculated according to efflux into the same buffer by worms with intact membrane.
- Group 1 are incubated with 35 S-labeled oligonudeotides for 16 hrs, followed by washing (3X) with cold PBS. The worms are then incubated in PBS buffer containing
- worms are vortexed (maximum power) for 2 X 1 min.
- the worms are free of the dissociated tegument and are then transferred to a sucrose/HEPES buffer for homogenization (15,000 rpm) for 1 min.
- the nuclei-rich fraction is separated from the cytoplasmic fraction by 2X centrifugation at 480x g.
- Schistosome worms in Group 2 have their tegument removed before incubation with 35 S-labeled oligonucleotides. The procedure for tegument removal is repeated at the end of the incubation period in view of minimal regeneration of the tegument. The results are presented in Figure 6. The uptake in different fractions is represented by percentage of cpm value.
- praziquantel Since the current therapeutic drug of choice, praziquantel, is thought to act by interfering with the integrity of the tegument, it is useful to investigate whether praziquantel affects the uptake of radiolabelled oligonucleotide by intact worms.
- a 250 mg/kg single dose of PZQ is given per os to mice that have been infected with schistosomes for a period of 49 days. Worms are recovered one hour after the administration of the drug and cultured with 35 S-labeled oligonucleotides at 37°C for 16 hrs. Male and female worms are washed (3X) separately with PBS and then measured. The results are present in Figure 5.
- the uptake is represented in cpm values.
- Figure 5 shows clearly that praziquantel treatment significantly increases the uptake of oligonucleotide. It is noted that paired female worms show little difference in uptake levels compared to the control with no praziquantel treatment. This is understandable in light of the fact that in worm pairs the smaller female is nearly entirely enveloped by the larger male. Thus, a large fraction of the female tegument is not exposed to praziquantel in the same way as is unpaired female tegument. Nonetheless, the effect on the male in a given pair may provide synergies with specific oligos where praziquantel is co-administered either before, with, or following administration of specific oligos.
- Schistosoma mansoni-specific antisense oligonucleotides targeted to two different regions of the Schistosoma mansoni female-specific eggshell protein mRNA are used to determine their efficacy against the parasite.
- administered I.V. is targeted to the putative proline hinge region of the folded protein.
- CD 1 female mice are each infected with 100 ⁇ schistosome cercariae by tail immersion.
- Specific and nonspecific antisense oligonucleotides, as well as water controls, are administered to mice 40 days post-infection.
- Oligonucleotides are administered either I.V. at a single dose of 30 mg/kg body wgt. or I.P. at a dose of 15 mg/kg body wgt. on three consecutive days. Fifteen days after last dosing mice are sacrificed, perfused for worm recovery, and the liver and intestines removed for egg examination. The results are presented in Table 4.
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Abstract
Antisense oligonucleotides effective at inhibiting protein synthesis of schistosome worms are disclosed. It is shown that these oligonucleotides are efficiently taken up by the schistosome worm and may effectively kill the worms in vivo. Accordingly, these antisense oligonucleotides and methods of their administration may be useful for treating schistosomiasis.
Description
ANTISENSE OLIGONUCLEOTIDES AND METHODS
FOR THE TREATMENT OF SCHISTOSOMIASIS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to the field of antisense oligonucleotides and their use in the study and treatment of schistosomiasis.
Summary of the Related Art
Schistosomiasis is a debilitating or fatal tropical disease that afflicts in excess of 250 million people in developing countries worldwide. The disease, a blood fluke infection, is caused by a parasite of the genus Schistosoma. The species responsible for the disease are S. haematobium, S. japonicum, S. mansoni, S. mekongi, and S. intercalation. Adult schistosome worms live in the human mesenteri venules of the bloodstream where they have been estimated to consume their dry weight in glucose every 5 hours. Both Rajkovic et al., Proc. Natl. Acad. Sci., USA 86, 8217 (1989), and
Chen et al., Exper. Parasit. 74, 82 (1991), as well as others, have shown that inhibition of 3-hydroxymethylglutaryl-coenzyme in S. mansoni results in cessation of egg laying and an inability of the adult to properly glycosylate their proteins. Eggs are the major cause of serious human pathology in schistosomiasis.
Currently there are no prophylactic or suppressive drugs, nor effective vaccines, available for clinical use in the control of schistosomiasis. The only usable drug for treatment is prazinquantel (PZQ), which has been quite effective against adult worms. The number of patients taking the drug that appear not to be cured is increasing, however. Work is being done to monitor these patients for the possible development of resistance to PZQ. Ongoing research suggests that resistance to PZQ may be developing. It is very important, therefore, that new drugs, or alternative agents, be developed for schistosomiasis treatment. New agents effective at regulating Schistosoma gene expression would also by useful to aid in the development of new ways to combat the parasite.
Antisense oligonucleotide technology may provide a novel approach to the treatment of schistosomiasis. See generally Agrawal, Trends in Biotech. 10, 152 (1992). By binding to the complementary nucleic acid sequence (the sense strand), antisense
oligonucleotides are able to inhibit splicing and translation of RNA. In this way, antisense oligonucleotides are able to inhibit protein expression. Antisense oligonucleotides have also been shown to bind to genomic DNA, forming a triplex, and inhibit transcription. Furthermore, a 17-mer base sequence statistically occurs only once in the human genome, and thus extremely precise targeting of specific sequences is possible with such antisense oligonucleotides.
In 1978 Zamecnik and Stephenson were the first to propose the use of synthetic antisense oligonucleotides for therapeutic purposes. Stephenson and Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 75, 285 (1978); Zamecnik and Stephenson, Proc. Natl Acad. Set U.S.A. 75, 280 (1978). They reported that the use of a oligonucleotide 13-mer complementary to the RNA of Rous sarcoma virus inhibited the growth of the virus in cell culture. Since then, numerous other studies have been published manifesting the in vitro efficacy of antisense oligonucleotide inhibition of viral growth, e.g., vesicular stomatitis viruses (Leonetti et al., Gene 72, 323 (1988)), herpes simplex viruses (Smith et al, Proc. Natl Acad. Set U.S.A. 83, 2787 (1986)), and influenza virus (Zerial et al., Nucleic Acids Res. 15, 9909 (1987)).
Antisense oligonucleotides have also been shown to inhibit protein expression in mammalian systems. For example, Burch and Mahan, J. Clin. Invest. 88, 1190 (1991), disclosed antisense oligonucleotides targeted to murine and human IL-1 receptors that inhibited IL-1-stimulated PGE2 synthesis in murine and human fibroblasts, respectively; Colige et al., Biochemistry 32, 7 (1993) disclosed antisense oligonucleotides that specifically inhibited expression of a mutated human procollagen gene in transfected mouse 3T3 cells without inhibiting expression of an endogenous gene for the same protein; and Monia et al., J. Biol Chem. 267, 19954 (1992), disclosed selective inhibition of mutant Ha-ras mRNA expression with phosphorothioate antisense oligonucleotide.
In most cases, however, unmodified antisense oligonucleotides are unsuitable for use in in vivo systems because of their susceptibility to attack by nucleases. Consequently, there has been much research in the area of modifying oligonucleotides to make them resistant to such attack, thereby stabilizing the molecules for in vivo use. See generally Uhlmann and Peymann, Chemical Reviews 90,
543 (1990) at pages 545-561 and references cited therein. Focus has been on modifying the internucleotide phosphate residues, modifying the nucleoside units, modifying the 2' position and substituting other moieties for the internucleotide phosphate. For example, Padmapriya and Agrawal, Bioorg. & Med. Chem. Lett. 3, 761 (1993) disclosed synthesis of oligodeoxynucleoside methlyphosphonothioates;
Temsamani et al., Ann. N.Y. Acad. Sci 660, 318 (1992) disclosed certain 3' end- capped oligodeoxynucleotide phosphorothioates; and Tang et al., Nucleic Acids Res. 21, 2729 (1993) disclosed self-stabilized antisense oligodeoxynucleotide phosphorothioates having a hair-pin loop structure at their 3' ends.
Many modified antisense oligonucleotides are capable of withstanding nucleolytic degradation, yet are still capable of hybridizing to target sequences and, thus, inhibiting protein expression. These modified oligonucleotides are better suited for in vivo applications. Tang et al., supra, showed that self-stabilized antisense oligonucleotides showed greater in vivo stability than their linear counterparts in mice. Simons et al. Nature 359, 67 (1992) reported the use of two antisense c-myb phosphorothioate oligonucleotides that suppressed intimal accumulation of rat carotid arterial smooth muscle cells in vivo.
The oligonucleotide structure disclosed by Pederson et al. in U.S. Patent No. 5,220,007 ('007) is another modified antisense oligonucleotide that may be particularly well-suited for both in vitro and in vivo inhibition of protein expression.
That molecule comprises an internal sequence having two or more consecutive, modified or unmodified, phosphodiester linkages. The internal sequence is flanked on both sides by modified nucleic acid sequences. The internal sequence activates RNase H, while the flanking sequences are unable to activate RNase H. The result is that when the oligonucleotide of the '007 patent is bound to the target mRNA sequence, RNase H will excise the region of the target sequence complementary to the internal sequence of the antisense oligonucleotide. The target mRNA is thereby inactivated and protein expression inhibited.
Similarly, 3' end-capped (Temsamani et al., supra) and self-stabilized 3' hair- pin loop (Tang et al., supra) antisense oligonucleotides have been shown to have increased stability to nucleolytic attack and therefore may be well suited for inhibition
of protein expression. The 3' hair-pin loop structure of Tang et al. is characterized as having a 3'-terminal sequence that is substantially complimentary to and anneals to an internal sequence.
Despite these developments, there has been no demonstration that antisense oligos can be taken up by shistosoma worms, and thus can be effective as therapeutic agents for treating schistosomiasis. There is therefore a need for antisense oligonucleotides and methods effective for the treatment of schistosomiasis.
SUMMARY OF THE INVENTION
As described more fully below, the present invention comprises antisense oligonucleotides and methods effective for the treatment of schistosomiasis. The antisense oligonucleotides of the present invention are targeted to nucleic acids that code proteins essential to the metabolism, and/or reproduction of the schistosome worm. Administration of the oligonucleotides of the present invention will reduce or eliminate infection by parasites of the Schistosoma genus. Accordingly, the present invention also comprises methods for using these antisense oligonucleotides to treat schistosomiasis.
Antisense oligonucleotides of the present invention are also useful for modulating gene expression in schistosome worms. Because they are targeted to a specific nucleic acid sequence, antisense oligonucleotides are able to selectively inhibit synthesis of the protein for which the targeted nucleic acid codes. Thus, antisense oligonucleotides of the present invention are useful for in vitro probing of the metabolic and reproductive processes of the schistosome parasite, leading to development of other compounds and methods for treating schistosomiasis. Antisense oligonucleotides of the present invention may also be useful in the reversal of possible drug resistance to PZQ through specific protein inhibition.
The foregoing merely summarizes some aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any way.
All patents and publications cited within this specification are hereby incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 displays the mass uptake of 35S-labeled oligonucleotides by schistosome worms in vitro.
Figure 2 displays the time course of 35S-labeled oligonucleotide by schistosome worms in vitro.
Figure 3 displays the efflux of 35S-labeled oligonucleotide by schistosome worms in vitro.
Figure 4 displays the efflux of 35S-labeled oligonucleotide by schistosome worms into the culture medium.
Figure 5 displays the effect of PZQ treatment on 35S-labeled oligonucleotide uptake by cultured schistosome worms.
Figure 6 displays the uptake 35S-labeled oligonucleotides by schistosome worm cell nucleus, cytoplasm and tegument membrane.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is ultimately directed to the treatment of schistosomiasis, a disease caused by a parasitic infection by a worm of the genus Schistosoma. One aspect of the present invention comprises antisense oligonucleotides targeted against the nucleic acids of schistosome worms. The target nucleic acid codes for a protein essential for a metabolic process and/or reproductive function of the parasite. The oligonucleotides of the present invention inhibit production of this protein by binding to the nucleic acid, thereby inhibiting transcription or translation.
As used herein, the term "antisense oligonucleotide" and "oligonucleotide" are interchangeable and mean a deoxyribonucleotide polymer having from 2 to about 100 nucleotide monomers. The nucleotide bases can be any base, modified or unmodified, that is able to form Watson-Crick or Hoogsteen base pairs. Oligonucleotides according to the present invention are non-naturally occurring.
As used herein, the term "modified," means any chemical modification of the nucleotide base or the sugar phosphate backbone. A modification may be of from one to all moieties. The only limitation on the type and number of modifications is
that they do not render the oligonucleotide unable to bind to the target nucleic acid and inhibit protein synthesis.
As used herein, the term "co-administered" means administered either before, with, or following administration of specific oligonucleotides.
The antisense oligonucleotides of the present invention are from about 5 to about 100 nucleotides long. Preferably, they are about 16 to 50 nucleotides in length.
In one embodiment of the present invention, the oligonucleotides are completely unmodified. In the preferred embodiment, however, the oligonucleotides of the present invention will have anywhere from one to all internucleotide linkages modified to enhance resistance to nuclease attack and/or enhance schistosome uptake.
Any modification that does not prevent or substantially diminish the ability of the oligonucleotide to inhibit protein production can be used in the present invention. Among the modifications contemplated include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidate, methylphosphonate, phosphomorpholidate, carbonate, and carbamate intemucleoside linkages. The olgionucleotides of the present invention may also have one or both ends capped to impart nuclease resistance. One or more types of modifications may be present in any particular oligonucleotide, as in, for instance, oligonucleotides having structural features as described by Pederson et al. in the '007 patent, supra. In a preferred embodiment, the oligonucleotides of the present invention have one or more modifications that render them resistant to nuclease attack and enhance their uptake by schistosome worms.
Resistance of the present oligonucleotides to nuclease attack may also be imparted by the appropriate choice of oligonucleotide sequence. For example, Tang et al., supra, demonstrated that an oligonucleotide having a 3' end sequence substantially complementary to an internal sequence of the oligonucleotide will form a structure in which the two regions hybridize, enhancing the oligonucleotide's resistance to nucleolytic degradation yet leaving the oligonucleotide with the ability to bind to its target nucleic acid sequence and inhibit protein synthesis. Such an oligonucleotide is a "self-stabilized" oligonucleotide.
Another example is foldback triplex-forming oligonucleotides, as described by
Agrawal and Kandimalla, Gene (in press). These oligonucleotides have a first region with a sequence substantially complementary to a target nucleic acid that binds to the target via Watson-Crick interactions, forming a duplex. A second region of the oligonucleotide has a sequence that folds back upon the duplex, forming a triplex via Hoogsteen base pairing. In another preferred embodiment, the oligonucleotides of the present are self-stabilized or triplex-forming oligonucleotides.
The oligonucleotides of the present invention may be obtained by any suitable method. In most instances the oligonucleotides are conveniently chemically synthesized. A variety of methods are well known to those skilled in the art and have been reviewed. E.g., Agrawal and Zamecnik, U.S. Patent No. 5,149,798; Goodchild et al., Bioconjugate Chem. 2, 165 (1990); Agrawal and Sarin, Adv. Drug Delivery Rev. 6, 251 (1991); Oligonucleotides and Analogues: A Practical Approach (F. Ekstein, Ed., IRL Press, 1991); and Uhlmann and Peyman, supra. 3'- and/or 5'-capped modifications may be prepared, for example, by the methods of Temsamani et al.,
New York Acad. Sci.
As is demonstrated below, oligonculeotides according to the invention are efficiently taken up by schistosome worms. Furthermore, the tegument (the outer coat of schistosome worms) interferes little, if at all, with oligonucleotide uptake. Accordingly, oligonucleotides according to the present invention may be useful for both in vitro gene modulation of parasites of the genus Schistosoma as well as for in vivo treatment of mammals infected with a Schistosoma parasite. The oligonucleotides find in vitro application in regulating schistosome genes essential for metabolism and reproduction. As used herein, the term "metabolism" means any process involved in the growth, development, and/or maintenance of the parasite.
Together, metabolism and reproduction are meant to encompass all processes essential to sustain a living worm and to propagate the species. Modulation of a gene allows for the determination of the role of the gene in metabolism and reproduction. Thus, oligonucleotides according to the invention act as probes for the physiological function of schistosoma genes. The use of oligonucleotides of the present invention as regulators of gene expression is also a powerful method of studying genetic
mutations without going through the laborious task of actually creating the mutant. Antisense oligonucleotides that inhibit protein production thereby create the functional equivalent of a mutation that results in the same effect. Such uses will aid in the development of still other compounds/compositions and methods for combating schistosomiasis, as well as in understanding the biology of schistosoma.
The oligonucleotides of the present invention may also be useful as an anti- schistosomiasis agent in vivo. Administration to an infected mammal of an antisense oligonucleotide targeted against a nucleic acid encoding a protein involved in some essential metabolic or reproductive process results in the decrease or elimination of the number of live, reproducing parasites within the infected mammal. As demonstrated below, the oligonucleotide 5'- GCCATAGGGGGCAGGGAAGGC-3' (SEQ. ID. NO. 1) significantly reduced the worm burden in S. mansoni-mfected mice and reduced the number of eggs per gram of liver.
It is a routine matter for those skilled in the art to obtain other oligonucleotides effective against schistosomiasis. The nucleic acid sequences for about 30 Schistosoma mansoni genes have been reported and are available in the literature. For example, Bobek et al., Mol Cell. Biol 8, 3008 (1988) disclose the sequence of the nucleic acid encoding a 14 kD eggshell (chorion) protein. Johnson et al., Parasit. 22, 89 (1987) disclose the sequence of the nucleic acid encoding a 48 kD eggshell protein. Skelly et al., J. Biol. Chem. 269, 1 (1994) disclose the sequence of cDNAs encoding adult worm glucose transporter proteins. Rajkovic et al., supra, disclose the sequence of the adult worm 3-hydroxymethylglutaryl-coenzyme A gene. See also Chen et al., supra. Rajkovic et al., Proc. Natl. Acad. Sei., USA 87, 8879 (1990) disclose adult worm 36-nucleotide spliced leader sequence specific to a subset of Schistosoma mansoni mRNAs not found in other organisms. Potential target sequences are preferably those lacking sequence homology to human sequences, which homology may be determined using the BLAST algorithm. Additionally, minimizing codon diversity is useful. mRNA start regions are also promising targets. In any event, once the sequence of a schistosome gene is known, oligonucleotides according to the present invention can be synthesized using standard techniques to have a sequence (comprising either the whole oligonucleotide or only a region of it)
complementary to that gene. Then, using the techniques disclosed herein, in particular in Example 6, one may determine in a routine manner the efficacy of the anti-worm agent.
In another aspect of the present invention, we provide methods for inhibiting the protein synthesis in schistosome worms. In a preferred embodiment, the protein is involved in a process essential for the metabolism, reproduction, or both, of the schistosome worm. In both in vitro and in vivo applications, the method comprises contacting the worm with an effective amount of the antisense oligonucleotide. Generally, in in vitro applications, this will be in an amount of from about 1 mg/ml to about 8 mg/ml. For in vivo applications, administration of from about 1 to about
30 mg oligonucleotide per kg body weight is appropriate. In vivo treatment of a mammal infected with a Schistosoma parasite is preferably accomplished by parental administration, most preferably by intravenous administration.
In another embodiment of the present invention, a method of inhibiting protein synthesis in a schistosome worm is provided comprising co-administering an oligonucleotide of the invention and praziquantel. It has unexpectedly been found that co-administration of a single 250 mg/kg body weight dose of praziquantel increases schistosome uptake of oligonucleotide in vitro by as much as 60%. The preferred amount of prazinquantel is any non-toxic amount that enhances the schistosome uptake of oligonucleotide. Accordingly, in a preferred embodiment, praziquantel may be optionally co-administered with an oligonucleotide of the invention, either in the same pharmaceutical composition or via separate administration.
The following Examples are intended for illustrative purposes only and should not be construed to limit the invention in any way.
EXAMPLES
Example 1
In vitro Mass Uptake of Antisense Oligonucleotides by Schistosome Worms
The in vitro mass uptake of nonspecific oligonucleotides can be determined by incubating worms in mixtures of cold and radiolabelled oligonucleotides in
concentrations ranging over nearly three orders of magnitude. Schistosome worms are incubated with different concentrations of 35S-labeled oligonucleotides for 24 hrs. The worms are then washed (3X) with PBS, solubilized, and measured. The results are presented in Figure 1. (Circles = males cultured in pairs; squares = females cultured separately; triangles = females cultured in pairs)
The data demonstrate an ever increasing uptake of oligonucleotides. Males are seen to take up around 10-fold more oligo than females, roughly consistent with the known ratio of male to female body mass (7:1 to 10:1). Uptake is seen to be as high as 0.1% of body weight.
The time course of in vitro uptake of nonspecific oligos by whole worms in culture wells can also be measured. Male and female worms are incubated with 35S-labeled oligonucleotides (1 μCi/ml). At the specified times they are washed (3X) with cold PBS, solubilized, and measured. The results are presented in Figure 2. The uptake per worm is represented by cpm values. As can be seen in Figure 2, the uptake per worm plateaus by 36 hours. These values for male and female worms again reflect nearly exactly the ratio of their body masses.
Example 2
In vitro Efflux of Oligonucleotides from Schistosome Worms The time dependent oligonucleotide efflux from Schistosome worms at two temperatures can be determined. Schistosome worms are incubated with 35S-Labelled oligonucleotides (1 μCi/ml). After 16 hrs. the worms are washed (3X) with cold PBS and incubated in oligonucleotide-free medium at 4°C and 37°C for different times. The results are presented in Figure 3. The efflux per worm pair is represented by the percentage of cpm remaining inside the worms. The temperature dependent behavior of the efflux is consistent with metabolically active processes.
The time dependent oligonucleotide efflux from Schistosome Worms at two temperatures can also be determined by measuring the amount of oligonucleotide in the culture medium. Schistosome worms are incubated with 35S-labeled oligonucleotides (1 μCi/ml). After 16 hrs. the worms are washed (3X) with cold PBS and incubated in oligonucleotide-free medium at 4°C and 37°C for different times.
The results are presented in Figure 4. The efflux of 35S-labeled oligonucleotides per worm pair into the medium is represented by the percentage of cpm recorded from 1 ml of culture medium. Efflux levels measured from radiolabel in the culture medium is consistent with those levels measured from radiolabel remaining in worms. Example 3
In vitro Toxicity of Non-specific Oligonucleotides to Schistosome Worms Five male and five female schistosome worms are incubated in 1 ml of culture medium with differing concentrations of non-specific oligonucleotides for up to 96 hrs. The worms are checked periodically for motility and viability. The results are presented in Table 1.
It is seen that toxicity is not evidenced until very high levels of the oligo (around 16.0 mg/ml) are reached. At lower levels of oligo (4.0 mg/ml and below) worm survival approximates that of control worms in culture. At 8.0 mg/ml, a low level of toxicity is evident as early as 48-60 hr. in culture, with toxicity being greater for the smaller mass female worms.
Example 4
Antisense Oligonucleotide Distribution in Schistosome Worms Schistosome worms are incubated with 35S-labeled oligonucleotides (0.6 μCi/ml) for 20 hrs, then washed 3X with cold PBS. These worms (either male/female separated or mixed) are transferred into PBS buffer containing 2.7 mM
KCI/0.9 mM CaCl2/0.5 mM MgCl2, incubated for 15 min at 37°C, and then high speed vortexed for 2 x 1 min. The worms and the buffer with tegument are measured separately. The results are presented in Table 2.
The uptake and amount of 35S-labeled oligonucleotides is represented by cpm remaining within worm body tissues and the buffer. The amount of 35S-labeled oligonucleotides in tegument membrane was calculated according to efflux into the same buffer by worms with intact membrane.
It is seen that for worm pairs and males and females cultured separately some
79 - 83% of CPM are found in the worm bodies, not in the tegument following its removal into the culture medium (17-21%). The control worm pairs were monitored to correct for efflux of oligos taking place simultaneously during the course of the tegument removal. Some 9.8% was found to efflux from the control worm pair with intact tegument. Therefore, the final efflux corrected column shows between 7-11% of the radiolabel to be contained in the tegument. Thus, the great majority of oligo appears to be taken up into the worm body interior tissue.
An experiment in which the tegument is removed prior to incubation with the radiolabelled non-specific oligonucleotide is also useful. Schistosome worms are incubated in PBS buffer with 2.7 mM KCl, 0.9 mM CaCl2, and 0.5 mM MgCl2 for 15 min. at 37°C followed by high speed vortexing for 2 x 1 min. These tegument membrane-free worms are quickly rinsed and then transferred into RPMI culture medium for incubation with 35S-labeled oligonucleotides (0.6 μCi/ml) for 20 hrs. at 37°C. Then, they are washed (3x) with cold PBS. The results are presented in Table 3.
The uptake per worm is represented by cpm values. It is seen clearly that nearly the
same level of radiolabelled oligonucleotide is taken up by intact worms as by those whose tegument had previously been removed. This agrees with the results presented in Table 2.
In order to demonstrate directly that the radiolabelled nonspecific oligonucleotides are taken up into worm body tissue, low shear mechanical lysis experiments are performed. Two groups of worms are studied, those incubated with radiolabelled oligo after tegument removal (Group 1) and those incubated with radiolabelled oligo before tegument removal (Group 2). Schistosome worms in
Group 1 are incubated with 35S-labeled oligonudeotides for 16 hrs, followed by washing (3X) with cold PBS. The worms are then incubated in PBS buffer containing
2.7 mM KCl , 0.9mM CaCl,, and 0.5 mM MgCl, at 37°C for 15 min. At that time worms are vortexed (maximum power) for 2 X 1 min. Following a rapid rinse with PBS, the worms are free of the dissociated tegument and are then transferred to a sucrose/HEPES buffer for homogenization (15,000 rpm) for 1 min. The nuclei-rich fraction is separated from the cytoplasmic fraction by 2X centrifugation at 480x g.
Schistosome worms in Group 2 have their tegument removed before incubation with 35S-labeled oligonucleotides. The procedure for tegument removal is repeated at the end of the incubation period in view of minimal regeneration of the tegument. The results are presented in Figure 6. The uptake in different fractions is represented by percentage of cpm value.
It is seen from Figure 6 that for tegument removal after (Group 1) and before (Group 2) incubation with radiolabel, the major percentage of the radiolabel (about 50-60%) is localized to the nuclei enriched pellet rather than to either cytosol or tegument containing fractions. Example 5
Effect of Praziquantel on Antisense Oligonucleotide Uptake by Schistosome Worms
Since the current therapeutic drug of choice, praziquantel, is thought to act by interfering with the integrity of the tegument, it is useful to investigate whether praziquantel affects the uptake of radiolabelled oligonucleotide by intact worms. A 250 mg/kg single dose of PZQ is given per os to mice that have been infected with
schistosomes for a period of 49 days. Worms are recovered one hour after the administration of the drug and cultured with 35S-labeled oligonucleotides at 37°C for 16 hrs. Male and female worms are washed (3X) separately with PBS and then measured. The results are present in Figure 5. The uptake is represented in cpm values.
Figure 5 shows clearly that praziquantel treatment significantly increases the uptake of oligonucleotide. It is noted that paired female worms show little difference in uptake levels compared to the control with no praziquantel treatment. This is understandable in light of the fact that in worm pairs the smaller female is nearly entirely enveloped by the larger male. Thus, a large fraction of the female tegument is not exposed to praziquantel in the same way as is unpaired female tegument. Nonetheless, the effect on the male in a given pair may provide synergies with specific oligos where praziquantel is co-administered either before, with, or following administration of specific oligos. Example 6
In vivo Administration of Schistosoma
mansoni-Targeted Antisense Oligonucleotides
Two Schistosoma mansoni-specific antisense oligonucleotides targeted to two different regions of the Schistosoma mansoni female-specific eggshell protein mRNA are used to determine their efficacy against the parasite. The specific oligo 2
(administered I.V.) is targeted to the putative proline hinge region of the folded protein.
CD1 female mice are each infected with 100± schistosome cercariae by tail immersion. Specific and nonspecific antisense oligonucleotides, as well as water controls, are administered to mice 40 days post-infection. Oligonucleotides are administered either I.V. at a single dose of 30 mg/kg body wgt. or I.P. at a dose of 15 mg/kg body wgt. on three consecutive days. Fifteen days after last dosing mice are sacrificed, perfused for worm recovery, and the liver and intestines removed for egg examination. The results are presented in Table 4.
A much reduced worm burden is found when the mouse is sacrificed. Interestingly, the female population is more adversely affected. Also, a much greater fraction of the worms are found in the liver, a sign of worm distress. Finally, the number of eggs per gram of liver is significantly reduced. This is the source of significant human pathology from this condition.
Claims
1. An oligonucleotide targeted against a Schistosoma nucleic acid essential for the metabolism, reproduction, or both, of Schistosome worms, which oligonucleotide effectively inhibits expression of the nucleic acid.
2. An oligonucleotide according to claim 1 being from about 20 to about
50 nucleotides long.
3. An oligonucleotide according to claim 2 in which the target nucleic acid codes for an eggshell protein.
4. An oligonucleotide according to claim 3 in which the target is the proline hinge region of the eggshell protein.
5. An oligonucleotide according to claim 3 being SEQ. ID. NO.: 2.
6. An oligonucleotide according to claim 2 having from one to all modified internucleotide linkage, or a 3'- or 5'-capped end, or any combination thereof.
7. An oligonucleotide according to claim 6 wherein the modified internucleotide linkages are chosen from the group consisting of phosphorothioates, phosphorodithioates, carbamates, carbonates, methylphosphonates, and phosphomorpholidates.
8. An oligonucleotide according to claim 6 that is a self-stabilized oligonucleotide.
9. An oligonucleotide according to claim 6 that is a foldback triplex- forming oligonucleotide.
10. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 1.
11. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 2.
12. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 3.
13. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 4.
14. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 5.
15. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 6.
16. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 7.
17. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 8.
18. A method of inhibiting Schistosome metabolism, reproduction, or both, comprising administering an effective amount of an oligonucleotide according to claim 9.
19. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 1 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
20. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 2 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
21. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 3 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
22. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 4 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
23. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 5 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
24. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 6 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
25. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 7 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
26. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 8 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
27. A method of treating schistosomiasis comprising administering to a mammal suffering from schistosomiosis a therapeutically effective amount of an oligonucleotide according to claim 9 and optionally co-administering an effective oligonucleotide uptake-increasing amount of prazinquantel.
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CN110564724A (en) * | 2019-06-11 | 2019-12-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | SiRNA of Schistosoma japonicum SjFrzb2 gene and application thereof |
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WO1993013740A2 (en) * | 1991-12-31 | 1993-07-22 | Worcester Foundation For Experimental Biology | Antiparasitic oligonucleotides active against drug resistant malaria |
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WO1993013740A2 (en) * | 1991-12-31 | 1993-07-22 | Worcester Foundation For Experimental Biology | Antiparasitic oligonucleotides active against drug resistant malaria |
Non-Patent Citations (2)
Title |
---|
Molecular and Biochemical Parasitology, Volume 52, 1992, LI-LY CHEN ET AL, "Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene" * |
Molecular and Cellular Biology, Volume 8, No. 8, August 1988, LIBUSE A. BOBEK ET AL, "Small Gene Family Encoding an Eggshell (Chorion) Protein of the Human Parasite Schistosoma mansoni" * |
Cited By (2)
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CN110564724A (en) * | 2019-06-11 | 2019-12-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | SiRNA of Schistosoma japonicum SjFrzb2 gene and application thereof |
CN110564724B (en) * | 2019-06-11 | 2023-01-06 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | SiRNA of Schistosoma japonicum SjFrzb2 gene and application thereof |
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