Description:FIELD OF THE DISCLOSURE
The present disclosure generally relates to treatment of Rheumatoid Arthritis (RA). In particular, the present disclosure relates to a siRNA sequence for alleviating symptoms of RA. The present disclosure also relates to a method for alleviating symptoms of RA by administering siRNA.
BACKGROUND
Rheumatoid Arthritis (RA) is an autoimmune disease, characterized by a chronic inflammation of various organs, mainly joints along with immune dysregulation. RA synovial fibroblasts are one of the major cell types involved in bone and cartilage destruction. Cytokines, including TNF alpha and IL-6, are involved in the pathogenesis and maintenance of the disease. There is no cure for RA till date.
There are many compositions available in the art to treat RA. However, such medications actually do not treat RA and tend to reduce effects of RA in joints. For example, reducing pain and stiffness in affected joints, preventing joint damage as much as possible, minimizing any disability caused by pain, joint damage, or deformity and reducing the risk of developing associated conditions such as heart disease. Medicines which are advised to ease pain and stiffness include non-steroidal anti-inflammatory drugs (NSAIDs). Role thereof is limited as such drugs do not have any effect on disease progression.
Second line of drugs include 'disease-modifying anti-rheumatic drugs' (DMARDs, such as methotrexate. Such drugs ease symptoms and work by reducing joint inflammation. There are many non-responders to these drugs making it a difficult choice for clinicians.
Next set of drugs called biological DMARDs have recently been developed and they include anti-TNF, IL6 blockers like etanercept, infliximab etc. Though such drugs show promise but long-term benefits thereof are still being evaluated as well as they are very expensive. Use of such drugs against cytokines such as TNF alpha have been shown to be associated with the risk of infection and malignancies.
Along with some serious side effects of the said medications, medication withdrawal after the remission has also not been proven beneficial for the patients, making RA a life-long condition that requires constant and expensive management. Therefore, search for causative agents and better therapeutics based on biologics like antibodies need to be done for developing a potential treatment for RA.
Therefore, there exists a need for developing alternatives to get rid of aforementioned issues.
OBJECTS OF THE EMBODIMENT
One object of the present disclosure is to disclose a siRNA sequence to combat RA.
Another object of the present disclosure is to disclose a siRNA sequence to combat RA by targeting Arl15 gene.
Another object of the present disclosure is to evaluate the efficacy of the siRNA against the Arl15 gene as an effective treatment in an appropriate RA animal model (in vivo).
Another object of the present disclosure is to evaluate if the Arl15 siRNA affects RA pathways involved in the related disease biology.
Another object of the present disclosure is to disclose the siRNA sequence which is non-toxic.
Another object of the present disclosure is to disclose the siRNA sequence which is administered in dosage range of 40-50 µg per kg body weight of a subject.
Another object of the present disclosure is to disclose the siRNA sequence which is administered intravenously.
Another object of the present disclosure is to disclose the siRNA sequence which is non-toxic to the subject at its effective dose.
Another object of the present disclosure is to disclose the siRNA sequence based therapeutic candidate that decreases synovitis formation thereby decreasing the flux of invading synovial fibroblasts.
Another object of the present disclosure is to disclose the siRNA sequence which minimizes bone degradation in RA.
In this respect, before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the disclosure is not limited to in its application to the details of processing and to the arrangements of the components set forth in the following description or illustrated in the drawings. The disclosure is capable of embodiments in addition to those described and of being practised and carried out in various ways. Also, it is to be understood that the phraseology terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
SUMMARY
In an embodiment, the present disclosure discloses a small-interference (si) RNA sequence that targets Arl15 gene to combat RA as set forth in SEQ ID No. 1. The sequence is administered twice in a week intravenously to the subject to combat RA.
In another embodiment, the present disclosure relates to a method to alleviate symptoms of RA by downregulating Arl15 gene by administering siRNA sequence as set forth in SEQ ID No. 1.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects, features, and advantages of the embodiment will be apparent from the following description when read with reference to the accompanying drawings. In the drawings, wherein like reference numerals denote corresponding parts throughout the several views:
Referring to Figure1, shows an effect of Arl15 siRNA on Clinical score on Day 14, in accordance with an illustrative embodiment of a present disclosure;
Referring to Figure 2, shows an effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-17, in accordance with the illustrative embodiment of the present disclosure;
Referring to Figure 3, shows an effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-6, in accordance with the illustrative embodiment of the present disclosure;
Referring to Figure 4, shows an effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines TNF-a, in accordance with the illustrative embodiment of the present disclosure;
Referring to Figure 5, shows an effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-1ß, in accordance with the illustrative embodiment of the present disclosure;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention as hereinbefore described with reference to the accompanying drawings.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the singular forms “a”, “an”, “the” include plural referents unless the context clearly dictates otherwise. Further, the terms “like”, “as such”, “for example”, “including” are meant to introduce examples which further clarify more general subject matter, and should be contemplated for the persons skilled in the art to understand the subject matter.
The present disclosure discloses a small-interference (si) RNA sequence that targets Arl15 gene to combat RA as set forth in SEQ ID No. 1. The sequence is 21 nucleotides long and double-stranded. The sequence is non-toxic upon administration to a subject. The sequence may be administered intravenously and twice in a week to the subject to combat RA. The sequence is administered to the subject to combat RA. The sequence may be administered in the dosage range of 50 µg per kg weight of the subject. The sequence may be administered along with saline. The ARL15 siRNA works independent of TNF pathway and affects RA pathways involved in the related disease biology.
In the embodiment, the subject may be human and a mammal similar to that of human.
In another aspect, the present disclosure also relates to a method to alleviate symptoms of RA by downregulating Arl15 gene by administering siRNA sequence as set forth in SEQ ID No. 1.
Experiment
Example1
DBA1/J female mice obtained for the study were randomly divided into four groups and each of them was marked by ear punch. The groups were termed healthy, vehicle control, Arl15 siRNA and Isotype control. On day 1 after acclimatization of 7 days, all DBA/1 mice except those in the healthy group were immunized by intradermal injections of an emulsion containing 100 µg of immunization grade bovine type II collagen in complete Freund's adjuvant containing heat-killed Mycobacterium butyricum the base of the tail. On day 21st, a booster containing 100 µg bovine CII in incomplete Freund's adjuvant was administered following previously described protocol (ref). The animals were being observed for disease development. Treatment was initiated after 12 days of the booster dose when disease severity of all groups was observed to be maximum. Drugs were administered intraperitoneally (IP) three times in a week. The healthy group did not receive any treatment. To assess the disease characteristics, all the mice were scored for disease extent at 4-5 days intervals for ~20 days following the booster immunization. both paws and joints of fore and hind limbs were scored as follows: 0- Normal; 1- swelling and redness of the paw or one digit; 2- swelling and redness of ankle and wrist; 3- severe redness and swelling of entire paw including digits and 4- severe arthritis of the limb including entire paw and digits.
Example 2
The levels of IL6 and TNFa in the sera samples of the experimental animals were measured using mouse TNFa and IL6 ELISA kits (BMS607HS & KMC0061, Thermo, USA). Briefly, standards in 500-7.8 pg/ml range were prepared using serial dilution of a 1000pg vial. The sera samples were diluted as per manufacturer’s instruction and 100µl of samples along with the diluted standards were loaded in the well of ELISA strips. Biotin conjugate was then added to each of the wells followed by incubation for 2 hours at room temperature. Color was developed in the wells by treatment of streptavidin-HRP followed by TMB substrate solution. OD readings in each of the wells were captured at 450nm on spectrophotometer and the concentrations of cytokines were derived based on the values of standards.
Figure 1 shows effect of Arl15 siRNA on Clinical score on Day 14; Data is represented as Mean ± SEM (n=3-12), *p<0.05, vs Isotope control, IP; $ p<0.05, vs Isotope control, IV; ANOVA followed by Dunnett's test
Figure 2 shows effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-17; Data is represented as Mean ± SEM (n=3-12), *p<0.05, vs Isotope control, IP; ANOVA followed by Dunnett’s multiple comparison test
Figure3 shows effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-6; Data is represented as Mean ± SEM (n=3-12), *p<0.05, vs Isotope control, IP; ANOVA followed by Dunnett’s multiple comparison test
Figure 4 shows effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines TNF-a; Data is represented as Mean ± SEM (n=3-12), *p<0.05, vs Isotope control, IP; ANOVA followed by Dunnett’s multiple comparison test
Figure 5 shows effect of Arl15 siRNA on Serum Pro-inflammatory Cytokines IL-1ß; Data is represented as Mean ± SEM (n=3-12), *p<0.05, vs Isotope control, IP; ANOVA followed by Dunnett’s multiple comparison test.
The foregoing descriptions of exemplary embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to best explain the principles of the disclosure and its practical application, to thereby enable others skilled in the art to best utilize the disclosure and various embodiments with various modifications as are suited to the particular use contemplated.

, Claims:We Claim
1. A small-interference (si) RNA sequence that targets Arl15 gene to combat RA as set forth in SEQ ID No. 1.
2. The siRNA sequence as claimed in claim 1, wherein the sequence is 21 nucleotides long.
3. The siRNA sequence as claimed in claim 1, wherein the sequence is double-stranded.
4. The siRNA sequence as claimed in claim 1, wherein the sequence is non-toxic upon administration to a subject.
5. The siRNA sequence as claimed in claim 1, wherein the sequence is administered intravenously to the subject to combat RA.
6. The siRNA sequence as claimed in claim 1, wherein the sequence is administered twice in a week to the subject to combat RA.
7. The siRNA sequence as claimed in claim 1, wherein the sequence is administered in the dosage range of 50 µg per kg weight of the subject.
8. The siRNA sequence as claimed in claim 1, wherein the subject is human and a mammal similar to that of human.
9. A method to alleviate symptoms of RA comprising downregulating Arl15 gene by administering siRNA sequence as set forth in SEQ ID No. 1.
10. The method as claimed in claim 9, wherein the siRNA sequence is administered intravenously in the dosage range of 50 µg per kg weight of the subject to the subject to combat RA.