1. A method to control geo-helminth parasite infection by probiotic mediated delivery of parasite target specific RNAi comprising of: i. Coproculturing of geo-helminths using a petri dish method ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite culture, iii. Designing DsRNA expression vectors cloning the essential target genes, and pairing with probiotic bacterial vectors by transformation, iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii), v. LAB colonization the gut of subject and subsequent colonization of the parasite gut during their feeding cycle resulting in internalization of the target DsRNA in the parasite gut leading to knock down of target essential genes by RNAi molecules.

2. The method to control geo-helminth parasite infection as claimed in claim 1, wherein the said essential target genes are selected from astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and, the said target enzymes responsible for metabolism and energy production are selected from malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome c oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde 3 Ph dehydrogenase, glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and phospholipase or a combination thereof.

3. The method to control geo helminth parasite infection as claimed in claim 1, wherein the said DsRNA vector is created by placing the said essential target gene between at least two opposable promoters selected from lactate dehydrogenase (Pldh), PslpA, Plaf, Ptac and P1 in L. acidophilous, P Dala, P ldh, Ppbg, Pfgs, P-HicDH, Pamy, P ccpA and P dlt P of L. casei, PxylA and Pxyl R, Prep of L. pentosus, PrepPC30il and Prep8014-2 of L. plantarum, Pusp 45 of L. lactis, PsapA of L. sakei, Pcbh of B.Subtilis , or a combination thereof, with complimentary strand having multiple cloning sites and at least two copies of restriction enzymes inserted in reverse directions on the said complimentary strand.

4. The method to control geo-helminth parasite infection as claimed in claim 1 wherein the said genetically engineered probiotic is selected from a group of different species of Lactic Acid Bacterium (LAB), Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus fermentum, Lactobacillus sakei, Lactococcus lactis, or Bacillus sp and/or a combination thereof.

5. The method to control geo-helminth parasite infection as claimed in claim 1 wherein the said genetically engineered probiotic can be used as food supplements in the endemic areas or as bio therapeutic in the form of tablet or capsules for direct oral gut delivery for human and veterinary application or as a combination therapy along with the deworming drugs to prevent re-infection, and reduce the resistance of the deworming drugs and/or a combination thereof. , Description:FIELD OF INVENTION The present invention relates to the method of controlling parasitic helminth infection using probiotic mediated RNA interference (RNAi). More particularly, the present invention relates to genetic constructs of double stranded RNA interference (dsRNAi) vector containing parasite specific targets paired with probiotic bacteria as vehicles, for delivery to the helminth gut, where the target helminth RNA complementary to the parasite mRNA sequence knocks down the gene by RNAi and blocks larval developmental process. BACKGROUND OF THE INVENTION Soil-transmitted helminth infections, commonly known as geo-helminth infections are among the most common parasitic infections that affect humans, plants and other animals of economic importance. In the case of humans, eggs present in human faeces, that contaminate the soil, water bodies and vegetation in areas with poor sanitation facilities, transmit the geo-helminths. The adult form of geo-helminths is the most harmful life cycle stage, causing soil-transmitted helminthiasis (STH). Adult worms live in the intestine in hundreds where they feed on the blood and produce thousands of eggs each day. These eggs give rise to the larval stage of the geo-helminths, the infective form and are internalized in the host from the soil through contaminated food or through direct penetration from cracks in the skin where they undergo tissue-migratory stages and invade vital organs such as lungs and liver. Thus, the disease manifestations can be both local and systemic. Geo-helminth parasites infect more than two billion humans particularly in poverty-stricken rural areas of in developing regions of Asia, sub Saharan Africa and the Americas. Over 80,000 species of helminths have been described, of which over 15,000 are parasitic. The parasitic worms that infect humans are Roundworms, Flatworms, Whipworms, Hookworms, Threadworms and Pinworms. The geo-helminth parasites of humans cause a variety of disease conditions and symptoms, ranging from lack of energy and vigor to blindness, anaemia, intestinal manifestations (diarrhea and abdominal pain), and malformations. Control of geo-helminth parasites presently relies primarily on the use of anthelmintic drugs combined with sanitation and pasture management. Such techniques have a number of drawbacks such as frequent administration of the drugs, which leads to the development of resistant strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the subjects being treated. Due to buildup of drug resistance larger doses are required leading to more severe side effects. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection itself. An alternative method to prevent geo-helminth infection includes administering a vaccine against a specific geo-helminth. Although the development of vaccines based on prominent antigen specific to the geo-helminths have been developed, the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting the subject from infection. In addition, application of new vaccines has resulted in urticarial reactions and hypersensitivity due to elevated levels of IgE because of previous geo-helminth infections. The geo-helminth are masters of immunomodulation that makes them difficult to target by vaccines and are also difficult to treat by drugs as they have complicated life cycles in various organs of the subject. In such a scenario, there is a need to develop new methods for control. As illustrated in U.S. Pat. No. 6,506,559, RNA interference (RNAi), also referred to as gene silencing, has been proposed as a method for controlling geo-helminths. When double-stranded RNA (dsRNA) corresponding to the sequence of a target gene is introduced into a cell, expression from the target gene is inhibited. Further, it demonstrates the effectiveness of RNAi against the prominent genes in a model laboratory nematode, C. elegans. Due to the labile nature of the RNAi molecules, the difficulty of delivering dsRNA to target cells has impeded the rapid expansion of RNAi based therapies. The use of vectors has been exploited to deliver RNAi to the specific target and improvements can be made to render RNAi therapy more effective. Probiotics are live bacteria which when administered in adequate amounts to the subject’s digestive system confer a health benefits. The role of probiotics in increasing the intestinal immunity and control of enteric organisms by the principle of mutual exclusion has been widely documented. Genetically engineered probiotics have also been used to deliver therapeutic immunomodulatory molecules and neutralizing antibodies to the site of action. The present invention proposes a method of controlling geo-helminth infection using genetically engineered probiotics containing inhibitory molecules targeting them in the gut stage of their life cycle. These probiotics colonizing the gut will clear out existing infections and prevent re-infection from the environmental source by RNA interference mediated helminth control. Continuous in situ production of the RNAi molecules, will have prophylactic effect and keep the gut sanitized and will find applications in both human and veterinary health. OBJECTIVES OF THE INVENTION The primary objective of the present invention is a method for developing potent anti-helminth molecules using RNA interference (RNAi) and probiotic bacteria as vehicles for targeted delivery of these molecules to the parasite gut. Another object of the present invention is to provide a targeted delivery of the RNAi vectors to the geo-helminth gut by using genetically engineered probiotic bacteria as delivery vehicle. Another object of the present invention is to develop genetically engineered probiotics continuously expressing parasite target specific RNAi molecules. Another object of the present invention is to target the genes present in geo-helminth parasite like the ones responsible for moulting and development, attachment, nutrient assimilation, locomotion and neuromuscular functions by specific selection of the targets to be cloned in the RNAi vectors. Yet another object of the present invention is monitoring the knockdown of the essential genes by RNAi in geo-helminth, and demonstrating its phenotypic effects on the worms by conducting in vitro and in vivo studies. Yet another object of the present invention is to provide a method of controlling and preventing geo-helminth infection by eliminating the worms and sanitizing the gut of the subjects. Yet another object of the present invention is to provide a method of controlling helminth infection having minimum off target effects in their hosts. Yet another object of the present invention is to provide a cost effective and error-free treatment for helminth infection. Yet another object of the present invention is to ameliorate one or more problems of the prior art and provide a useful alternative. SUMMARY OF THE INVENTION The embodiments of the present invention described herein below are illustrative only and should not be construed to limit the scope of the present invention in any manner. Unless stated to the contrary, any use of the words such as "including”, "containing”, "comprising", "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but can be implemented in various combinations. The term "geo-helminth" is used to denote the parasite that resides in the gut of the subject. The term “subject” used herein means human or veterinary animal infected with geo-helminth. The term "probiotic" denotes the bacterium that delivers the RNAi vector containing the DsRNA producing target to the host. The term “host” used herein means a geo-helminth parasite that resides in subject's gut and acts as host for probiotic bacteria. The present invention describes a method of controlling geo-helminth infection using genetically engineered probiotics containing gene inhibitory RNA interference (RNAi) vector. In this method, a RNA interference (RNAi) vector is developed that can be delivered by Lactic Acid Bacteria (also referred as LAB) to the subject i.e. human/animal gut. The essential genes responsible for the development of geo-helminth larval stage or maintenance of the adult stage are cloned in the RNAi vector. This RNAi vector targets the geo-helminth in their gut stage of life cycle. These RNAi vectors are transformed into probiotic commensal bacteria i.e. Lactic Acid Bacteria (LAB), which are fed to the host and colonizes the gut from where they deliver the targeted RNAi therapy into the helminth gut in human or veterinary subject. This RNAi LAB strain can be used as a probiotic to control geo-helminths in endemic areas, prevent reinfections or can be used as a passive vaccine. According to the present invention, the method of controlling geo-helminth infection using RNAi comprises of: i. Coproculturing of the geo-helminths using a petri dish method; ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite culture, iii. Designing DsRNA expression vectors containing the isolated essential target genes with suitable constitutive promoters, and pairing them with the probiotic bacteria. iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii), v. Colonization of the gut of subject with the probiotics, which then in turn are internalized in the parasite gut during their feeding cycle resulting in release of the RNAi molecules in the parasite gut and knock down of target genes. The knockdown of essential target genes/enzymes, responsible for developmental and energy metabolism, nutrient assimilation or tissue penetration of geo-helminth parasite by RNAi results in anti-parasitic effects. The said essential target genes includes astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and the said essential target enzymes includes malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome C oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and/or phospholipase. The said RNAi vector can be either single hairpin RNA (hairpin of about 20 bases) or double stranded RNA (DsRNA) of 100-200 bp against the target sequences. The said double stranded RNA (DsRNA) are used in the present invention as DsRNA are very stable and shows minimal off target activity as they have been selected in such a way so that they have no homology with their human counterparts and will easily be destroyed by immune response if taken up by mammalian cells accidently. The said probiotic bacteria are selected from different species Lactobacilli preferably Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis or Bacillus sp. Further, the DsRNAi expression vectors are created using at least two opposite constitutive promoters cloned in a reversible direction flanking the target gene. These promotors are selected from lactate dehydrogenase (Pldh) as in the present disclosure or PslpA, Plaf, Ptac and P1 in L. acidophilous, PDala, Pldh, Ppbg, Pfgs, P-HicDH, Pamy , PccpA and Pdlt P of L. casei, PxylA and Pxyl R, Prep of L. pentosus, PrepPC30il and Prep8014-2 of L. plantarum, Pusp 45 of L. lactis. PsapA of L.sakei, Pcbh of B.subtilis or a combination thereof. The complimentary strands of the DsRNA has multiple cloning sites for insertion of the parasite targets and consists of at least two copies of restriction enzymes inserted in reverse directions. The said RNAi expression vector containing probiotic is orally administered to the infected subject and on reaching the subject’s gut colonizes there, which is further engulfed by the geo-helminth parasite present in the subject’s gut. The said probiotic upon degradation in the geo-helminth parasite’s gut releases the said DsRNA, which is taken up by the intestinal cells and processed to siRNA that exerts the RNAi mechanism by formation of RISC complex as illustrated in Figure 4,. RNA interference is carried out when the said siRNA binds with argonaute protein molecule to form RNA induced silencing complex (AGO/RISC) by recognizing complementary messenger RNA (mRNA) and cleaving them. The said RNAi mechanism enables the knockdown of the essential target genes by blocking the production of the said essential enzymes responsible in various developmental and energy metabolism pathways eventually leading to killing of geo-helminth. The said dsRNA expression vector suitable for cloning includes different target genes against variety of helminths namely Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Flatworms (family Schistosoma mansoni and Schistosoma japonicum), Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis, and pinworms ( family Enterobius sp). The said genetically engineered probiotic can be used as both food supplements, and as bio therapeutic form of tablet or capsules for direct oral gut delivery for human and veterinary application. The said genetically engineered probiotic can be used as a combination therapy along with the deworming drugs to prevent re-infection, and reduce the dose of the deworming drugs and subsequent resistance along with the gut colonizing probiotic benefits. This method of geo-helminth control results in an economical, safe and sustainable anti helminthic therapeutic strategy that augments current control. These curative and prophylactic methods of controlling helminth infection confers continuous protection against new helminth infection as well as eliminates the established adult worms, sanitizes the gut and provides all round immunity against geo-helminth infections. Other objects, features and advantages of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings wherein like characters of reference designate corresponding parts throughout the several views. BRIEF DESCRIPTION OF THE DRAWINGS A complete understanding of the present invention may be by reference to the following detailed description which taken in conjugation with the accompanying drawings. The accompanying drawings, which is incorporated into and constitutes a part of the specification, illustrates one or more embodiments of the present invention and, together with the detailed description, it serves to explain the principles and implementations of the invention. Figure 1: illustrates the flowchart of the method of controlling helminth infection using genetically engineered probiotics containing gene inhibiting RNA interference (RNAi) vector targeting the helminth Figure 2: illustrates the construction of vector for generation of dsRNA, Figure 3(a): illustrates schematic diagram of the dsRNAi vector and the sequence with the target gene (astacin) inserted between opposable ldh promoters, Figure 3(b): illustrates The results of a PCR reaction using a single ldh primer in the genetically engineered probiotic giving a prominent band indicating the cloning of target gene in between the same opposable promoters, Figure 3(c) illustrates electrophoretic representation of DsRNA astacin fragments in in the genetically engineered probiotic an agarose gel. The fragment is present in the transformed bacteria only. Figure 4: illustrates diagrammatic representation of the mechanism of action effecting target gene knockdown by the genetically engineered probiotic in the geo-helminth gut, Figure 5(a): illustrates electrophoretic representation of a PCR isolation of cDNA of the targets cathepsin D, astacin and FAR of A caninum, Figure 5(b): illustrates electrophoretic representation of a PCR isolation of FAR a target of the human hookworm A. duodenalis, Figure 6(a): illustrates microscopic image of moulting and transformation of the larvae to the feeding stage in induced hookworms, Figure 6(b): illustrates fluorescent microscopic image of FITC labelled LAB internalized by the feeding stages of the hookworm larvae, Figure 7: illustrates live dead assay using hookworm larvae fed with LAB, where C is untreated worms, and M is worms fed with dsRNA producing bacteria, Ll is Lactococcus lactis and Ds is double stranded RNA produced in vitro. Figure 8: illustrates skin migration assay using hookworm larvae fed with DsRNAi LAB, where C is untreated worms, and M is worms fed with dsRNA producing bacteria, Ll is Lactococcus lactis and Ds is double stranded RNA produced in vitro. Figure 9: illustrates electrophoretic representation of knockdown of astacin gene by reverse transcription PCR reaction, where M is worms fed with DsRNAi LAB, P is worms fed with unmodified LAB, The right hand panel is the 60S rRNA control. Figure 10: illustrates tissue migration of hookworm larvae in unfed mice, LAB RNAi fed and in vitro produced dsRNA fed mice 24 hours and 72 hours post challenge, Figure 11: Liver and kidney function tests in mice in normal (no challenge), control (challenge with unfed worms), and after challenge with LAB (LlM) and dsRNA (DsT) fed worms. DETAILED DESCRIPTION OF THE INVENTION The foregoing objects of the invention are accomplished and the problems and shortcomings associated with prior art techniques and approaches are overcome by the present invention described in the present embodiment. Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Before the present invention is described, it is to be understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, it is to be understood that the present invention is not limited to the methodologies and materials similar, equivalent to those described herein can be used in the practice, or testing of the present invention, the preferred methods and materials are described, as these may vary within the specification indicated. Unless stated to the contrary, any use of the words such as "including," "containing," "comprising," "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose of illustration rather than limitation of the invention as set forth the appended claims. Further, the terms disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms. The present invention describes a method of controlling geo-helminth infection using genetically engineered probiotics containing gene inhibitory RNA interference (RNAi) vector. In this method, a RNA interference (RNAi) vector is developed that can be delivered by Lactic Acid Bacteria (LAB) to the subject i.e. human/animal gut. The essential genes responsible for the development of geo-helminth larval stage or those responsible for food assimilation and energy metabolism are cloned in the RNAi vector. These RNAi vectors are transformed into probiotic commensal bacteria i.e. Lactic Acid Bacteria (LAB), which are further used as delivery vehicles to deliver the targeted RNAi vector into the helminth gut via human or veterinary subject from where they are picked up by the hookworms cohabiting the same niche. This RNAi LAB strain can be used as a probiotic to control geo-helminths in endemic areas, either by itself or with existing drugs, prevent reinfections and can be used as a passive vaccine. According to the present invention, the method of controlling geo-helminth infection using RNAi comprises of: i. Coproculturing the geo-helminths using a petri dish method; ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite , iii. Designing DsRNA expression vectors and cloning the isolated essential target genes, and pairing them with probiotic bacterial vectors, iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii), v. LAB colonization the gut of subject and subsequent colonization the parasite gut during their feeding cycle resulting in internalization of the target DsRNA in the parasite gut leading to knock down of target l genes by RNAi. Figure 1 illustrates the method for controlling helminth infection. It illustrates various steps for controlling the helminth infection, such as step of isolation of the essential target genes from geo helminth parasite and the designing of DsRNA expression vectors and inserting them in probiotics by transformation. Further, it illustrates that the genetically engineered probiotics expressing the RNAi molecules, when administered with the feed, will colonize the subject’s gut. The parasitic helminths present in the subject's gut feed on these probiotics that are degraded, resulting in delivering of the RNAi molecules to the helminth gut, which when taken up by the parasite intestinal cells transforms the DsRNA to siRNA that targets the complimentary mRNA, like the ones responsible for nutrient assimilation, development and metabolic pathways and knocks them out, debilitating the parasites thereby breaking their lifecycle. According to the one of the embodiments of the present invention, the initial step includes coproculturing the helminth by petri dish method. For this, helminth's eggs removed from the subject's fecal matter are hatched in the laboratory by a petri-dish culture method. The said method for growth and culture of human and veterinary helminth involves mixing the fecal sample with 3 times the amount of sterile sand, and incubating in a moist damp place for 10-14 days followed by isolation of the worms that hatch out and mixing 2 ml of 3.3% agarose at 37-40°C with 4 ml of the parasite containing solution obtained from coproculture, pouring them on a petriplate and topping it with 4 ml. of a buffer solution and incubating them at 37°C for 1 hr. The contaminants are stuck in the agar and the larvae rise up and collect in the buffer layer from where they are isolated for further use. Another embodiment of the present invention describes how the essential target genes/enzymes are selected from the genes which are responsible for developmental and energy metabolism, or gut digestion or tissue penetration of geo-helminth parasite. Knockdown of such target genes/enzymes using RNAi results in anti-parasitic effects. The said essential target genes selected for knockdown by detailed bioinformatics study includes astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and the said essential target enzymes includes malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome C oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and/or phospholipase. Further, the selection of the target genes/enzymes is crucial step in the present invention. The knockdown effects of the target gene/enzyme are immediate and detectable by in vitro assays. The target sites selected for the knockdown of essential target gene/enzyme are preferably from beta tubulin, ligand gated Ca+ and Na+ ion channels, glutamate gated chloride channels, gut neuropeptides, and G protein coupled receptors. Further, it may also include signaling pathways like Wnt/Frizzled signaling, FaRPergic signaling, FMRF amide like peptide signaling, and allatostatin like neuropeptide that are present in a majority of geo-helminths. From these RNAi target genes, complimentary sequences are isolated from helminth for the said essential parasite genes and cDNA are prepared using conventional reverse transcriptase polymerase chain reaction (RT PCR). Another embodiment of the present invention describes, the said RNAi vector used in the present invention. The RNAi can be either single hairpin RNA (hairpin of about 20 bases) or double stranded RNA (DsRNA) of 100-200 bp against the target sequences. The said double stranded RNA (DsRNA) are used in the present invention as DsRNA are very stable and shows minimal off target activity as they have been selected from areas having no homology to the human genes and can be easily destroyed by immune response if taken up by mammalian cells accidently. Further, the lactate dehydrogenase promoter (Pldh) was used for the construction of the DsRNA vector. Since lactate dehyrogenase is a predominant secretory enzyme in lactic acid bacteria, this vector is expected to work in the other lactobacilli as well. However, other promoters can be used as well and DsRNA will be produced if they are cloned in a reversible direction flanking the target gene. The DsRNAi expression vectors are created using at least two opposite constitutive promoters cloned in a reversible direction flanking the target gene. Other promotors are selected from PslpA, Plaf, Ptac and P1 in L. acidophilous, PDala, , Ppbg,P fgs, PD-HicDH, Pamy , PccpA and PdltP of L. casei, PxylA and PxylR, Prep p353-2 of L. pentosus, Prep PC30il and Prep 8014-2 of L. plantarum, Pusp45 of L. lactis. PsapA of L. sakei, Pcbh of B.subtilis or a combination thereof can be used in LAB as well. In addition to these, synthetic promoters can be generated using the consensus sequences of the promoters. Depending on the requirement like expression levels and suitability of the promoters in different hosts, these promoters can be used interchangeably in the LAB as well. The complimentary strands of the DsRNA have multiple cloning sites and consist of at least two copies of restriction enzymes inserted in reverse directions for insertion of the target between the opposable promoters. Further, the said RNAi expression vector containing genetically engineered probiotic is orally administered to the infected subject and on reaching the subject’s gut, the said genetically engineered probiotic is engulfed by the geo-helminth parasite present in the subject’s gut as illustrated in Figure 4. The said probiotic upon degradation in the geo-helminth parasite’s gut releases the said DsRNA, which is taken up by receptor mediated endocytosis in the intestinal cells and further processed to siRNA that exerts the RNAi mechanism by formation of RISC complex; The RNA interference is carried out when the said siRNA binds with argonaut protein molecule to form RNA induced silencing complex (AGO/RISC) by recognizing complementary messenger RNA (mRNA) and cleaving them. The said RNAi mechanism enables the knockdown of the said essential target genes thereby blocking the production of the said essential target enzymes eventually leading to killing of the parasite. Another aspect of the present invention is the design of dsRNA expression vector and its pairing with probiotic LAB strains. As illustrated in Figure 2, the dsRNA producing probiotic LAB vector is created by placing the target gene under two opposable constitutive promoters (P) in complimentary strands with multiple cloning sites (1/2/3) inserted between them. The restriction enzymes (RE) namely Eco RI/Sac I/Sma I (1/2/3) on both sides are inserted in reverse directions to prevent re-ligation. Cloning of the target genes is accomplished by a single restriction digestion with any of the three said restriction enzymes, followed by ligation of the target gene flanked by a single inverted restriction enzyme site in a reversed orientation. Inversion of the restriction enzyme site prevents recircularization of the vector or the target during the ligation reaction. The target can be inserted in either direction but that would not have any effect on the production of the dsRNA as promoters are present in both the directions and in frame cloning is not required, as translation does not take place. Numerous origins of replications exist for different LAB species namely Rep A, Rep C, Rep D, Rep E, Rep F and Rep G. Not all the origin of replications may be necessary for the maintenance of the plasmids in the LAB. Rep D, E, F and G that has a wider host range and works in different species of LAB were selected for designing of the DsRNAi vector. The selection cassette used is Ery, a gene that confers resistance to erythromycin, but a Choramphenicol Acetyltransferase gene (Cm/CAT) that confers resistance to Chloramphenicol can also be used as well. For example, as Figure 3 (a) illustrates the schematic diagram of the dsRNA vector and the sequence with the target gene (astacin) inserted between the opposable constitutive Lactate Dehydrogenase (LDH) promoters. PCR with a single LDH primer gave a prominent band indicating the target gene had been cloned in between the opposable promoters and the DsRNA astacin fragment is clearly visible in the DNA gel as illustrated in Figure 3(b) and 3(c). The said dsRNA expression vector suitable for cloning includes different target genes against variety of helminths. The medically important helminths where this will be applicable are Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Flatworms (family Schistosoma mansoni and Schistosoma japonicum), Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis, and Pinworms (family Enterobius sp) or a combination thereof. These vectors can be paired with a suitable probiotic LAB strain for delivery and continuous production of the RNAi molecules in the gut of the helminth. Another embodiment of the present invention describes, the said probiotic bacteria of the present invention are selected from different species of Lactobacilli preferably Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis or Bacillus sp. According to another embodiment of the present invention, the genetically engineered probiotics that produce DsRNAi molecules in situ can be used as value added food supplements that can be administered to subject in endemic areas to control helminth infestation. In addition, the genetically engineered probiotics can be used to produce dsRNA that can be purified and used as bio-therapeutic molecules that can be formulated as a tablet or capsule for direct oral delivery to the gut. According to another embodiment of the present invention, the genetically engineered probiotics can be used either singly or as a passive vaccine for human and veterinary applications, in helminth endemic areas. If administered immediately after the use of deworming drugs such as Albendazole, the probiotics will colonize the subject gut and prevent reinfection by the helminth that have survived in the environment. If used as a combination therapy with the existing deworming drugs, a reduction in the dosage of deworming drug can brought about, decreasing the ability of the helminth to develop drug resistance. Further, the Lactobacillus vectors have a gram positive origin of replication and is expected to work in other species of Lactobacilli commonly used as probiotics like Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis as well as Bacillus sp. These strains can be used to produce the value added probiotics that can be used as food supplements in endemic areas to control geo-helminth infestation. They can be used to produce double stranded RNA, be purified and used as bio-therapeutic molecules that can be formulated as a tablet or capsule for direct oral delivery to the gut. ADVANTAGES

1. The RNAi vector so constructed is a replacement vector suitable for cloning different target genes against other geohelminth parasites. This vector can be used against other common soil transmitted parasites like Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis . This vector can be paired with a suitable Lactobacillus strain by transformation for delivery and continuous production of the RNAi molecules in the gut.

2. Apart for use in humans this invention can have wide applications in the area of animal health and can be used to control parasite infestations reducing the need for small molecule drugs like Albendazole and Ivermectin against which resistance can be generated quickly by the parasites. This invention can be tailored for a specific group of animals by selecting an appropriate animal specific target and pairing it with a suitable probiotic and used singly or in combination with other antiparasitic molecules, like Ivermectin for effective parasite control in pets, farm animals and poultry.

3. The anti-parasitic value added probiotics can be used either singly as a passive vaccine for human applications, particularly for children in hookworm endemic areas. If used immediately after the application of an antiparasitic molecule like Albendazole, the probiotics will colonize the gut and prevent reinfection by the parasite that has survived in the soil. If used for a sufficiently long period, a combination therapy with the existing antiparasitics like Albendazole and the probiotics can wipe out all traces of the parasites from endemic areas.

5. The value added probiotic will also result in regulating the dose of Albendazole to children, many of who are affected by the extreme toxicity of the molecule and have to be hospitalized. With a combination therapy and a reduction of the dose of Albendazole, the ability of the parasites to develop drug resistance to the small molecule drugs will be considerably reduced.

6. Targeted delivery of the RNAi expression vector at the site of action utilizing genetically engineered probiotics: The DsRNA is released in the geo-helminth gut and not the subject’s gut so the off target effects if any are minimized and the RNAi expression vector containing the target is enclosed by live genetically engineered probiotics so their production can be modulated specifically at the site of action by using specific promoters that will be inoperative once the probiotics leave the site of action. EXAMPLES The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations, which are evident because of the teaching provided herein. Example 1: Growth and culture of hookworm Ancylostoma duodenale and Ancylostoma caninum by coproculture technique: Method of controlling Hookworm infection by LAB mediated RNA interference was carried out on two different species of hook worms namely Ancylostoma duodenale (affecting humans) and Ancylostoma caninum (affecting canines). The method comprises of the steps of: Hook worm eggs were extracted out of subject fecal matter and hatched in the laboratory by novel petri-dish culture method. The eggs hatch into the L1 larvae in 4-5 days and are transformed to L3 larvae rapidly in 3 - 4 days following which they are collected and maintained in RPMI 1640 medium or BU buffer with antibiotics (Amphotericin B, Penicillin, Streptomycin). Example 2: Isolation of RNAi targets from the hookworms Three targets were shortlisted for studying Ancylostoma caninum and Ancylostoma duodenalis: i. Astacin: it is a zinc metalloprotease responsible for tissue migration, moulting, predominantly found in larval stages, and is induced prior to migration. The homologue of astacin is absent in humans and so the chance of off target activity is expected to be low; ii. Fatty Acid Receptor (FAR): A nutrient sensor for regulating energy metabolism, found in adults; and iii. Cathepsin D: Responsible for hemoglobin digestion, found in adults. 200- 400 bp fragments of the cDNA of Cathepsin D, Astacin and FAR of A caninum and FAR of A. duodenalis were isolated by RT-PCR as illustrated in Figures 5(a) and (b). Example 3: Design of DsRNA expression vectors and paired probiotic bacterial strains LAB is known to colonize the gut and are used as probiotics. A vector backbone containing the gram-positive origin of Rep D, E, F and G was developed that would work in both Lactococcus sp and Lactobacillus sp and used to generate dsRNA as illustrated in Figure 3 (a). A dsRNA producing Lactococcus lactis strain was created by placing the astacin gene under two opposable constitutive Lactate Dehydrogenase (LDH) promoters in the vector and transforming the Lactococcus strain with it. PCR with a single LDH primer gave a prominent band indicating the target gene had been cloned in between the opposable promoters as illustrated in Figure 3 (b) and the Ds astacin RNA fragment in a DNA gel was clearly visible as illustrated in Figure 3 (c). Example 4: In vitro studies to establish RNAi activity in Ancylostoma caninum by feeding: Feeding stimulation and moulting studies were conducted with FITC labelled LAB. The Ancylostoma L3 larval stage is non-feeding, so were stimulated to the feeding L4 stage by the addition of 5 % dog serum and reduced glutathione. As illustrated in Figure 6(a) within 2 hours after stimulation, moulting was seen in the larvae along with the development of mouthparts and buccal funnel following which the larvae are converted to the feeding stage and were ready for tissue migration. Copious amounts of the protease astacin are secreted during this stage that help shed cuticle, penetrate the gut musculature and helps in subsequent tissue migration. These stimulated L3 larvae were fed with the LAB containing the astacin DsRNA. The LAB was internalized in the hookworm tissues and processed to siRNA, which results in knockdown of the levels of astacin by RNAi mechanism and stop moulting and larval migration through the tissues. The induced larvae were incubated with FITC labelled LAB overnight and observed under a fluorescent microscope as illustrated in Figure 6(b). The bacteria were internalized and visible in the gut as bright fluorescent spots in the feeding induced Ancylostoma. The combination of moulting and feeding indicated that the induction had successfully transformed the hookworm to the next larval stage. Example 5: In vitro larval motility and mortality studies In vitro larval motility and mortality studies were carried out to observe the effect of the RNAi knockdown of essential genes. A phenotypic behavioral effect was observed resulting in sickness and eventual death of the worms. The worms were fed with the RNAi LAB and motility assays performed after 72 hours. Fifty worms were pre-fed with astacin DsRNA producing LAB and were analyzed for thrashing movement (motility) by adding lukewarm water at 37°C. The control worms without any treatment immediately started trashing but the bacteria fed worms were sick and did not show a significant thrashing movement. These sick worms started dying and breaking apart as illustrated in Figure 7 after another 24 hours, indicating that feeding the double stranded RNA had the desired anti-parasitic effect. Example 6: Skin migration assays Skin migration assays were performed to study larval penetration into the subject body through the skin. Induced Ancylostoma larvae rapidly penetrate the skin and migrate through the tissues eventually reaching the lungs from where they are coughed into the alimentary canal and finally settle in the intestines as egg laying adults. The skin migration assay was performed using chicken skin (since fresh dog skin is unavailable in India) that was stretched out on a small culture plate filled with BU buffer (Na2HPO4 7%, KH2PO4 3%, NaCl 4% pH 6.8 ) and 200 µl of dog serum was added on the skin (to simulate conditions of the dog gut). After 30 mins, Ancylostoma L3 larvae pre-fed with DsRNA producing bacteria, were carefully placed and incubated overnight at room temperature. Next day the larvae that migrated through the skin and fell in the buffer underneath were counted under the microscope by briefly spinning them down to concentrate it. No difference in the migration patterns was seen in the control worms whereas in the DsRNA treated worms more than 80% of the worms failed to migrate possibly because Astacin was knocked out and failed to penetrate through the skin as illustrated in Figure 8. Example 7: Detection of target RNA levels post RNA interference: Establishment of the DsRNA and its effect in knocking down essential genes was done in two sequential steps: a) detection of DsRNA in bacteria after induction; and, b) effect of knockdown of the target gene in hookworms by Reverse Transcription PCR. For detection of DsRNA, the extracted total RNA was run in a DNA gel after digestion with RNAseA to remove the single stranded RNA. A band at 200 bp is clearly visible in LAB that harbors the RNAi plasmid as illustrated in Figure 3(c). Sequencing of the plasmids confirmed the creation of a two novel DsRNA vectors. The opposable promoters (ldh in LAB) flanking the multiple cloning site were placed on a stable backbone containing a selection marker. The knockdown effect of the target gene was seen using RT PCR; 72 hours post feeding of the in vitro transcribed dsRNA. The astacin gene was knocked down considerably as illustrated in Figure 9. Example 8: In vivo studies with mice challenged with RNAi bacteria fed Ancylostoma: Swiss albino mice were used to study the tissue migration patterns of hookworms fed with Ds astacin RNAi (herein referred as DsT) produced in vitro and Ds astacin RNAi producing LAB (referred to as LlM). The mice were challenged by a bolus dose with Ancylostoma pre fed 48 hours earlier with the LlM. 500 pre-fed worms were fed to freshly weaned 3 week old mice andthe migration of the worms was observed by necroscopy after dissecting the mice and collecting the organs, intestines and liver (24hours) and lungs and forelimbs (72hours). The organs were tied up in cheesecloth to prevent disintegration and immersed in beakers containing artificial gastric juice (5gm pepsin + 0.7 ml conc. HCl + 1000ml D/W + 8.5gm NaCl) at 37°C for 3 hours. The larvae are released from the organs and settle at the bottom of beaker, after concentrating the worms by a brief spin, the worms were counted under a microscope. After 24 hours more number of worms were recovered from stomach and liver of the control mice as compared to the mice fed with worms treated with the in vitro produced DsRNA (DsT) and DsRNA producing LAB ( LlM) as illustrated in Figure 10. After another 48 hours during which the worms had a chance of migrating further, the lungs and forelimbs were analyzed and the same trend was seen. This indicated that the DsRNA produced in vitro or delivered through the LAB had the desired phenotypic effect, i.e. inhibiting tissue migration by knocking down of the proteases required for burrowing through the skin. Immuno-histological studies also showed the presence of dead worms in the organs (data not shown). The difference of the worm migration to the vital organs was also reflected in the organ damage. Biochemical assays like the liver and kidney function tests were performed to determine the extent of damage caused by hookworms pre and post RNAi treatment. Liver and kidney damage was indicated by an increase in the levels of SGPT (Serum Glutamic-Pyruvic Transaminase), and BUN (Blood Urea Nitrogen) respectively that are release in the blood from the damaged organs. As illustrated in Figure 11 considerable organ damage was observed by a rise in the level of the enzymes in the control mice that were challenged with the untreated hookworms as compared to normal mice that were challenged with the worms pre-fed with the genetically modified LAB or the astacin produced in vitro. While considerable emphasis has been placed herein on the specific elements of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Claims:1. A method to control geo-helminth parasite infection by probiotic mediated delivery of parasite target specific RNAi comprising of:
i. Coproculturing of geo-helminths using a petri dish method
ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite culture,
iii. Designing DsRNA expression vectors cloning the essential target genes, and pairing with probiotic bacterial vectors by transformation,
iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii),
v. LAB colonization the gut of subject and subsequent colonization of the parasite gut during their feeding cycle resulting in internalization of the target DsRNA in the parasite gut leading to knock down of target essential genes by RNAi molecules.
2. The method to control geo-helminth parasite infection as claimed in claim 1, wherein the said essential target genes are selected from astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and, the said target enzymes responsible for metabolism and energy production are selected from malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome c oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde 3 Ph dehydrogenase, glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and phospholipase or a combination thereof.
3. The method to control geo helminth parasite infection as claimed in claim 1, wherein the said DsRNA vector is created by placing the said essential target gene between at least two opposable promoters selected from lactate dehydrogenase (Pldh), PslpA, Plaf, Ptac and P1 in L. acidophilous, P Dala, P ldh, Ppbg, Pfgs, P-HicDH, Pamy, P ccpA and P dlt P of L. casei, PxylA and Pxyl R, Prep of L. pentosus, PrepPC30il and Prep8014-2 of L. plantarum, Pusp 45 of L. lactis, PsapA of L. sakei, Pcbh of B.Subtilis , or a combination thereof, with complimentary strand having multiple cloning sites and at least two copies of restriction enzymes inserted in reverse directions on the said complimentary strand.
4. The method to control geo-helminth parasite infection as claimed in claim 1 wherein the said genetically engineered probiotic is selected from a group of different species of Lactic Acid Bacterium (LAB), Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus fermentum, Lactobacillus sakei, Lactococcus lactis, or Bacillus sp and/or a combination thereof.
5. The method to control geo-helminth parasite infection as claimed in claim 1 wherein the said genetically engineered probiotic can be used as food supplements in the endemic areas or as bio therapeutic in the form of tablet or capsules for direct oral gut delivery for human and veterinary application or as a combination therapy along with the deworming drugs to prevent re-infection, and reduce the resistance of the deworming drugs and/or a combination thereof. , Description:FIELD OF INVENTION
The present invention relates to the method of controlling parasitic helminth infection using probiotic mediated RNA interference (RNAi). More particularly, the present invention relates to genetic constructs of double stranded RNA interference (dsRNAi) vector containing parasite specific targets paired with probiotic bacteria as vehicles, for delivery to the helminth gut, where the target helminth RNA complementary to the parasite mRNA sequence knocks down the gene by RNAi and blocks larval developmental process.
BACKGROUND OF THE INVENTION
Soil-transmitted helminth infections, commonly known as geo-helminth infections are among the most common parasitic infections that affect humans, plants and other animals of economic importance. In the case of humans, eggs present in human faeces, that contaminate the soil, water bodies and vegetation in areas with poor sanitation facilities, transmit the geo-helminths. The adult form of geo-helminths is the most harmful life cycle stage, causing soil-transmitted helminthiasis (STH). Adult worms live in the intestine in hundreds where they feed on the blood and produce thousands of eggs each day. These eggs give rise to the larval stage of the geo-helminths, the infective form and are internalized in the host from the soil through contaminated food or through direct penetration from cracks in the skin where they undergo tissue-migratory stages and invade vital organs such as lungs and liver. Thus, the disease manifestations can be both local and systemic. Geo-helminth parasites infect more than two billion humans particularly in poverty-stricken rural areas of in developing regions of Asia, sub Saharan Africa and the Americas.
Over 80,000 species of helminths have been described, of which over 15,000 are parasitic. The parasitic worms that infect humans are Roundworms, Flatworms, Whipworms, Hookworms, Threadworms and Pinworms.
The geo-helminth parasites of humans cause a variety of disease conditions and symptoms, ranging from lack of energy and vigor to blindness, anaemia, intestinal manifestations (diarrhea and abdominal pain), and malformations.
Control of geo-helminth parasites presently relies primarily on the use of anthelmintic drugs combined with sanitation and pasture management. Such techniques have a number of drawbacks such as frequent administration of the drugs, which leads to the development of resistant strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the subjects being treated. Due to buildup of drug resistance larger doses are required leading to more severe side effects. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection itself.
An alternative method to prevent geo-helminth infection includes administering a vaccine against a specific geo-helminth. Although the development of vaccines based on prominent antigen specific to the geo-helminths have been developed, the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting the subject from infection. In addition, application of new vaccines has resulted in urticarial reactions and hypersensitivity due to elevated levels of IgE because of previous geo-helminth infections.
The geo-helminth are masters of immunomodulation that makes them difficult to target by vaccines and are also difficult to treat by drugs as they have complicated life cycles in various organs of the subject. In such a scenario, there is a need to develop new methods for control.
As illustrated in U.S. Pat. No. 6,506,559, RNA interference (RNAi), also referred to as gene silencing, has been proposed as a method for controlling geo-helminths. When double-stranded RNA (dsRNA) corresponding to the sequence of a target gene is introduced into a cell, expression from the target gene is inhibited. Further, it demonstrates the effectiveness of RNAi against the prominent genes in a model laboratory nematode, C. elegans. Due to the labile nature of the RNAi molecules, the difficulty of delivering dsRNA to target cells has impeded the rapid expansion of RNAi based therapies. The use of vectors has been exploited to deliver RNAi to the specific target and improvements can be made to render RNAi therapy more effective.
Probiotics are live bacteria which when administered in adequate amounts to the subject’s digestive system confer a health benefits. The role of probiotics in increasing the intestinal immunity and control of enteric organisms by the principle of mutual exclusion has been widely documented. Genetically engineered probiotics have also been used to deliver therapeutic immunomodulatory molecules and neutralizing antibodies to the site of action.
The present invention proposes a method of controlling geo-helminth infection using genetically engineered probiotics containing inhibitory molecules targeting them in the gut stage of their life cycle. These probiotics colonizing the gut will clear out existing infections and prevent re-infection from the environmental source by RNA interference mediated helminth control. Continuous in situ production of the RNAi molecules, will have prophylactic effect and keep the gut sanitized and will find applications in both human and veterinary health.
OBJECTIVES OF THE INVENTION
The primary objective of the present invention is a method for developing potent anti-helminth molecules using RNA interference (RNAi) and probiotic bacteria as vehicles for targeted delivery of these molecules to the parasite gut.
Another object of the present invention is to provide a targeted delivery of the RNAi vectors to the geo-helminth gut by using genetically engineered probiotic bacteria as delivery vehicle.
Another object of the present invention is to develop genetically engineered probiotics continuously expressing parasite target specific RNAi molecules.
Another object of the present invention is to target the genes present in geo-helminth parasite like the ones responsible for moulting and development, attachment, nutrient assimilation, locomotion and neuromuscular functions by specific selection of the targets to be cloned in the RNAi vectors.
Yet another object of the present invention is monitoring the knockdown of the essential genes by RNAi in geo-helminth, and demonstrating its phenotypic effects on the worms by conducting in vitro and in vivo studies.
Yet another object of the present invention is to provide a method of controlling and preventing geo-helminth infection by eliminating the worms and sanitizing the gut of the subjects.
Yet another object of the present invention is to provide a method of controlling helminth infection having minimum off target effects in their hosts.
Yet another object of the present invention is to provide a cost effective and error-free treatment for helminth infection.
Yet another object of the present invention is to ameliorate one or more problems of the prior art and provide a useful alternative.
SUMMARY OF THE INVENTION
The embodiments of the present invention described herein below are illustrative only and should not be construed to limit the scope of the present invention in any manner. Unless stated to the contrary, any use of the words such as "including”, "containing”, "comprising", "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but can be implemented in various combinations.
The term "geo-helminth" is used to denote the parasite that resides in the gut of the subject.
The term “subject” used herein means human or veterinary animal infected with geo-helminth.
The term "probiotic" denotes the bacterium that delivers the RNAi vector containing the DsRNA producing target to the host.
The term “host” used herein means a geo-helminth parasite that resides in subject's gut and acts as host for probiotic bacteria.
The present invention describes a method of controlling geo-helminth infection using genetically engineered probiotics containing gene inhibitory RNA interference (RNAi) vector. In this method, a RNA interference (RNAi) vector is developed that can be delivered by Lactic Acid Bacteria (also referred as LAB) to the subject i.e. human/animal gut. The essential genes responsible for the development of geo-helminth larval stage or maintenance of the adult stage are cloned in the RNAi vector. This RNAi vector targets the geo-helminth in their gut stage of life cycle. These RNAi vectors are transformed into probiotic commensal bacteria i.e. Lactic Acid Bacteria (LAB), which are fed to the host and colonizes the gut from where they deliver the targeted RNAi therapy into the helminth gut in human or veterinary subject. This RNAi LAB strain can be used as a probiotic to control geo-helminths in endemic areas, prevent reinfections or can be used as a passive vaccine.
According to the present invention, the method of controlling geo-helminth infection using RNAi comprises of:
i. Coproculturing of the geo-helminths using a petri dish method;
ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite culture,
iii. Designing DsRNA expression vectors containing the isolated essential target genes with suitable constitutive promoters, and pairing them with the probiotic bacteria.
iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii),
v. Colonization of the gut of subject with the probiotics, which then in turn are internalized in the parasite gut during their feeding cycle resulting in release of the RNAi molecules in the parasite gut and knock down of target genes.
The knockdown of essential target genes/enzymes, responsible for developmental and energy metabolism, nutrient assimilation or tissue penetration of geo-helminth parasite by RNAi results in anti-parasitic effects. The said essential target genes includes astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and the said essential target enzymes includes malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome C oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and/or phospholipase.
The said RNAi vector can be either single hairpin RNA (hairpin of about 20 bases) or double stranded RNA (DsRNA) of 100-200 bp against the target sequences. The said double stranded RNA (DsRNA) are used in the present invention as DsRNA are very stable and shows minimal off target activity as they have been selected in such a way so that they have no homology with their human counterparts and will easily be destroyed by immune response if taken up by mammalian cells accidently.
The said probiotic bacteria are selected from different species Lactobacilli preferably Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis or Bacillus sp.
Further, the DsRNAi expression vectors are created using at least two opposite constitutive promoters cloned in a reversible direction flanking the target gene. These promotors are selected from lactate dehydrogenase (Pldh) as in the present disclosure or PslpA, Plaf, Ptac and P1 in L. acidophilous, PDala, Pldh, Ppbg, Pfgs, P-HicDH, Pamy , PccpA and Pdlt P of L. casei, PxylA and Pxyl R, Prep of L. pentosus, PrepPC30il and Prep8014-2 of L. plantarum, Pusp 45 of L. lactis. PsapA of L.sakei, Pcbh of B.subtilis or a combination thereof. The complimentary strands of the DsRNA has multiple cloning sites for insertion of the parasite targets and consists of at least two copies of restriction enzymes inserted in reverse directions.
The said RNAi expression vector containing probiotic is orally administered to the infected subject and on reaching the subject’s gut colonizes there, which is further engulfed by the geo-helminth parasite present in the subject’s gut. The said probiotic upon degradation in the geo-helminth parasite’s gut releases the said DsRNA, which is taken up by the intestinal cells and processed to siRNA that exerts the RNAi mechanism by formation of RISC complex as illustrated in Figure 4,. RNA interference is carried out when the said siRNA binds with argonaute protein molecule to form RNA induced silencing complex (AGO/RISC) by recognizing complementary messenger RNA (mRNA) and cleaving them. The said RNAi mechanism enables the knockdown of the essential target genes by blocking the production of the said essential enzymes responsible in various developmental and energy metabolism pathways eventually leading to killing of geo-helminth.
The said dsRNA expression vector suitable for cloning includes different target genes against variety of helminths namely Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Flatworms (family Schistosoma mansoni and Schistosoma japonicum), Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis, and pinworms ( family Enterobius sp).
The said genetically engineered probiotic can be used as both food supplements, and as bio therapeutic form of tablet or capsules for direct oral gut delivery for human and veterinary application. The said genetically engineered probiotic can be used as a combination therapy along with the deworming drugs to prevent re-infection, and reduce the dose of the deworming drugs and subsequent resistance along with the gut colonizing probiotic benefits. This method of geo-helminth control results in an economical, safe and sustainable anti helminthic therapeutic strategy that augments current control. These curative and prophylactic methods of controlling helminth infection confers continuous protection against new helminth infection as well as eliminates the established adult worms, sanitizes the gut and provides all round immunity against geo-helminth infections.
Other objects, features and advantages of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings wherein like characters of reference designate corresponding parts throughout the several views.
BRIEF DESCRIPTION OF THE DRAWINGS
A complete understanding of the present invention may be by reference to the following detailed description which taken in conjugation with the accompanying drawings. The accompanying drawings, which is incorporated into and constitutes a part of the specification, illustrates one or more embodiments of the present invention and, together with the detailed description, it serves to explain the principles and implementations of the invention.
Figure 1: illustrates the flowchart of the method of controlling helminth infection using genetically engineered probiotics containing gene inhibiting RNA interference (RNAi) vector targeting the helminth
Figure 2: illustrates the construction of vector for generation of dsRNA,
Figure 3(a): illustrates schematic diagram of the dsRNAi vector and the sequence with the target gene (astacin) inserted between opposable ldh promoters,
Figure 3(b): illustrates The results of a PCR reaction using a single ldh primer in the genetically engineered probiotic giving a prominent band indicating the cloning of target gene in between the same opposable promoters,
Figure 3(c) illustrates electrophoretic representation of DsRNA astacin fragments in in the genetically engineered probiotic an agarose gel. The fragment is present in the transformed bacteria only.
Figure 4: illustrates diagrammatic representation of the mechanism of action effecting target gene knockdown by the genetically engineered probiotic in the geo-helminth gut,
Figure 5(a): illustrates electrophoretic representation of a PCR isolation of cDNA of the targets cathepsin D, astacin and FAR of A caninum,
Figure 5(b): illustrates electrophoretic representation of a PCR isolation of FAR a target of the human hookworm A. duodenalis,
Figure 6(a): illustrates microscopic image of moulting and transformation of the larvae to the feeding stage in induced hookworms,
Figure 6(b): illustrates fluorescent microscopic image of FITC labelled LAB internalized by the feeding stages of the hookworm larvae,
Figure 7: illustrates live dead assay using hookworm larvae fed with LAB, where C is untreated worms, and M is worms fed with dsRNA producing bacteria, Ll is Lactococcus lactis and Ds is double stranded RNA produced in vitro.
Figure 8: illustrates skin migration assay using hookworm larvae fed with DsRNAi LAB, where C is untreated worms, and M is worms fed with dsRNA producing bacteria, Ll is Lactococcus lactis and Ds is double stranded RNA produced in vitro.
Figure 9: illustrates electrophoretic representation of knockdown of astacin gene by reverse transcription PCR reaction, where M is worms fed with DsRNAi LAB, P is worms fed with unmodified LAB, The right hand panel is the 60S rRNA control.
Figure 10: illustrates tissue migration of hookworm larvae in unfed mice, LAB RNAi fed and in vitro produced dsRNA fed mice 24 hours and 72 hours post challenge,
Figure 11: Liver and kidney function tests in mice in normal (no challenge), control (challenge with unfed worms), and after challenge with LAB (LlM) and dsRNA (DsT) fed worms.
DETAILED DESCRIPTION OF THE INVENTION
The foregoing objects of the invention are accomplished and the problems and shortcomings associated with prior art techniques and approaches are overcome by the present invention described in the present embodiment. Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention.
Before the present invention is described, it is to be understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, it is to be understood that the present invention is not limited to the methodologies and materials similar, equivalent to those described herein can be used in the practice, or testing of the present invention, the preferred methods and materials are described, as these may vary within the specification indicated. Unless stated to the contrary, any use of the words such as "including," "containing," "comprising," "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose of illustration rather than limitation of the invention as set forth the appended claims. Further, the terms disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms.
The present invention describes a method of controlling geo-helminth infection using genetically engineered probiotics containing gene inhibitory RNA interference (RNAi) vector. In this method, a RNA interference (RNAi) vector is developed that can be delivered by Lactic Acid Bacteria (LAB) to the subject i.e. human/animal gut. The essential genes responsible for the development of geo-helminth larval stage or those responsible for food assimilation and energy metabolism are cloned in the RNAi vector. These RNAi vectors are transformed into probiotic commensal bacteria i.e. Lactic Acid Bacteria (LAB), which are further used as delivery vehicles to deliver the targeted RNAi vector into the helminth gut via human or veterinary subject from where they are picked up by the hookworms cohabiting the same niche. This RNAi LAB strain can be used as a probiotic to control geo-helminths in endemic areas, either by itself or with existing drugs, prevent reinfections and can be used as a passive vaccine.
According to the present invention, the method of controlling geo-helminth infection using RNAi comprises of:
i. Coproculturing the geo-helminths using a petri dish method;
ii. Isolating the essential target RNAi targets genes/enzymes from geo-helminth parasite ,
iii. Designing DsRNA expression vectors and cloning the isolated essential target genes, and pairing them with probiotic bacterial vectors,
iv. Feeding the subjects with DsRNA paired probiotic produced in above step (iii),
v. LAB colonization the gut of subject and subsequent colonization the parasite gut during their feeding cycle resulting in internalization of the target DsRNA in the parasite gut leading to knock down of target l genes by RNAi.
Figure 1 illustrates the method for controlling helminth infection. It illustrates various steps for controlling the helminth infection, such as step of isolation of the essential target genes from geo helminth parasite and the designing of DsRNA expression vectors and inserting them in probiotics by transformation.
Further, it illustrates that the genetically engineered probiotics expressing the RNAi molecules, when administered with the feed, will colonize the subject’s gut. The parasitic helminths present in the subject's gut feed on these probiotics that are degraded, resulting in delivering of the RNAi molecules to the helminth gut, which when taken up by the parasite intestinal cells transforms the DsRNA to siRNA that targets the complimentary mRNA, like the ones responsible for nutrient assimilation, development and metabolic pathways and knocks them out, debilitating the parasites thereby breaking their lifecycle.
According to the one of the embodiments of the present invention, the initial step includes coproculturing the helminth by petri dish method. For this, helminth's eggs removed from the subject's fecal matter are hatched in the laboratory by a petri-dish culture method. The said method for growth and culture of human and veterinary helminth involves mixing the fecal sample with 3 times the amount of sterile sand, and incubating in a moist damp place for 10-14 days followed by isolation of the worms that hatch out and mixing 2 ml of 3.3% agarose at 37-40°C with 4 ml of the parasite containing solution obtained from coproculture, pouring them on a petriplate and topping it with 4 ml. of a buffer solution and incubating them at 37°C for 1 hr. The contaminants are stuck in the agar and the larvae rise up and collect in the buffer layer from where they are isolated for further use.
Another embodiment of the present invention describes how the essential target genes/enzymes are selected from the genes which are responsible for developmental and energy metabolism, or gut digestion or tissue penetration of geo-helminth parasite. Knockdown of such target genes/enzymes using RNAi results in anti-parasitic effects. The said essential target genes selected for knockdown by detailed bioinformatics study includes astacin, fatty acid receptor, cathepsin D, hyaluronidases, cuticulin, aspartic acid, cysteine, serine protease; and the said essential target enzymes includes malate dehydrogenase, adenosine monophosphate dehydrogenase, fructose biphosphate, aldolases, cytochrome P 450, cytochrome C oxidase, alcohol dehydrogenase, cytochrome oxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol hydrolase, choline kinase, sugar hydrogen symporter, lipoprotein biogenesis proteins and/or phospholipase.
Further, the selection of the target genes/enzymes is crucial step in the present invention. The knockdown effects of the target gene/enzyme are immediate and detectable by in vitro assays. The target sites selected for the knockdown of essential target gene/enzyme are preferably from beta tubulin, ligand gated Ca+ and Na+ ion channels, glutamate gated chloride channels, gut neuropeptides, and G protein coupled receptors. Further, it may also include signaling pathways like Wnt/Frizzled signaling, FaRPergic signaling, FMRF amide like peptide signaling, and allatostatin like neuropeptide that are present in a majority of geo-helminths. From these RNAi target genes, complimentary sequences are isolated from helminth for the said essential parasite genes and cDNA are prepared using conventional reverse transcriptase polymerase chain reaction (RT PCR).
Another embodiment of the present invention describes, the said RNAi vector used in the present invention. The RNAi can be either single hairpin RNA (hairpin of about 20 bases) or double stranded RNA (DsRNA) of 100-200 bp against the target sequences. The said double stranded RNA (DsRNA) are used in the present invention as DsRNA are very stable and shows minimal off target activity as they have been selected from areas having no homology to the human genes and can be easily destroyed by immune response if taken up by mammalian cells accidently.
Further, the lactate dehydrogenase promoter (Pldh) was used for the construction of the DsRNA vector. Since lactate dehyrogenase is a predominant secretory enzyme in lactic acid bacteria, this vector is expected to work in the other lactobacilli as well. However, other promoters can be used as well and DsRNA will be produced if they are cloned in a reversible direction flanking the target gene. The DsRNAi expression vectors are created using at least two opposite constitutive promoters cloned in a reversible direction flanking the target gene. Other promotors are selected from PslpA, Plaf, Ptac and P1 in L. acidophilous, PDala, , Ppbg,P fgs, PD-HicDH, Pamy , PccpA and PdltP of L. casei, PxylA and PxylR, Prep p353-2 of L. pentosus, Prep PC30il and Prep 8014-2 of L. plantarum, Pusp45 of L. lactis. PsapA of L. sakei, Pcbh of B.subtilis or a combination thereof can be used in LAB as well. In addition to these, synthetic promoters can be generated using the consensus sequences of the promoters. Depending on the requirement like expression levels and suitability of the promoters in different hosts, these promoters can be used interchangeably in the LAB as well. The complimentary strands of the DsRNA have multiple cloning sites and consist of at least two copies of restriction enzymes inserted in reverse directions for insertion of the target between the opposable promoters.
Further, the said RNAi expression vector containing genetically engineered probiotic is orally administered to the infected subject and on reaching the subject’s gut, the said genetically engineered probiotic is engulfed by the geo-helminth parasite present in the subject’s gut as illustrated in Figure 4. The said probiotic upon degradation in the geo-helminth parasite’s gut releases the said DsRNA, which is taken up by receptor mediated endocytosis in the intestinal cells and further processed to siRNA that exerts the RNAi mechanism by formation of RISC complex; The RNA interference is carried out when the said siRNA binds with argonaut protein molecule to form RNA induced silencing complex (AGO/RISC) by recognizing complementary messenger RNA (mRNA) and cleaving them. The said RNAi mechanism enables the knockdown of the said essential target genes thereby blocking the production of the said essential target enzymes eventually leading to killing of the parasite.
Another aspect of the present invention is the design of dsRNA expression vector and its pairing with probiotic LAB strains. As illustrated in Figure 2, the dsRNA producing probiotic LAB vector is created by placing the target gene under two opposable constitutive promoters (P) in complimentary strands with multiple cloning sites (1/2/3) inserted between them. The restriction enzymes (RE) namely Eco RI/Sac I/Sma I (1/2/3) on both sides are inserted in reverse directions to prevent re-ligation. Cloning of the target genes is accomplished by a single restriction digestion with any of the three said restriction enzymes, followed by ligation of the target gene flanked by a single inverted restriction enzyme site in a reversed orientation. Inversion of the restriction enzyme site prevents recircularization of the vector or the target during the ligation reaction. The target can be inserted in either direction but that would not have any effect on the production of the dsRNA as promoters are present in both the directions and in frame cloning is not required, as translation does not take place. Numerous origins of replications exist for different LAB species namely Rep A, Rep C, Rep D, Rep E, Rep F and Rep G. Not all the origin of replications may be necessary for the maintenance of the plasmids in the LAB. Rep D, E, F and G that has a wider host range and works in different species of LAB were selected for designing of the DsRNAi vector. The selection cassette used is Ery, a gene that confers resistance to erythromycin, but a Choramphenicol Acetyltransferase gene (Cm/CAT) that confers resistance to Chloramphenicol can also be used as well. For example, as Figure 3 (a) illustrates the schematic diagram of the dsRNA vector and the sequence with the target gene (astacin) inserted between the opposable constitutive Lactate Dehydrogenase (LDH) promoters. PCR with a single LDH primer gave a prominent band indicating the target gene had been cloned in between the opposable promoters and the DsRNA astacin fragment is clearly visible in the DNA gel as illustrated in Figure 3(b) and 3(c).
The said dsRNA expression vector suitable for cloning includes different target genes against variety of helminths. The medically important helminths where this will be applicable are Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Flatworms (family Schistosoma mansoni and Schistosoma japonicum), Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis, and Pinworms (family Enterobius sp) or a combination thereof. These vectors can be paired with a suitable probiotic LAB strain for delivery and continuous production of the RNAi molecules in the gut of the helminth.
Another embodiment of the present invention describes, the said probiotic bacteria of the present invention are selected from different species of Lactobacilli preferably Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis or Bacillus sp.
According to another embodiment of the present invention, the genetically engineered probiotics that produce DsRNAi molecules in situ can be used as value added food supplements that can be administered to subject in endemic areas to control helminth infestation. In addition, the genetically engineered probiotics can be used to produce dsRNA that can be purified and used as bio-therapeutic molecules that can be formulated as a tablet or capsule for direct oral delivery to the gut.
According to another embodiment of the present invention, the genetically engineered probiotics can be used either singly or as a passive vaccine for human and veterinary applications, in helminth endemic areas. If administered immediately after the use of deworming drugs such as Albendazole, the probiotics will colonize the subject gut and prevent reinfection by the helminth that have survived in the environment. If used as a combination therapy with the existing deworming drugs, a reduction in the dosage of deworming drug can brought about, decreasing the ability of the helminth to develop drug resistance.
Further, the Lactobacillus vectors have a gram positive origin of replication and is expected to work in other species of Lactobacilli commonly used as probiotics like Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus crispatus Lactobacillus fermentum, Lactobacillus sakei, Lactococcus lactis as well as Bacillus sp. These strains can be used to produce the value added probiotics that can be used as food supplements in endemic areas to control geo-helminth infestation. They can be used to produce double stranded RNA, be purified and used as bio-therapeutic molecules that can be formulated as a tablet or capsule for direct oral delivery to the gut.
ADVANTAGES
1. The RNAi vector so constructed is a replacement vector suitable for cloning different target genes against other geohelminth parasites. This vector can be used against other common soil transmitted parasites like Roundworms (family Ascarididae), e.g. Ascaris lumbricoides, Whipworms (family Trichuridae), e.g. Trichuris trichiura, Hookworms (family Ancylostomatidae), e.g. Ancylostoma duodenale and Necator americanus, Threadworms (family Strongyloididae), e.g. Strongyloides stercoralis . This vector can be paired with a suitable Lactobacillus strain by transformation for delivery and continuous production of the RNAi molecules in the gut.
2. Apart for use in humans this invention can have wide applications in the area of animal health and can be used to control parasite infestations reducing the need for small molecule drugs like Albendazole and Ivermectin against which resistance can be generated quickly by the parasites. This invention can be tailored for a specific group of animals by selecting an appropriate animal specific target and pairing it with a suitable probiotic and used singly or in combination with other antiparasitic molecules, like Ivermectin for effective parasite control in pets, farm animals and poultry.
3. The anti-parasitic value added probiotics can be used either singly as a passive vaccine for human applications, particularly for children in hookworm endemic areas. If used immediately after the application of an antiparasitic molecule like Albendazole, the probiotics will colonize the gut and prevent reinfection by the parasite that has survived in the soil. If used for a sufficiently long period, a combination therapy with the existing antiparasitics like Albendazole and the probiotics can wipe out all traces of the parasites from endemic areas.
5. The value added probiotic will also result in regulating the dose of Albendazole to children, many of who are affected by the extreme toxicity of the molecule and have to be hospitalized. With a combination therapy and a reduction of the dose of Albendazole, the ability of the parasites to develop drug resistance to the small molecule drugs will be considerably reduced.
6. Targeted delivery of the RNAi expression vector at the site of action utilizing genetically engineered probiotics: The DsRNA is released in the geo-helminth gut and not the subject’s gut so the off target effects if any are minimized and the RNAi expression vector containing the target is enclosed by live genetically engineered probiotics so their production can be modulated specifically at the site of action by using specific promoters that will be inoperative once the probiotics leave the site of action.
EXAMPLES
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations, which are evident because of the teaching provided herein.
Example 1: Growth and culture of hookworm Ancylostoma duodenale and Ancylostoma caninum by coproculture technique:
Method of controlling Hookworm infection by LAB mediated RNA interference was carried out on two different species of hook worms namely Ancylostoma duodenale (affecting humans) and Ancylostoma caninum (affecting canines). The method comprises of the steps of:
Hook worm eggs were extracted out of subject fecal matter and hatched in the laboratory by novel petri-dish culture method. The eggs hatch into the L1 larvae in 4-5 days and are transformed to L3 larvae rapidly in 3 - 4 days following which they are collected and maintained in RPMI 1640 medium or BU buffer with antibiotics (Amphotericin B, Penicillin, Streptomycin).
Example 2: Isolation of RNAi targets from the hookworms
Three targets were shortlisted for studying Ancylostoma caninum and Ancylostoma duodenalis:
i. Astacin: it is a zinc metalloprotease responsible for tissue migration, moulting, predominantly found in larval stages, and is induced prior to migration. The homologue of astacin is absent in humans and so the chance of off target activity is expected to be low;
ii. Fatty Acid Receptor (FAR): A nutrient sensor for regulating energy metabolism, found in adults; and
iii. Cathepsin D: Responsible for hemoglobin digestion, found in adults.
200- 400 bp fragments of the cDNA of Cathepsin D, Astacin and FAR of A caninum and FAR of A. duodenalis were isolated by RT-PCR as illustrated in Figures 5(a) and (b).
Example 3: Design of DsRNA expression vectors and paired probiotic bacterial strains
LAB is known to colonize the gut and are used as probiotics. A vector backbone containing the gram-positive origin of Rep D, E, F and G was developed that would work in both Lactococcus sp and Lactobacillus sp and used to generate dsRNA as illustrated in Figure 3 (a). A dsRNA producing Lactococcus lactis strain was created by placing the astacin gene under two opposable constitutive Lactate Dehydrogenase (LDH) promoters in the vector and transforming the Lactococcus strain with it. PCR with a single LDH primer gave a prominent band indicating the target gene had been cloned in between the opposable promoters as illustrated in Figure 3 (b) and the Ds astacin RNA fragment in a DNA gel was clearly visible as illustrated in Figure 3 (c).
Example 4: In vitro studies to establish RNAi activity in Ancylostoma caninum by feeding:
Feeding stimulation and moulting studies were conducted with FITC labelled LAB. The Ancylostoma L3 larval stage is non-feeding, so were stimulated to the feeding L4 stage by the addition of 5 % dog serum and reduced glutathione. As illustrated in Figure 6(a) within 2 hours after stimulation, moulting was seen in the larvae along with the development of mouthparts and buccal funnel following which the larvae are converted to the feeding stage and were ready for tissue migration. Copious amounts of the protease astacin are secreted during this stage that help shed cuticle, penetrate the gut musculature and helps in subsequent tissue migration. These stimulated L3 larvae were fed with the LAB containing the astacin DsRNA. The LAB was internalized in the hookworm tissues and processed to siRNA, which results in knockdown of the levels of astacin by RNAi mechanism and stop moulting and larval migration through the tissues.
The induced larvae were incubated with FITC labelled LAB overnight and observed under a fluorescent microscope as illustrated in Figure 6(b). The bacteria were internalized and visible in the gut as bright fluorescent spots in the feeding induced Ancylostoma. The combination of moulting and feeding indicated that the induction had successfully transformed the hookworm to the next larval stage.
Example 5: In vitro larval motility and mortality studies
In vitro larval motility and mortality studies were carried out to observe the effect of the RNAi knockdown of essential genes. A phenotypic behavioral effect was observed resulting in sickness and eventual death of the worms. The worms were fed with the RNAi LAB and motility assays performed after 72 hours. Fifty worms were pre-fed with astacin DsRNA producing LAB and were analyzed for thrashing movement (motility) by adding lukewarm water at 37°C. The control worms without any treatment immediately started trashing but the bacteria fed worms were sick and did not show a significant thrashing movement. These sick worms started dying and breaking apart as illustrated in Figure 7 after another 24 hours, indicating that feeding the double stranded RNA had the desired anti-parasitic effect.
Example 6: Skin migration assays
Skin migration assays were performed to study larval penetration into the subject body through the skin. Induced Ancylostoma larvae rapidly penetrate the skin and migrate through the tissues eventually reaching the lungs from where they are coughed into the alimentary canal and finally settle in the intestines as egg laying adults. The skin migration assay was performed using chicken skin (since fresh dog skin is unavailable in India) that was stretched out on a small culture plate filled with BU buffer (Na2HPO4 7%, KH2PO4 3%, NaCl 4% pH 6.8 ) and 200 µl of dog serum was added on the skin (to simulate conditions of the dog gut). After 30 mins, Ancylostoma L3 larvae pre-fed with DsRNA producing bacteria, were carefully placed and incubated overnight at room temperature. Next day the larvae that migrated through the skin and fell in the buffer underneath were counted under the microscope by briefly spinning them down to concentrate it. No difference in the migration patterns was seen in the control worms whereas in the DsRNA treated worms more than 80% of the worms failed to migrate possibly because Astacin was knocked out and failed to penetrate through the skin as illustrated in Figure 8.
Example 7: Detection of target RNA levels post RNA interference:
Establishment of the DsRNA and its effect in knocking down essential genes was done in two sequential steps: a) detection of DsRNA in bacteria after induction; and, b) effect of knockdown of the target gene in hookworms by Reverse Transcription PCR.
For detection of DsRNA, the extracted total RNA was run in a DNA gel after digestion with RNAseA to remove the single stranded RNA. A band at 200 bp is clearly visible in LAB that harbors the RNAi plasmid as illustrated in Figure 3(c). Sequencing of the plasmids confirmed the creation of a two novel DsRNA vectors. The opposable promoters (ldh in LAB) flanking the multiple cloning site were placed on a stable backbone containing a selection marker. The knockdown effect of the target gene was seen using RT PCR; 72 hours post feeding of the in vitro transcribed dsRNA. The astacin gene was knocked down considerably as illustrated in Figure 9.
Example 8: In vivo studies with mice challenged with RNAi bacteria fed Ancylostoma:
Swiss albino mice were used to study the tissue migration patterns of hookworms fed with Ds astacin RNAi (herein referred as DsT) produced in vitro and Ds astacin RNAi producing LAB (referred to as LlM). The mice were challenged by a bolus dose with Ancylostoma pre fed 48 hours earlier with the LlM. 500 pre-fed worms were fed to freshly weaned 3 week old mice andthe migration of the worms was observed by necroscopy after dissecting the mice and collecting the organs, intestines and liver (24hours) and lungs and forelimbs (72hours). The organs were tied up in cheesecloth to prevent disintegration and immersed in beakers containing artificial gastric juice (5gm pepsin + 0.7 ml conc. HCl + 1000ml D/W + 8.5gm NaCl) at 37°C for 3 hours. The larvae are released from the organs and settle at the bottom of beaker, after concentrating the worms by a brief spin, the worms were counted under a microscope. After 24 hours more number of worms were recovered from stomach and liver of the control mice as compared to the mice fed with worms treated with the in vitro produced DsRNA (DsT) and DsRNA producing LAB ( LlM) as illustrated in Figure 10. After another 48 hours during which the worms had a chance of migrating further, the lungs and forelimbs were analyzed and the same trend was seen. This indicated that the DsRNA produced in vitro or delivered through the LAB had the desired phenotypic effect, i.e. inhibiting tissue migration by knocking down of the proteases required for burrowing through the skin.
Immuno-histological studies also showed the presence of dead worms in the organs (data not shown). The difference of the worm migration to the vital organs was also reflected in the organ damage. Biochemical assays like the liver and kidney function tests were performed to determine the extent of damage caused by hookworms pre and post RNAi treatment. Liver and kidney damage was indicated by an increase in the levels of SGPT (Serum Glutamic-Pyruvic Transaminase), and BUN (Blood Urea Nitrogen) respectively that are release in the blood from the damaged organs. As illustrated in Figure 11 considerable organ damage was observed by a rise in the level of the enzymes in the control mice that were challenged with the untreated hookworms as compared to normal mice that were challenged with the worms pre-fed with the genetically modified LAB or the astacin produced in vitro.
While considerable emphasis has been placed herein on the specific elements of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.