Novel Paratransgenic System for the Biocontrol of Disease-Transmitting Mosquitos

Abstract

The inventive technology relates to novel paratransgenic strategies for the control of pathogens. The inventive technology may specifically include a novel paratransgenic system configured to deliver one or more inhibitory RNA molecules to pathogen/disease-transmitting organisms. In a preferred embodiment, the invention may include one or more genetically engineered enteric bacteria configured to deliver one or more interfering RNA molecules to pathogen/disease-transmitting mosquitos.

1 - 126 . (canceled) 
     
     
         127 . A method of controlling a viral pathogen in a mosquito comprising the steps of:
 introducing into the mosquito a genetically modified bacteria that colonizes the mosquito, wherein the bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a heterologous double stranded RNA (dsRNA) polynucleotide that inhibits expression of an essential gene of an arbovirus pathogen; and   wherein the genetically modified bacteria is a mosquito symbiont selected from the group consisting of: at least one biotyped mosquito symbiont identified as Ae001-263; an autotropic symbiotic enteric bacteria, an RNase III deficient symbiotic enteric bacteria,  E. coli  strain HT115,  E. coli  strain HT27,  E. coli  strain JC8031,  Pantoea  strain Ael6,  Serratia  strain AeS1, and  Serratia  strain MS5.   
     
     
         128 . The method of  claim 127 , wherein the expression of the dsRNA polynucleotide within the mosquito inhibits expression of an essential gene of an arbovirus pathogen is selected from the group consisting of: an essential gene of a Zika virus, or an essential gene of a Dengue virus. 
     
     
         129 . The method of  claim 127 , wherein the dsRNA polynucleotide is selected from one of the nucleotide sequences of SEQ ID NOs. 1, 2, 3, 4 and 5. 
     
     
         130 . The method of  claim 127 , wherein the genetically modified symbiotic bacteria further expresses a heterologous nucleotide encoding at least one of the following: a VrrA small-non-coding RNA, SIDa, SID2, Ago2, YmdB, HlyA, a sec-secretory signal peptide, a tat-secretory signal peptide, a cell-penetrating peptide, and a dsRNA-binding protein. 
     
     
         131 . The method of  claim 127 , wherein the mosquito is: a female mosquito that transmits the viral pathogen to a mammalian organism, a mosquito larvae, a third instar larva, a pupa, or an adult mosquito. 
     
     
         132 . The method of  claim 130 , wherein the introducing to a mosquito the genetically modified bacteria comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution. 
     
     
         133 . A system for the bio-control of an arbovirus-carrying mosquito comprising the steps of:
 identifying a mosquito symbiotic bacteria that persists in at least two of the following: the mosquito's midgut, the mosquito's gut epithelial cells, the mosquito's ovaries, the mosquito's rectum, an egg laid by a female mosquito, the chorion of eggs laid by a female mosquito, salivary glands, fat bodies, malpighian tubules and haemolymph;   genetically modifying the mosquito symbiotic bacteria to include an RNase III deficiency and further genetically modifying the mosquito symbiotic bacteria to include an expression control sequence operably linked to at least one heterologous nucleotide encoding at least one inhibitory RNA polynucleotide that downregulates expression of at least one arbovirus gene; and   introducing into the mosquito an effective amount of the genetically modified symbiotic bacteria that colonizes the mosquito and expresses the at least one inhibitory RNA polynucleotide that downregulates expression of the at least one arbovirus gene.   
     
     
         134 . The system of  claim 133 , wherein the mosquito is:  Aedes aegypti, Aedes albopictus , or  Anopheles gambiae.    
     
     
         135 . The system of  claim 134 , wherein the mosquito is: a female mosquito that transmits the arbovirus to a mammalian organism, a mosquito larvae, a third instar larva, a pupa, or an adult mosquito. 
     
     
         136 . The system of  claim 135 , wherein the step of introducing to the mosquito an effective amount of the genetically modified symbiotic bacteria comprises a method selected from the group consisting of: administering an effective amount of the genetically modified symbiotic bacteria through one or more of the following methods selected from the groups consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the genetically modified symbiotic bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution. 
     
     
         137 . The system of  claim 133 , wherein the genetically modified symbiotic bacteria is selected from the group consisting of: at least one biotyped mosquito symbiont identified as Ae001-263, an autotropic symbiotic enteric bacteria, an RNase III deficient symbiotic enteric bacteria,  E. coli  strain HT115,  E. coli  strain HT27,  E. coli  strain JC8031,  Pantoea  strain Ael6,  Serratia  strain AeS1, and  Serratia  strain MS5. 
     
     
         138 . The system of  claim 137 , wherein said at least one inhibitory RNA polynucleotide is a dsRNA polynucleotide molecule. 
     
     
         139 . The system of  claim 138 , wherein the dsRNA polynucleotide is selected from the nucleotide sequences of SEQ ID Nos. 1, 2, 3, 4 and 5. 
     
     
         140 . The system of  claim 133 , wherein the genetically modified mosquito symbiotic bacteria further expresses a heterologous nucleotide encoding at least one of the following: a VrrA small-non-coding RNA, SIDa, SID2, Ago2, YmdB, HlyA, a sec-secretory signal peptide, a tat-secretory signal peptide, a cell-penetrating peptide, and a dsRNA-binding protein. 
     
     
         141 . A system for the control of an arbovirus-carrying mosquito comprising the steps of:
 identifying a mosquito symbiotic bacteria on which mosquito or mosquito larva feed, that persists in at least two of the following: the mosquito's midgut, the mosquito's gut epithelial cells, the mosquito's ovaries, the mosquito's rectum, an egg laid by a female mosquito, the chorion of eggs laid by a female mosquito, salivary glands, fat bodies, malpighian tubules and haemolymph;   genetically modified the mosquito symbiotic bacteria to include an expression control sequence operably linked to at least one heterologous nucleotide encoding at least one inhibitory RNA polynucleotide that downregulates expression of at least one arbovirus gene;   incorporating said genetically modified mosquito symbiotic bacteria into a mosquito ingestible feed; and   introducing the mosquito ingestible feed to the mosquito such that it receives an effective amount of the genetically modified symbiotic bacteria that colonizes the mosquito and expresses the at least one inhibitory RNA polynucleotide.   
     
     
         142 . The system of  claim 141 , wherein said at least one inhibitory RNA polynucleotide is a dsRNA polynucleotide molecule. 
     
     
         143 . The system of  claim 141 , wherein the arbovirus is selected from the group consisting of: a Zika virus or a Dengue virus. 
     
     
         144 . The system of  claim 142 , wherein the dsRNA polynucleotide is selected from the nucleotide sequences of SEQ ID Nos. 1, 2, 3, 4 and 5. 
     
     
         145 . The system of  claim 141 , wherein the genetically modified mosquito symbiotic bacteria further expresses a heterologous nucleotide encoding at least one of the following: a VrrA small-non-coding RNA, SIDa, SID2, Ago2, YmdB, HlyA, a sec-secretory signal peptide, a Tat-secretory signal peptide, a cell-penetrating peptide, and a dsRNA-binding protein. 
     
     
         146 . The system of  claim 141 , wherein the mosquito ingestible feed is fed to: a female mosquito that transmits the viral pathogen to a mammalian organism, a mosquito larvae, a third instar larva, a pupa, or an adult mosquito. 
     
     
         147 . The system of  claim 141 , wherein the genetically modified mosquito symbiotic bacteria is a mosquito symbiont selected from the group consisting of: at least one biotyped mosquito symbiont identified as Ae001-263; an autotropic symbiotic enteric bacteria, an RNase III deficient symbiotic enteric bacteria,  E. coli  strain HT115,  E. coli  strain HT27,  E. coli  strain JC8031,  Pantoea  strain Ael6,  Serratia  strain AeS1, and  Serratia  strain MS5. 
     
     
         148 . The system of  claim 141 , and further comprising the step of genetically modifying said mosquito symbiotic bacteria to include an RNase III deficiency.