Paratransgenic system for the biocontrol of disease-transmitting mosquitos
Inventors
Sayre, Richard • Xu, Jiaannong
Assignees
Pebble Labs Inc • Colorado State University Research Foundation
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Publication Number
US-10294475-B2
Publication Date
2019-05-21
Expiration Date
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.
Core Innovation
The invention relates to controlling Zika virus in a mosquito using a paratransgenic approach in which a genetically modified bacteria colonizes the mosquito. The bacteria comprises an expression control sequence operably linked to a nucleotide sequence encoding a heterologous double stranded RNA (dsRNA) polynucleotide selected from SEQ ID NOs 1, 2, 3, 4 and 5, and expression of the dsRNA polynucleotide within the mosquito inhibits expression of an essential gene of the Zika virus.
The paratransgenic system further comprises generating a genetically modified endosymbiotic bacteria that is paratransgenic with the mosquito and colonizes the mosquito. The endosymbiotic bacteria includes 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 Zika virus gene, including essential genes selected from NS2B, NS3, or NS4.
In some implementations, the disclosed platform includes genetically modified endosymbiotic bacteria expressing a heterologous nucleotide encoding a VrrA small non-coding RNA. The partial content also characterizes helper-gene concepts used with bacterial systems, including VrrA and SID transporters, and related RNAi factors such as Dicer 2, RISC, and Argonaute/Ago2.
Claims Coverage
The independent claims are directed to paratransgenic mosquito bio-control using genetically modified bacteria that colonize the mosquito and express heterologous inhibitory RNA polynucleotides selected from SEQ ID NOs 1-5 to inhibit or downregulate Zika virus gene expression. Across the independent claims, the core inventive features are an expression control sequence linked to inhibitory RNA, selection of specific SEQ ID NOs, and inhibition or downregulation of essential Zika virus genes.
Genetically modified colonizing bacteria expressing heterologous dsRNA from SEQ ID NOs 1-5
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 the Zika virus; wherein the dsRNA polynucleotide is selected from SEQ ID NOs 1, 2, 3, 4 and 5; and wherein expression of the dsRNA polynucleotide within the mosquito inhibits expression of the essential gene of the Zika virus.
Paratransgenic endosymbiotic bacteria colonizing the mosquito and downregulating Zika virus genes using inhibitory RNA from SEQ ID NOs 1-5
generating a genetically modified endosymbiotic bacteria comprising 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 Zika virus gene, and wherein the genetically modified endosymbiotic bacteria is paratransgenic with the mosquito; introducing into the mosquito an effective amount of the genetically modified endosymbiotic bacteria that colonizes the mosquito and expresses the at least one inhibitory RNA polynucleotide that downregulates expression of the at least one Zika virus gene; and wherein the at least one inhibitory RNA polynucleotide is selected from SEQ ID NOs 1, 2, 3, 4 and 5.
Both independent claims cover paratransgenic mosquito bio-control by expressing inhibitory RNA polynucleotides selected from SEQ ID NOs 1-5 from genetically modified bacteria that colonize, including endosymbiotic bacteria that are paratransgenic with the mosquito. The inhibitory RNA downregulates or inhibits expression of essential Zika virus genes in mosquito tissues.
Stated Advantages
Bio-control of a Zika virus-carrying mosquito.
Inhibition or downregulation of expression of essential Zika virus genes.
Paratransgenic colonization of the mosquito by genetically modified endosymbiotic bacteria that express inhibitory RNA polynucleotides.
Documented Applications
Paratransgenic control or bio-control of Zika virus-carrying mosquitoes using bacteria that express dsRNA and are processed into siRNA in mosquitoes.
Target selection of ZIKV dsRNA targets based on sequence conservation for use as inhibitory RNA polynucleotides (SEQ ID NOs 1-5).
Characterization of mosquito-associated bacterial strains or endosymbionts suitable for paratransgenesis.
Demonstrations of dsRNA production, processing, and reductions in viral loads or infection rates in the context of the described paratransgenic system.
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