Transkingdom platform for therapeutic nucleic acid delivery
Inventors
Linke, Lyndsey M. • Mora, Darcy
Assignees
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Publication Number
US-11312954-B2
Publication Date
2022-04-26
Expiration Date
Abstract
A transkingdom platform for the delivery of therapeutic nucleic acids to epithelial tissues where the nucleic acids are designed to have enhanced stability. The platform offers numerous improvements to prior delivery platforms including expression of the double-stranded RNA binding domain (dsRBD) domains of TAR RNA binding protein (TRBP), knockout of RNase R activity in the bacterial delivery vehicle, and expression of the methyltransferase gene, HEN1, for simultaneous packaging with a therapeutic nucleic acid delivery vehicle.
Core Innovation
The disclosed invention relates to transkingdom therapeutic nucleic acid delivery to a eukaryotic cell using a nonpathogenic bacterium or yeast as a delivery vehicle. The bacterium is engineered to express at least one invasion factor from a sequence on the chromosome of the bacterium, and the delivery system comprises therapeutic nucleic acids encoded on a plasmid having a length less than 4,700 base pairs and including one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter to control transcription.
The invention addresses stability and persistence of nucleic acids by enhancing protection of dsRNA and siRNA/shRNA and by reducing nucleic-acid decay. The platform includes expression of a dsRNA-binding protein with a dsRNA-binding domain for dsRNA protection, reduction of bacterial RNase R by knocking out the rnr gene to reduce dsRNA decay, and siRNA 3′ terminal methylation by expressing the HEN1 methyltransferase or a domain to protect against 3′-5′ degradation and 3′ uridylation.
Targeting and expression stability are further supported by engineered invasion and nucleic-acid interference against viruses or viral binding domains. Invasion can be enhanced by surface expression of influenza HA-1 and optionally invasin/LLO, and the constructs can be described as chromosomally integrated to increase expression stability while avoiding antibiotic resistance genes by using auxotrophic selection. The therapeutic nucleic acids described include siRNA/shRNA and RNA interference, directed to mucosal epithelial tissues and applicable to mucosal-associated diseases and virus targets.
Claims Coverage
The provided claim set includes four independent claims. Across these claims, the inventive coverage centers on engineered nonpathogenic bacterial nucleic-acid delivery with chromosome-encoded invasion factors and small plasmids under prokaryotic promoter control, with additional limitations on plasmid content and, in some independent claims, the specific invasion-factor selection and plasmid architecture.
Chromosome-encoded invasion factor and sub-4700 bp therapeutic plasmid
A nonpathogenic bacterium engineered to express at least one invasion factor from a sequence on the chromosome of the bacterium, wherein the bacterium comprises a plasmid having a length less than 4,700 base pairs, the plasmid comprising one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter to control transcription of the therapeutic nucleic acids.
Invasion factor selected from LLO, INV, or HA-1 with sub-4700 bp therapeutic plasmid
A nonpathogenic bacterium engineered to express LLO, INV, or HA-1 from a sequence on the chromosome of the bacterium and wherein the bacterium comprises a plasmid having a length less than 4,700 base pairs, the plasmid comprising one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter to control transcription of the therapeutic nucleic acids.
Nonpathogenic engineered bacterium plus plasmid cloning site under prokaryotic promoter control
A nucleic acid delivery kit comprising a nonpathogenic bacterium engineered to express at least one invasion factor from a sequence on the chromosome of the bacterium; and a plasmid having a length less than 4,700 base pairs, the plasmid comprising a cloning site for one or more sequences encoding one or more therapeutic nucleic acids, and a prokaryotic promoter, wherein the prokaryotic promoter controls transcription for the cloning site.
Essential plasmid architecture for therapeutic nucleic acids under prokaryotic promoters
A nonpathogenic bacterium engineered to express at least one invasion factor from a sequence on the chromosome of the bacterium and wherein the bacterium comprises a plasmid consisting essentially of one or more sequences encoding one or more therapeutic nucleic acids, an origin of replication, a sequence encoding a selection marker, and one or more prokaryotic promoters to control transcription of the nucleic acids.
The independent claims collectively cover engineered nonpathogenic bacterial nucleic-acid delivery to a eukaryotic cell by combining chromosome-encoded invasion factors with small plasmids under prokaryotic promoter control, with additional independent-claim-specific limitations on invasion-factor selection and plasmid structure including cloning-site or essential components.
Stated Advantages
Enhanced stability of nucleic acids, including enhanced stability and persistent RNAi.
Targeting mucosal epithelial tissues.
Multiple administration routes.
Reduced manufacturing cost and toxicity.
Documented Applications
Delivering shRNA/other NA therapeutics against mucosal-associated diseases/viruses.
Interference against viruses, including influenza, HSV-1, HSV-2, HPV, RSV, MERS, SARS, and other viruses listed in the disclosure.
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