Compositions of and methods for in vitro viral genome engineering
Inventors
Cady, Kyle C. • Barbu, E. Magda • DiPetrillo, Christen G.
Assignees
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Publication Number
US-10711253-B2
Publication Date
2020-07-14
Expiration Date
Abstract
The present disclosure relates to a method of in vitro engineering of nucleic acids. This disclosure further relates to in vitro engineering of viral genomes and to the improvement of viral properties by in vitro genomic engineering of viral genomes. Specifically, the disclosure relates to in vitro viral genomic digestion using RNA-guided Cas9, the assembly of a recombinant genome by the insertion of a DNA or RNA fragment into the digested viral genome and transformation of a host cell with the recombinant genome. This method also related to in vitro engineering for error correction of nucleic acids.
Core Innovation
The disclosed invention relates to engineering complete viral genomes in vitro using an RNA-guided nuclease and a digestion-and-assembly workflow. An isolated viral genome is digested with guide RNAs, followed by recombinant genome assembly to insert DNA or RNA fragments into the viral genome. The workflow enables cell-free assembly of engineered viral nucleic acids and engineered viruses, including bacteriophages.
The invention also provides engineered viruses and engineered viral nucleic acids having improved viral properties. Improvements are described in terms of expanded host range, increased viral lytic activity, early biofilm disruption or biofilm dispersion, antibiotic sensitization, anti-phage resistance, and immune stimulation/deactivation.
The engineered phage embodiments include modifications of viral genome features that affect adsorption, attachment, injection, replication, assembly, lysis, lytic cycle, and burst size. Specific examples include alterations of LUZ19 genomic regions and payload incorporation for biofilm control, lysin-mediated antibiotic sensitization, anti-phage resistance proteins, and reporter engineering, with optional in vivo assembly, transformation to host cells, a kit formulation, and use in packaging contexts.
Claims Coverage
The independent claims in the provided set cover recombinant phages that express multiple payloads, with dependent refinements narrowing payload identity, host specificity, phage identity, LUZ19-derived genome similarity, and defined gp mutation sets. The claim set presents seven inventive features.
Recombinant phage expressing two or more payloads
A recombinant phage that expresses two or more payloads selected from DNase, an exopolysaccharide depolymerase, and one or more surfactant phenol soluble modulins.
Payload combinations including DNase, EPS depolymerase, and phenol soluble modulins
The recombinant phage further specifies which payload categories are included, including combinations selected from DNase, EPS depolymerase, and one or more surfactant phenol soluble modulins.
Phenol soluble modulin selection for the recombinant phage
The recombinant phage includes a phenol soluble modulin chosen from PSMa, PSMa3, or PSMb2.
Host-specific recombinant phage infecting Pseudomonas aeruginosa
The recombinant phage is a bacteriophage that infects Pseudomonas aeruginosa.
A recombinant phage identified as ΦKMV
The recombinant phage is the ΦKMV phage.
LUZ19-derived genome with a defined sequence identity threshold
The phage comprises a viral genome having at least 85% sequence identity to the LUZ19 genome.
LUZ19 gp modifications for improved host range and/or lytic activity
A phage comprising gp13, gp18, gp38, gp40, and gp34 mutations or substitutions/deletions, with modifications intended to improve host range and/or increase lytic activity compared to wild type LUZ19.
Across the independent claim coverage provided, the core inventive theme is recombinant phage payload expression, with dependent features narrowing payload identity, specific phenol soluble modulins, host specificity, specific phage identity, LUZ19-derived genome similarity, and defined gp mutation sets aimed at improved host range and/or increased lytic activity.
Stated Advantages
Expanded host range.
Increased viral lytic activity.
Early biofilm disruption or biofilm dispersion.
Antibiotic sensitization.
Anti-phage resistance.
Improved viral properties, including altered lytic activity and burst size.
Immune stimulation/deactivation.
Documented Applications
Improving host range and lytic activity, enabling early biofilm disruption or biofilm dispersion, and providing antibiotic sensitization and anti-phage resistance.
Using targeted Cas9 cleavage to map terminally redundant viral ends.
Engineered phage embodiments including reporter engineering, region swapping, replacement, iterative co-engineering, and use in packaging contexts.
Engineered bacteriophages for biofilm-related disruption, including dispersing bacterial biofilms.
Engineered phage embodiments intended to modify viral infection-related characteristics such as adsorption, attachment, injection, replication, assembly, lysis, and host range.
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