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-10221398-B2
Publication Date
2019-03-05
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 disclosure provides a modified LUZ19 podovirus phage engineered to exhibit improved viral properties relative to LUZ19. The engineered phage includes specified genetic alterations that improve host range and/or increase lytic activity compared to wild type LUZ19.
The described genetic alterations include one or more gp mutations selected from gp13 C17Y, gp18 D36Y, gp38 I83S, and gp40 N253D, and an embodiment in which a wildtype LUZ19 gp18 is replaced with gp18 from virus LKD16. The disclosure also includes a gp34 leucine at position 55 deletion (gp34L55Δ) that shows increased lytic activity compared to wild type LUZ19.
The disclosure further describes replacement of the gp49 gene with an expression cassette containing a gene of interest, configured within a LUZ19 regulatory context. The engineered viruses express biofilm-dispersing payloads, antibiotic sensitizing payloads, and/or species-specific antimicrobial payloads, with the overall objective of improved phage efficacy through host range expansion, increased lytic activity, and payload delivery.
Claims Coverage
The document contains one independent claim directed to a modified LUZ19 podovirus phage. The claim requires one or more of four specific modification categories: gp mutations for improved host range, gp18 swap with LKD16, gp34L55Δ for increased lytic activity, and gp49 replacement with an expression cassette carrying a gene of interest.
Engineered LUZ19 phage with gp point mutations for improved host range
A modified LUZ19 podovirus phage comprising one or more mutations selected from gp13 C17Y, gp18 D36Y, gp38 I83S, and gp40 N253D, wherein the phage shows improved host range compared to LUZ19.
Replacement of wildtype LUZ19 gp18 with LKD16 gp18
A modified LUZ19 podovirus phage wherein a wildtype LUZ19 gp18 is replaced with gp18 from virus LKD16.
Gp34 leucine-55 deletion for increased lytic activity
A modified LUZ19 podovirus phage comprising a deletion of leucine at position 55 of gp34 (gp34L55Δ mutation), wherein the phage shows increased lytic activity compared to wild type LUZ19.
Gp49 gene replacement with expression cassette carrying gene of interest
A modified LUZ19 podovirus phage wherein the gp49 gene is replaced with an expression cassette containing a gene of interest.
Overall, the claim set focuses on engineering a LUZ19 podovirus chassis through specified gp point mutations tied to improved host range, a wildtype gp18 replacement sourced from LKD16, a gp34L55Δ deletion tied to increased lytic activity, and replacement of gp49 with an expression cassette to carry a gene of interest.
Stated Advantages
Improved host range compared to LUZ19.
Increased lytic activity compared to wild type LUZ19.
Documented Applications
Engineered viruses implementing biofilm-dispersing payloads, antibiotic sensitizing payloads, and species-specific antimicrobial payloads are described in the document.
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