US 2025/0051796 A1Published application

Engineered viral nucleic acids for directed evolution and uses thereof

AssigneeAlphinia PTY LTD

InventorMinh Thuan Nguyen Tran

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Abstract

The present invention provides nucleic acids sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest. The nucleic acid sequences of the invention have been developed primarily for use in evolution of biomolecules of interest. Using a split, non-competent viral vector, the gene of interest can be stably and recombinantly integrated into the viral vector via in-frame insertion of the open reading frame of the gene of interest with an aspect of the native but attenuated viral genome. This configuration of the viral genome, leveraging a split viral vector and an aspect of a viral factor that has been shown to robustly interact with the split viral vector, enables the serial passaging of the recombinant viral particle in an indel and recombination-averse manner.

Claims17

Dependent claims

A nucleic acid sequence selected from the group consisting of:

a first nucleic acid sequence that comprises 5′ to 3′:
at least part of a 5′ UTR sequence,
at least part of one or more non-structural protein(s) coding sequence,
at least part of a sub-genomic promoter coding sequence,
at least part of a capsid coding sequence,
at least part of a first protease cleavage signal coding sequence,
a gene of interest coding sequence,
at least part of a second protease cleavage signal coding sequence,
at least part of an E3 coding sequence,
at least part of an E2 coding sequence,
at least part of a 6K coding sequence,
at least part of an E1 coding sequence, and
at least part of a 3′ UTR sequence,
wherein the 5′ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3′ UTR sequence are sequences of a Togaviridae virus;
a second nucleic acid sequence that comprises 5′ to 3′:
at least part of a 5′ UTR sequence,
at least part of one or more non-structural protein(s) coding sequence,
at least part of a sub-genomic promoter coding sequence,
at least part of a capsid protein coding sequence,
at least part of one or more structural protein(s) coding sequence(s), and
at least part of a 3′ UTR sequence,
wherein the 5′ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid protein coding sequence, the one or more structural protein(s) coding sequence(s), and the 3′ UTR sequence are coding sequences of a Togaviridae virus;
and
a third nucleic acid sequence that comprises 5′ to 3′:
at least part of a capsid protein coding sequence,
at least part of one or more structural genes coding sequence(s), and
at least part of a 3′ UTR sequence,
wherein the capsid protein coding sequence, the one or more structural proteins coding sequence(s), and the 3′ UTR sequence are sequences of a Togaviridae virus.

25 more elements

A method of evolution of a gene product of a gene of interest, the method comprising:

providing one or more viral vector(s) comprising 5′ to 3′ at least part of a 5′ UTR sequence, at least part of one or more non-structural protein(s) coding sequence, at least part of a sub-genomic promoter coding sequence, at least part of a capsid coding sequence, at least part of a first protease cleavage signal coding sequence, a gene of interest coding sequence, at least part of a second protease cleavage signal coding sequence, at least part of an E3 coding sequence, at least part of an E2 coding sequence, at least part of a 6K coding sequence, at least part of an E1 coding sequence, and at least part of a 3′ UTR sequence, wherein the 5′ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3′ UTR sequence are sequences of a Togaviridae virus;
providing one or more first complement nucleic acid sequence(s) comprising 5′ to 3′ at least part of a 5′ UTR sequence, at least part of one or more non-structural protein(s) coding sequence, at least part of a sub-genomic promoter coding sequence, at least part of a capsid protein coding sequence, at least part of one or more structural protein(s) coding sequence(s), and at least part of a 3′ UTR sequence into the population of suitable host cells, wherein the 5′ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid protein coding sequence, the one or more structural genes coding sequence(s), and the 3′ UTR sequence are sequences of a Togaviridae virus, and wherein expression of the one or more first complement nucleic acid sequence(s) is linked to a function of a gene product of the gene of interest;
introducing the one or more viral vector(s) to a population of suitable host cells;
introducing the one or more first complement nucleic acid sequence(s) to the population of suitable host cells;
allowing maturation and egress of one or more mature virus(es) that comprise the gene of interest or a variant thereof from the population of suitable host cells;
recovering the one or more mature virus(es);
introducing the one or more mature virus(es) and one or more first complement nucleic acid sequence(s) into a population of naïve suitable host cells;
allowing further maturation and egress of further one or more mature virus(es) that comprise the gene of interest or a variant thereof from the population of suitable host cells;
recovering the further one or more mature virus(es);
isolating one or more nucleic acid sequence(s) from the further one or more mature virus(es) to provide one or more isolated nucleic acid sequence(s);
and
isolating the gene of interest or a variant thereof from the one or more isolated nucleic acid sequence(s).

9 more elements

Dependencies are inferred from the claim text.