ENGINEERED VIRAL NUCLEIC ACIDS FOR DIRECTED EVOLUTION AND USES THEREOF

FIELD OF THE INVENTION

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 and will be described hereinafter with reference to this application. It will, however, be appreciated that the invention is not limited to this particular field of use.

BACKGROUND OF THE INVENTION

Directed evolution is an effective strategy for developing one or more gene products of a gene of interest with desirable characteristics. During typical directed evolution, a library of genetic variants is established via mutagenesis of an initial gene of interest. Subsequently, expressed gene products of the gene of interest are selected and assayed for a specific activity and/or function.

Known methods of cell culture-based directed evolution using, for example, complementing mutator polymerases suffer from low, non-dynamic mutation rates, and may not produce sufficient mutagenic diversity to effectively explore the available sequence space of an evolving gene of interest. The use of a mutator polymerase with a fixed mutation rate may also suffer from an inability to modulate its mutagenic rate based on the fitness of the evolving gene product without manual experimenter intervention.

In cases wherein attenuated RNA viruses are used with an inserted gene of interest, significant genomic instability and a tendency towards viral recombination occurs. Here, a gene of interest is often inserted into the viral genome, rendering it non-competent in the absence of a viral factor that has been segmented in trans. Viral propagation is therefore dependent on the reconstitution of the complete viral genome to mediate viral egress. Split viral vector systems fail to propagate in an activity-specific manner to the gene of interest, and either fail to produce sufficient viral titres amenable to serial passaging or show loss of system integrity before variants of interest can become fixed in the gene pool. In the former case, the properties of the gene product of the gene of interest have negligible effects on the propagation of the viral particle, as reconstitution of the segmented viral factor fails to efficiently complement the viral particle. In the latter case, loss of system integrity is hallmarked by viral recombination in which the gene of interest becomes unstable and entirely lost from the split viral vector.

The present invention seeks to ameliorate the shortcomings of the known methods of the directed evolution of a gene product of a gene of interest at least partially or to at least provide a useful alternative.

It is to be understood that, if any prior art information is referred to herein, such reference does not constitute an admission that the information forms part of the common general knowledge in the art, in Australia or any other country.

SUMMARY OF THE INVENTION

There is a need to ameliorate the issue of low and non-dynamic mutation rates by using mutator RNA viruses as a basis for directed evolution and the serial passaging of the gene products thereof. 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. This allows for the steady accumulation of mutations in the gene of interest and allows for its gene products to have improved function and protein solubility. Thus, the present invention discloses a configuration that is amenable to the purposes of directed evolution as sufficiently high viral titres can be achieved without loss of system integrity.

The present invention provides nucleic acid sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest.

According to an aspect of the invention there is provided a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence that includes 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 includes 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 includes 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.

According to another aspect of the invention, there is provided a method of stabilising a recombinant non-competent genome adjacent a 3′ end, the method including inserting a gene of interest in frame with an open reading frame of at least a part of at least one structural protein coding sequence in a non-competent viral vector, wherein the at least one structural protein coding sequence is a Togaviridae virus structural protein coding sequence.

According to another aspect of the invention, there is provided a method of increasing viral titre selected from the group consisting of a first method of increasing viral titre, the first method including: providing a suitable host cell; introducing a viral vector including 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 coding sequences of a Togaviridae virus, into the suitable host cell; introducing one or more first complement nucleic acid sequence including 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 genes coding sequence(s), and the 3′ UTR sequence are sequences of a Togaviridae virus into the suitable host cell; enabling expression of the viral vector in the suitable host cell; and enabling expression of the nucleic acid sequence in the suitable host cell; and a second method of increasing viral titre, the second method including: providing a suitable host cell; introducing a viral vector including 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, and the E1 coding sequence, and the 3′ UTR sequence are sequences of a Togaviridae virus, into the suitable host cell; introducing one or more second complement nucleic acid sequence including 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 genes coding sequence(s), and the 3′ UTR sequence coding sequences of a Togaviridae virus into the suitable host cell; enabling expression of the viral vector in the suitable host cell; and enabling expression of the nucleic acid sequence in the suitable host cell. It will be appreciated that the order of introduction and expression of the viral vector and the nucleic acid to the suitable host cell may occur in any order or simultaneously.

According to another aspect of the invention, there is provided a method of function-dependent propagation of a Togaviridae virus in a suitable host cell, the method includes providing a viral vector including 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; linking expression of the at least part of an in-frame Togaviridae virus capsid nucleic acid sequence and expression of the at least part of one or more in-frame Togaviridae virus structural protein(s) encoding nucleic acid sequence(s) to a function of a gene product of the gene of interest, wherein the at least part of the Togaviridae virus capsid gene and the gene of interest are segmented in trans.

According to any aspect of the invention, there is provided a method of evolution of a gene product of a gene of interest, the method including: 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).

Another aspect of the invention provides a method of evolution of a gene product of a gene of interest selected from the group consisting of: a first method of evolution of a gene product of a gene of interest, the first method including: providing one or more viral vector(s) including 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) including 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 include 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 include 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); and a second method of evolution of a gene product of a gene of interest, the second method including: providing one or more viral vector(s) including 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, and the E1 coding sequence, and the 3′ UTR sequence are sequences of a Togaviridae virus; providing one or more second complement nucleic acid sequence(s) including 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, and 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 second 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 complement nucleic acid(s) to the population of suitable host cells; allowing maturation and egress of one or more mature virus(es) that include 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 second 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 include 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). It will be appreciated that the steps may be repeated to further evolve the gene product of the gene of interest. It will also be appreciated that the order of introducing the one or more viral vector(s) to a population of suitable host cells and introducing the one or more first and/or second complement nucleic acid(s) to a population of suitable host cells may occur in any order or simultaneously.

In some embodiments, the Togaviridae virus is an Alphavirus. In some embodiments, the Alphavirus is Sindbis virus.

BRIEF DESCRIPTION OF THE DRAWINGS

Notwithstanding any other forms which may fall within the scope of the present invention, a preferred embodiment/preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

FIG. 1 is a schematic overview outlining a mechanism of action for an RNA biosensor according to the invention. Modified SinCapsid mRNA complement (SINV biosensor) containing an mCherry fluorophore transgene downstream from a 26S promoter strongly expresses the mCherry fluorophore within 4 hpi. SINV biosensor mRNA is introduced either simultaneously with viral mRNA components via nucleofection at 0 hpi, or via lipid transfection 1 hpi following application of Sindbis viral particles to naive BHK-21 cells. mCherry signal appears inversely proportional to the formation of replication complexes (RC), which could preclude interaction between the SINV biosensor RNA complement and the Sindbis virus non-structural genes (nsp1-4) required to induce transcription from the 26S promoter. SINV, Sindbis virus; RdRp, RNA-dependent RNA polymerase (nsp1-4 complex); RC, replication complex; red barrel shapes objects, mCherry.

FIG. 2 is a graphical representation of results obtained depicting utility of the RNA biosensor. Strongest interaction between Sindbis viral particles and RNA biosensor complement occurs upon cointroduction or after one hour post infection (hpi).

FIG. 3 is a schematic representation of a genomic arrangement of split Sindbis viral nucleic acid sequences which have had either the capsid gene fragment, or the structural glycoproteins removed. A gene-of-interest (GOI) is inserted either downstream of a secondary sub-genomic promoter (dsSIN Capsid or dsSIN Envelope) or inserted in-frame with the capsid gene (SIN2A Capsid). A 2A protease signal derived from foot-and-mouth disease virus (FMD) was used downstream of the GOI to separate it from other aspects of the structural proteins.

FIG. 4 is a schematic representation of passaging steps according to the invention. Schematic showing the passaging method for the determination of cytopathic effects (CPE) indicating viral recombination and competency. Viral variants were passaged serially for five rounds under genetic drift in which the complement component of the recombinant virus is provided in trans, and two rounds of ‘orthogonal passaging’ on naïve cells at the exclusion of their complement components.

FIG. 5 is a heatmap showing non-cytopathic-effects-(CPE)-mediated recombination and competency for dsSIN-Envelope and SIN2A-Capsid, which shows robust GFP expression on the second round of orthogonal passaging as a ratio of GFP+ to brightfield-imaged cells according to the invention. A heatmap showing non-CPE-mediated recombination and competency for dsSIN-Envelope and SIN2A-Capsid, which shows robust GFP expression on the second round of orthogonal passaging as a ratio of GFP+ to brightfield-imaged cells.

FIG. 6 is a representative heatmap displaying the CPE of four viral redesigns following serial passaging, except for an dsSIN-Envelope condition, according to the invention. Representative heatmap displaying the CPE of the four viral redesigns following serial passaging, except for the dsSIN-Envelope condition.

FIG. 7 is schematic representation of engineering of a 26S sub-genomic region of an SIN2A-Envelope variant according to the invention. Engineering of the 26S sub-genomic region of the SIN2A-Envelope variant.

FIG. 8 is a heatmap that depicts effects of removing a packaging signal (PS) from the complement component intended for an SIN2A-Envelope viral variant, as well as a negligible CPE observed during serial passaging upon provision of a complement from a plasmid according to the invention. The heatmap displays the effects of removing the packaging signal (PS) from the complement component intended for the SIN2A-Envelope viral variant, as well as the negligible CPE observed during serial passaging upon provision of the complement from a plasmid. The plasmid expresses the proteinaceous capsid with no 5′UTR or PS elements.

FIG. 9 is heatmap that depicts effects of deletion of a partial capsid fragment results in robust transgene expression but mediates persistent, non-CPE indicating viral recombination and competency according to the invention. Deletion of the partial capsid fragment results in robust transgene expression but mediates persistent, non-CPE indicating viral recombination and competency. At round 3 of orthogonal passaging in the absence of the viral complement, GFP foci are evident, as measured by the ratio of GFP+ cells to a brightfield imaged cells for the same field of view.

FIG. 10 is an image of representative wells of a plaque assay performed with associated GFP images of recombinant viral redesigns for indicated passage round. Representative wells of the plaque assay performed with their GFP images of the recombinant viral redesigns for indicated passage round.

FIG. 11 is a graphical representation of mutation frequency as determined across five rounds of serial passaging under genetic drift conditions according to the invention. Variants were called using LoFreq and setting a minimal allele frequency (MAF) of 0.0025 followed by the subtraction of background sequencing and PCR errors as determined by “Plasmid” sample of the SIN2A-Envelope, which underwent the same pipeline. Mutation frequency is reported as nucleotide mutation per base pair per round of passaging.

FIG. 12 is a graphical representation depicting an SIN2-envelope construct used to screen for nonsense mutations according to the invention. Blasticidin resistance gene (BSR) was inserted into the SIN2A-Envelope construct with and without the introduction of a stop codon (C56X).

FIG. 13 is a graphical representation of passaging steps under permissive, genetic drift conditions for three rounds using mRNA transfection according to the invention. Viral variants were passaged under permissive, genetic drift conditions for three rounds using mRNA transfection. Briefly, R0 (Round 0) virus was applied at an approximate MOI of 1 following titration of viral variants using qPCR. Subsequent passages (R1, R2, and R3; Round 1, Round 2, and Round 3, respectively) were applied by taking 1 mL of clarified, undiluted virus from the previous passage and applying the viral supernatant to BHK-21 cells.

FIG. 14 is a graphical representation of selection of a SIN2A-Envelope variant of SINV against introduction of nonsense mutations in an open reading frame (ORF) of a gene-of-interest (GOI) in accordance with Muller's Ratchet principle according to the invention. Viral variants containing the full BSR ORF were serially propagated with amplifying viral titres, whereas the C56X variant was unable to recover due to disruption of the structural glycoprotein ORF (n=2). NTC, no template control.

FIG. 15 is a graphical representation of preliminary testing of an SP6 circuit; SP6 RNA polymerase (SP6 RNAP), which natively accepts an SP6 promoter, expressed under a CMV promoter following transfection in naive BHK-21 cells according to the invention. Preliminary testing of the SP6 circuit; SP6 RNA polymerase (SP6 RNAP), which natively accepts an SP6 promoter, was expressed under a CMV promoter following transfection in naive BHK-21 cells. A cytosolic plasmid (pUC57-SP6-mCherry) bearing an SP6 promoter driving an mCherry fluorophore was co-transfected and fluorescence was observed.

FIG. 16 is a graphical representation of recombinant SP6 RNAP incubated with a complement plasmid expressing SinCapsid under an SP6 promoter, SP6/T7 hybrid promoter, or a T7 promoter for one hour and visualized on a 1% agarose gel to confirm loss of activity on a non-native SP6 promoter in vitro according to the invention. Recombinant SP6 RNAP was incubated with the complement plasmid expressing SinCapsid under either an SP6 promoter, SP6/T7 hybrid promoter or a T7 promoter for one hour and visualized on a 1% agarose gel to confirm loss of activity on a non-native SP6 promoter in vitro.

FIG. 17 is a schematic representation of a SIN2A-Envelope expressing mutagenized SP6 RNA polymerase (SIN2A-SP6-Envelope) serially passaged on a transfected BHK-21 cell line expressing a viral complement under a modified T7 promoter (TC86-T7) according to the invention. SIN2A-Envelope expressing mutagenized SP6 RNA polymerase (SIN2A-SP6-Envelope) was serially passaged on a transfected BHK-21 cell line expressing the viral complement under a modified T7 promoter (TC86-T7). SP6 RNAP natively accepts an SP6 promoter at round 5 of serial passaging, an increase in viral titre as measured by qPCR against the E2 glycoprotein was observed, which increased progressively until cytopathic effect (CPE) was observed at round 7.

FIG. 18 is a graphical representation of successive rounds of serially passaged SIN2A-SP6-Envelope, which results in enrichment of mutations throughout an open reading frame of SP6 according to the invention. Mutations are mapped for viral variants serially passaged under permissive, genetic drift conditions, along with those mutations mapped following two and seven rounds of selection (R3R2R2). Representative amino acids are coloured and mapped onto the surface representation of the predicted structure for SP6 RNAP as determined by AlphaFold2. Active site (AS) residues are represented in orange.

FIG. 19 is a schematic representation of an SIN2A-Envelope polynucleotide according to the invention. Virus design showing the simplified construct map of recombinant, split Sindbis virus (SIN2A-Envelope). A gene-of-interest (GOI) is inserted in-frame between partial capsid fragment and the complete open reading frame of structural glycoproteins (E3, E2, 6K, E1) intervened by bipartite 2A protease cleavage signal from foot-and-mouth disease virus (FMD).

FIG. 20 is a schematic representation of an SIN2A-Envelope complement polynucleotide in a relaxed form and a stringent form according to the invention. Circuitous amplification of the SIN2A-Envelope construct is dependent on the provision of the in trans complement, of which two circuits are possible. The ‘relaxed’ circuit permits some degree of co-packaging between the complement and the SIN2A-Envelope component owing to the inclusion of the packaging signal (PS), whereas the ‘stringent’ circuit almost entirely abrogates co-packaging and places a higher selective burden for circuitous amplification.

FIG. 21 is a schematic representation of a Capsid expression in a relaxed circuit according to the invention. Relaxed circuit leveraging the expression of the RNA complement from a cytosolic pUC19 plasmid expressing RNA (IncRNA) from a T7 promoter. Induction of the T7 promoter produces the RNA complement for SIN2A-Envelope, enabling interaction between the in trans 26SP from the complement and the RdRp component of SIN2A-Envelope. Refractive amplification enables productive expression of the proteinaceous capsid component, complementing the split virus and allowing for viral egress. 5′UTR, 5′ untranslated region; nsp1, non-structural gene 1; nsp4, non-structural gene 4; 26SP, 26 sub-genomic promoter; SL1234, stem loops 1,2,3,4; 3′UTR, 3′ untranslated region; HDV ribozyme, Hepatitis delta virus ribozyme; and pA, polyA signal.

FIG. 22 is a schematic representation of a circuitous amplification of a split Sindbis virus according to the invention. BHK-21 cells can be engineered to express recombinant T7 RNA polymerase (RNAP) and amplification of the split Sindbis virus (SIN2A-Envelope) is achieved by coupling the expression of the in trans complement with a gated selection circuit.

FIG. 23 is a schematic representation disruption and restoration of a T7 RNA polymerase (RNAP) according to the invention. One-hybrid circuit leveraging the restoration of the open reading frame (ORF) of T7 RNAP drives the expression of the complement allowing for interaction with the non-structural genes of the SIN2A-Envelope component. Disruption of the ORF of T7 requires correction that can be enabled by a packaged gene-of-interest (GOI) in the SIN2A-Envelope, which accumulates necessary mutations to impart improved functionality.

FIG. 24 is a schematic representation of circuitous amplification of split SINV virus (SIN2A-Envelope) achieved by coupling expression of the complement polynucleotide of FIG. 1.2 with activation of a tetracycline-transactivator (tTa) RNA polymerase (RNAP) according to the invention. Engineered BHK-21 cells were lentivirally integrated with tTa. Expression of tTA induces the expression of the viral complement under the ‘strict’ circuit in which proteinaceous capsid is expressed for nucleocapsid assembly for viral egress. Open reading frame (ORF) of tTa is disrupted; introduction of SIN2A-Envelope carrying a gene-of-interest (GOI) capable of reverting the disrupted ORF enables expression of tTa, which drives expression of capsid complement from a nuclear-localized plasmid.

FIG. 25 is a schematic representation of viral entry into and egress from a host cell in an activity gated manner according to the invention. Expression of capsid complement facilitates nucleocapsid assembly, which enables viral budding. Relevant mutations in the GOI meant to enhance the reversion of the disrupted tTa ORF accumulates and is packaged as part of viral genome encapsidation prior to viral egress.

FIG. 26 is a general overview of a use of a viral vector in circuitous evolution of a gene of interest according to the invention. A construct expressing the SIN2A-Envelope viral variant containing ae gene-of-interest (GOI) undergo randomized in vitro mutagenesis using GOI-specific primers and subsequent transformation and selection in competent E. coli cells. Extraction of mutagenic library is followed by appropriate linearization and in vitro transcription, followed by purification of in vitro transcribed mRNA. Mutagenized mRNA is nucleofected with the complement component enabling production of Sindbis viral particles that undergo iterative serial passaging in a circuitous manner. Viral supernatant is harvested and the GOI is amplified via RT-PCR and gel extracted. UMI-specific primers assign randomized nucleotides to the gel-extracted GOI to assign a unique molecular barcode to each PCR amplicon allowing for the elimination of background sequencing artefacts associated with nanopore sequencing.

FIG. 27 is a graphical representation and photomicrographs of a split, recombinant Sindbis virus containing an EGFP transgene reporter (pTSIN-EGFP) generated using nucleofection of in vitro transcribed SinCapsid, SinHelper, and pTSIN-EGFP mRNA, as described using the VEGAS platform. In total, five conditions were evaluated for their capacity to enable the serial passaging of the VEGAS system under genetic drift condition. Briefly, 250,000 BHK-21 cells were plated in a T25 flask, and transfected using pCMV-SSG, which is a mammalian expression plasmid enabling the overexpression Sindbis virus structural genes and envelope proteins (Capsid, E3, E2, 6K and E1, respectively) under a constitutive CMV promoter. The transfected cells were subsequently transduced with undiluted viral supernatant generated from the previous round, and after 24 hours, the cells were observed for viral transduction efficiency using EGFP fluorescence as a reporter. As compared to RNA transfection of cells in which in vitro transcribed SinCapsid and SinHelper mRNA were introduced, a significant drop in viral titre was observed as measured by the number of EGFP foci within a limited field of view. As pCMV-SSG contains no upstream elements to the structural genes, such as 5′UTR or non-structural genes, three conditions were performed in parallel in which in vitro transcribed SinCapsid and SinHelper had their upstream 5′ elements removed from one or both of the IncRNAs to evaluate the necessity of these 5′ upstream elements to enable serial passaging of the split, recombinant Sindbis virus. Deletion of these upstream elements showed that they were necessary for sufficient viral propagation of a split, recombinant Sindbis virus under genetic drift conditions.

FIG. 28 shows the tetracycline transactivator (tTA) open reading frame integrated into the SIN2A-vector and evolved against varying concentrations of doxycycline and defective interfering particles across several rounds. Variations to the circuit set-up in which the induction of the tTA-inducible PspCas13b, which knockdowns defective interfering RNA, were tested in a plasmid set-up (A, B) and in a cell line set-up (C, D). Mutational profile was evaluated with variations to the concentration of defective interfering RNA spiked into each round, with stringent (A, C, D) and relaxed selective (B) pressures tuned with respect to low multiplicity of infection (A, C) and high multiplicity of infection (B, D). The mutational landscape revealed the prevalence of several putative mutations that conferred doxycycline insensitivity that did not occur in the initial round 0 virus, or in the genetic drift condition in the absence of doxycycline addition (Negative).

FIG. 29 is a graphical representation relating to the characterisation of several mutants identified from several rounds of evolving a doxycycline-resistant mutant of a tetracycline transactivator (tTA) protein incorporated into the SIN2A-vector. Single nucleotide mutations enriched in the non-genetic drift conditions were re-synthesised into a wildtype pCMV-tTA plasmid upon identification of mutants following next generation deep sequencing of the viral supernatant derived from several trials of selection. Next, the mutant variants of tTA were incubated in the presence or absence of 100 nM of doxycycline to evaluate the induction of an mCherry reporter construct driven by a TETO7 promoter. Apart from some mutants, the induction of tTA is mostly unchanged in the absence of doxycycline. However, when media containing 100 nM of doxycycline is supplemented, several mutants show significant induction of the mCherry reporter relative to the wildtype variant of tTA, with modest induction evident by several other mutants.

FIG. 30 shows the tetracycline transactivator (tTA) open reading frame integrated into the SIN2A-vector (SIN2A-tTA-ENV) and evolved against varying concentrations of doxycycline, which was supplemented into the media (2 nM DOX, 20 nM DOX, 200 nM DOX, 2 μM DOX, and 20 μM DOX). Briefly, per T25 flask, 250,000 BHK21 cells were plated and equimolar amounts of defective helper (DH) and defective interfering (DIP) RNA were supplemented in the presence of viral supernatant from the preceding round. Viral supernatant containing the SIN2A-tTA-ENV recombinant viral particles were transduced at a multiplicity of infection of 1, and qPCR was performed tiled against the packaging signal (PS), which provides a global measure of selection strength, and the viral glycoprotein (E2), which provides a measure of viral particles.

DESCRIPTION OF EMBODIMENTS

Notwithstanding any other forms which may fall within the scope of the present invention, preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures, as relevant.

The present invention employs methods and techniques that are known to a person having ordinary skill in the art. Such methods and techniques as employed in the present invention include conventional molecular biology, microbiology, and recombinant DNA methods and techniques as disclosed and explained fully in the relevant literature, for example only, Becker's World of the Cell, 9^thedition, Hardin, J., et al., Pearson (2015); Essential Cell Biology, 5^thedition, Alberts, B, et al., T&F/Garland (2019); Freshney's Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Freshney, R. I., and Capes-Davis, A., Wiley-Blackwell (2021); Gel Electrophoresis: Nucleic Acids, Martin, R., Garland Science (2020); Karp's Cell and Molecular Biology, 9^thedition, Karp, G., et al., Wiley (2020); Lewin's Genes, 12^thedition, Krebs, J. E., et al., Jones & Bartlett Learning (2017); Molecular Biology of the Cell, 6^thedition, Alberts, B., et al., Garland Science (2014); Molecular Biology of the Gene, 7^thedition, Watson, J., et al., Pearson (2013); Molecular Biology, 5^thedition, Weaver, R., McGraw-Hill Education (2011); Molecular Biology: Principles of Genome Function, 2^ndedition, Craig, N., et al., Oxford University Press (2014); Molecular Cell Biology, 8^thedition, Lodish, H, W. H. Freeman (2016): Molecular Cloning: A Laboratory Manual, Volumes 1, 2, and 3, 4^thedition, Green, M. R., and Sambrook, J., Cold Spring Harbour Laboratory Press (2014); and Nucleic Acid Hybridization, Anderson, M. L. M., Garland Science (2020).

Abbreviations of amino acids and nucleic acids, and analogs and derivatives of amino acids and nucleic acids will be known to a person having ordinary skill in the art. Such abbreviations may be found as published as the International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry and Molecular Biology (IUBMB) recommendation (see, for example only, Amino Acids and Peptides, 1985, 16, 387-410; Arch. Biochem. Biophys. 1971, 145, 425-436; Biochem. J., 1971, 120, 449-454; Biochem. J., 1984, 219, 345-373; Biochem. J., 1985, 229, 281-286; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69, 109-114, and 122-126; Biochemistry, 1971, 9, 4022-4027; Biochim. Biophys. Acta 1971, 247, 1-12; Eur. J. Biochem., 1970, 15, 203-208; 1972, 25, 1; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Eur. J. Biochem., 1985, 150, 1-5; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1970, 245, 5171-5176; J. Biol. Chem., 1985, 260,14-42; J. Biol. Chem., 1986, 261, 13-17; J. Mol. Biol., 1971, 55, 299-310; Mol. Biol. Evol., 1986, 3, 99-108; Nucl. Acids Res., 1985, 13, 3021-3030; Proc. Nat. Acad. Sci. (U. S.), 1986, 83, 4-8; Pure Appl. Chem., 1974, 40, 277-290; and Pure Appl. Chem., 1984, 56, 595-624 with respect to abbreviations of this nature.

The terms “biomolecule”, “biomacromolecule”, and “biological macromolecule” as used herein should be understood to refer to naturally occurring macromolecular compounds and synthetic macromolecular compounds such as nucleic acids, proteins, peptides, lipids, and carbohydrates.

Nucleic acids, also termed “polynucleotides”, as used herein should be understood to include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), artificial nucleic acid analogs, and any combination thereof. Artificial nucleic acid analogs include, for example, glycol nucleic acid, locked nucleic acid, morpholino nucleic acid, peptide nucleic acid, threose nucleic acid, and any combination of such analogs. Nucleic acids should also be understood to include, for example, modified nucleobases and artificial nucleobases.

The terms “nucleic acid” and “nucleic acid sequence” and variations of each term should be understood as used herein to interchangeably refer to a nucleic acid molecule or to a series of letters that indicate the order of nucleotides in a nucleic acid as appropriate in the context of the relevant term.

The term “gene of interest” as used herein should be understood to refer to a nucleotide sequence encoding a gene product of interest. Such a gene product of interest should be understood to refer to a gene product intended to be evolved in a circuitous evolution process as disclosed herein. It will be appreciated that the term “gene of interest” includes variations of the gene of interest that are a result of the circuitous evolution process disclosed herein. A person of ordinary skill will appreciate that a gene of interest could be any nucleic acid encoding a gene product to be evolved.

The terms “protein” and “peptide” and derivatives thereof as used herein should be understood to refer to, for example, molecules including amino acids, and analogs, and derivatives of amino acids.

The term “protein” as used herein should be understood to mean proteins, polypeptides, and peptide of any size, structure, or function. The term as used herein should be understood to refer to a plurality of amino acids linked by peptide bonds. Proteins, as understood herein, include naturally occurring and non-naturally occurring amino acids. Such naturally occurring and non-naturally occurring amino acids may also be modified by addition of one or more chemical entity. Furthermore, proteins may include naturally occurring and non-naturally occurring amino acids analogs and/or derivatives. A person of ordinary skill will understand that proteins may be a single molecule or a plurality of molecules in a complex. Proteins as understood herein include protein fragments, naturally occurring molecules, synthetic molecules, recombination molecules, and any combination of the afore mentioned molecules.

The term “lipid” should be understood as used herein to refer to, for example, diglycerides, fat-soluble vitamins (such as vitamins A, D, E, and K), fatty acids, glycerolipids, glycerophospholipids, monoglycerides, phospholipids, polyketides, prenols, saccharolipids, sphingolipids, sterols, triglycerides, waxes, and analogs and derivatives of the afore mentioned.

The term “carbohydrate” should be understood as used herein to refer to, for example, monosaccharides, disaccharides, oligosaccharides, polysaccharides, and analogs and derivatives of the afore mentioned.

Nucleic Acid Sequences

One aspect of the invention provides a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence that includes 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 includes 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 includes 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.

Promoters useful in the present invention include, but are not limited to, a polynucleotide motif related to the recruitment of VP64-p65-Rta elements, miniCMV promoter, a motif ten element and downstream promoter element, a polynucleotide motif encompassing a TATA box, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of Rta, a polynucleotide motif related to the recruitment of VP64, SP6 promoter in a mammalian expression vector system, T3 promoter in a mammalian expression vector system, T7 promoter in a mammalian expression vector system, a tetracycline response element, and a tetracycline-inducible promoter.

In some embodiments, the Togaviridae virus is an Alphavirus.

In some embodiments, the Alphavirus is Sindbis virus.

In some embodiments, the 5′ UTR sequence is SEQ ID NO. 1.

In some embodiments, the one or more non-structural protein(s) coding sequence(s) is any one or more of SEQ ID No. 2, 3, 4, and 5.

In some embodiments, the gene of interest coding sequence and the sub-genomic promoter coding sequence are operably linked.

In some embodiments, the sub-genomic promoter coding sequence is 26S promoter coding sequence.

In some embodiments, the 26S promoter coding sequence is SEQ ID NO. 6.

In some embodiments, the capsid protein coding sequence is SEQ ID NO. 7.

In some embodiments, each of the first protease cleavage signal coding sequence and the second protease cleavage signal is a self-cleaving peptide coding sequence.

In some embodiments, each of the first protease cleavage signal coding sequence and the second protease cleavage signal is independently selected from the group consisting of equine rhinitis A virus E2A coding sequence, foot-and-mouth disease virus F2A coding sequence, porcine teschovirus-1 2A P2A coding sequence, and those assigned as virus 2A T2A coding sequence.

In some embodiments, the self-cleaving peptide coding sequence is SEQ ID NO. 8.

A person of ordinary skill in the art will appreciate that the first self-cleaving peptide coding sequence and/or the second self-cleaving peptide coding sequence can be substituted with any coding sequence that induces ribosomal skipping.

In some embodiments, the one or more structural protein(s) coding sequence(s) is at least a part of the E3 structural protein coding sequence.

In some embodiments, the E3 structural protein coding sequence is SEQ ID NO. 9.

In some embodiments, the one or more structural protein(s) coding sequence(s) is at least a part of the E2 structural protein gene coding sequence.

In some embodiments, the E2 coding sequence is SEQ ID NO. 10.

In some embodiments, the one or more structural protein(s) coding sequence(s) is at least a part of the 6K structural gene coding sequence.

In some embodiments, the 6K coding sequence is SEQ ID NO. 11.

In some embodiments, the one or more structural protein(s) coding sequence(s) is at least a part of the E1 structural gene coding sequence.

In some embodiments, the E1 coding sequence is SEQ ID NO. 12.

In some embodiments, the 3′ UTR sequence is SEQ ID NO. 13.

In some embodiments, the gene of interest encodes a biomolecule.

In some embodiments, the gene of interest encodes at least one precursor of a biomolecule.

In some embodiments, the gene of interest encodes at least one enzyme of a biosynthetic pathway of a biomolecule.

In some embodiments, the second nucleic acid sequence further includes at least a part of an RNA polynucleotide that encodes a non-coding RNA necessary for viral replication coding sequence.

In some embodiments, the third nucleic acid sequence further includes at least a part of an RNA polynucleotide that encodes a non-coding RNA necessary for viral replication coding sequence.

In some embodiments, the RNA polynucleotide that encodes a non-coding RNA necessary for viral replication coding sequence is hepatitis delta virus (HDV) ribozyme coding sequence.

In some embodiments, the hepatitis delta virus (HDV) ribozyme coding sequence is SEQ ID NO. 17.

In an embodiment, the RNA polynucleotide that encodes an RNA necessary for viral replication coding sequence is any one of SEQ ID NO. 7 to 13.

In an embodiment, the second nucleic acid sequence further includes at least a part of a polyadenylation signal coding sequence.

In an embodiment, the third nucleic acid sequence further includes at least a part of a polyadenylation signal coding sequence.

In some embodiments, the polyadenylation signal coding sequence is a bGH PolyA coding sequence.

In some embodiments, the bGH PolyA coding sequence SEQ ID No. 18.

In some embodiments, the polyadenylation signal coding sequence is any one of SEQ ID NO. 16 and 18.

In some embodiments, the second nucleic acid sequence further includes at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA.

In some embodiments, the third nucleic acid sequence further includes at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA.

In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE).

In some embodiments, the constitutive transport element (CTE) is a type D retrovirus constitutive transport element.

In some embodiments, the constitutive transport element (CTE) is an RNA hairpin motif that binds transporter associated with antigen processing (TAP) host factors.

In some embodiments, the constitutive transport element (CTE) is SEQ ID NO. 14.

In some embodiments, the constitutive transport element (CTE) is SEQ ID NO. 15.

Method of Stabilising a Recombinant Non-Competent Alphavirus Genome Adjacent a 3′ End

Another aspect of the invention provides a method of stabilising a recombinant non-competent genome adjacent a 3′ end, the method includes inserting a gene of interest in frame with an open reading frame of at least a part of at least one structural protein coding sequence in a non-competent viral vector, wherein the at least one structural protein coding sequence is a Togaviridae virus structural protein coding sequence. It will be appreciated that a method of stabilising a recombinant non-competent genome adjacent a 3′ end, the method including inserting a gene of interest in frame with an open reading frame of at least a part of at least one non-structural protein coding sequence in a non-competent viral vector, wherein the at least one non-structural protein coding sequence is a Togaviridae virus non-structural protein coding sequence is also contemplated.

As used herein, the term “viral vector” should be understood to refer to a nucleic acid that includes a viral genome which, when introduced into a suitable host cell or used as a template for in vitro transcription, can produce viral RNA which can be replicated and packaged into viral particles. Such viral particles transfer the viral genome into another host cell. It will be appreciated that the term “viral vector” extends to at least part of a viral genome, i.e., truncated and/or partial viral genomes. In some embodiments, a viral vector is provided that lacks one or more gene encoding a protein essential for generation of an infectious viral particle. As used herein the term “viral particle” should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope. As used herein, the term “infectious viral particle” as herein should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.

A person skilled in the art will appreciate, as an example, that the viral vector can be linearized via a restriction digest and used as a template for in vitro transcription if the viral coding sequence is downstream of an SP6 or T7 promoter using ordinary kits (i.e., mMESSAGE mMACHINE™ SP6 Transcription Kit, Thermo Fisher, #AM1340).

In some embodiments, a non-competent viral vector is provided that lacks one or more gene(s) encoding at least one protein essential for generation of an infectious viral particle.

As used herein the term “viral particle” should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope. As used herein, the term “infectious viral particle” as herein should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.

In some embodiments, the Togaviridae virus structural protein coding sequence is selected from the group consisting of the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, and the E1 coding sequence of a Togaviridae virus.

In some embodiments, the capsid coding sequence is SEQ ID NO. 7.