Chimeric structural proteins, polycistronic architectures for enhanced acoustic reporter genes and related constructs, genetic circuits, vectors, cells, hosts, compositions, methods and systems

Abstract

Genetically engineered gas vesicle expression systems (GVES), polycistronic architectures, chimeric structural proteins, and engineered assembly complexes configured for enhanced nonlinear acoustic detection. An engineered configurable gas vesicle architecture comprising a multi-tier stoichiometric hierarchy wherein structural gvp genes are expressed at a higher Dosage Index (DI) than assembly factor genes, and wherein the assembly factor gene modules follow a descending DI hierarchy (AF1 > AF2 > AF3). Also provided is a chimeric gas vesicle structural protein comprising a core structural shell domain fused to a stabilizing tail domain, conferring a right-shifted critical acoustic collapse pressure, and a yield-enhancing GvpN-GvpU assembly protein complex. Combinations of these modules produce order-of-magnitude increases in intracellular volumetric yield and enable high-resolution, multiplexed volumetric nonlinear acoustic imaging and dynamic biosensing of target cells in deep-tissue environments.

Description

Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCTCHIMERIC STRUCTURAL PROTEINS, POLYCISTRONIC ARCHITECTURES FOR ENHANCED ACOUSTIC REPORTER GENES AND RELATED CONSTRUCTS, GENETIC CIRCUITS, VECTORS, CELLS, HOSTS, COMPOSITIONS, METHODS AND SYSTEMSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 788,467 entitled “Updated Mammalian Gas Vesicle Genes for Enhanced Contrast, Expression And Multiplex” filed on April 14, 2025, with docket number CIT 9303-P, incorporated herein by reference in its entirety.

[0002] The present application may also be related to US application S / N 19 / 377,725 entitled “Stoichiometric Gas Vesicle Expression System And Related Constructs, Genetic Circuits, Vectors, Cells, Hosts, Compositions, Methods And Systems” filed on November 3, 2025 with Docket number P3286-US, which in turn claims priority to US Provisional Application No.63 / 715,040, entitled “Ultrasound Imaging Of Gene Expression And Activation In Immune Cells” filed on November 1, 2024, with docket number CIT 9230-P, the contents of each of which are incorporated herein by reference in their entirety.

[0003] The present application may further be related to U.S. Patent Application Serial Number 18 / 066,814 filed on December 15, 2022 and published with publication number US 2023 / 0210926, to U.S. Patent Application Serial Number 16 / 736,683 filed on January 7, 2020, (now U.S. Patent No. 11,761,008), to US Application S / N 16 / 736,581 filed on January 7, 2020 (now US Patent 11,786,218), and to PCT Application Number PCT / US2020 / 012557 filed on January 7, 2020, the contents of each of which are also incorporated by reference in their entirety.STATEMENT OF GOVERNMENT GRANT

[0004] This invention was made with government support under Grant No. EB018975 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE STATEMENT FOR SEQUENCE LISTINGTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0005] Further, the computer readable form of the sequence listing of the ST26 XML file P3343-US-Seq-ID created on April 13,2026, and having size615, 899 bytes measured on Windows Server 2019 Datacenter is incorporated herein by reference in its entirety.FIELD

[0006] The present disclosure relates generally to the fields of synthetic biology, genetic engineering, and non-invasive imaging. More specifically, the disclosure relates to chimeric structural proteins, polycistronic architectures for enhanced acoustic reporter genes and related constructs, genetic circuits, vectors, cells, hosts, compositions, methods and systems.BACKGROUND

[0007] Non-invasive imaging of cellular function within deep-tissue environments is highly desirable in biomedical research and clinical applications. Acoustic imaging provides a widely available, cost-effective imaging modality that offers sub-millimeter spatial resolution at substantial tissue depths.

[0008] While these systems are useful, adapting complex, multi-gene prokaryotic operons for efficient expression and multiplexed imaging in eukaryotic or mammalian hosts is still challenging in particular when related to optimizing the stoichiometric expression of assembly factors to increase overall yield, and engineering structurally reinforced gas vesicle chassis that possess distinct acoustic signatures.SUMMARY

[0009] Provided herein are engineered gas vesicle expression systems, polycistronic architectures, chimeric structural proteins, engineered assembly complexes, and related methods. These components provide a systems-level, multiparametric toolbox for gas vesicle configuration directed to independently or synergistically optimize at least one of the stoichiometric expression of the gas vesicle system, the mechanical stability of the gas vesicle shell, and the functional assembly of the gas vesicle complex. This systems-level optimization is configured to increase overall intracellular expression yield, reduce cellular toxicity, and enable non-destructive, multiplexed acoustic imaging.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0010] According to a first aspect, the genetic architectures described herein provide a systems-level approach configured to optimize the stoichiometric expression of the assembly factor components of a gas vesicle system to provide an Assembly Factor Hierarchical Gas Vesicle Expression System (GVES), herein also referred to as an AF-hierarchical GVES.

[0011] In the AF-hierarchical GVES herein described, a gas vesicle gene cluster is partitioned into discrete operational modules which are configured to be expressed according to a hierarchical stoichiometry defined in terms of a Dosage Index (DI) ratio. The Dosage Index is a quantitative parameter representing the normalized expression output of each distinct gene module throughout a target cell population where the GVES is expressed. In particular, in the AF-hierarchical GVES herein described, the hierarchical expression of the assembly factor genes results in increased yield and a reduction of metabolic burden for the target cells where the GVES is expressed.

[0012] Accordingly, and according to the first aspect of the present disclosure, a genetically engineered AF-hierarchical GVES is described, configured for expressing in a target cell within a target site a gene cluster of gvp genes (GVGC) encoding GV proteins capable of forming a GV type. The AF-hierarchical GVES comprises:a) at least one structural gvp differentially expressed gene module comprising a chimeric gvpA / B gene of the GV gene cluster under control of a promoter and additional regulatory regions in a configuration allowing expression of a GvpA / B protein in the target cell with a structural Dosage Index (DI);b) at least one first assembly factor (AF1) gvp differentially expressed gene module comprising an AF 1 set of gp genes of the GV gene cluster encoding for a first set of GV assembly factors AF1 comprising at least a gvpG gene and a gvpF gene, under control of a promoter and additional regulatory regions in a configuration allowing expression of the GV assembly factors AF 1 in the target cell with an AF1 Dosage Index (DI);c) at least one second assembly factor (AF2) gp differentially expressed gene module comprising an AF2 set of gvp genes of the GV gene cluster encoding for a second set of GV assembly factors AF2 comprising at least a gvpN gene, and optionally a gvpW gene, under controlTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT of a promoter and additional regulatory regions in a configuration allowing expression of the GV assembly factors AF2 in the target cell with an AF2 Dosage Index (DI); andd) at least one third assembly factor (AF3) gp differentially expressed gene module comprising an AF3 set of gvp genes of the GV gene cluster encoding for a third set of GV assembly factors AF3 comprising at least a gvpK gene, and optionally a gvpj gene, under control of a promoter and additional regulatory regions in a configuration allowing expression of the GV assembly factors AF3 in the target cell with an AF3 Dosage Index (DI).

[0013] In the AF -hierarchical GVES, the configuration and composition of the gvp gene modules can vary. Each of the structural gvp gene expression module, the AF 1 gvp gene expression module, the AF2 gvp gene expression module, and the AF3 gvp gene expression module, when comprising two or more gvp genes, further comprises a separation element between the two or more gvp genes configured to provide a separate expression of the corresponding GV protein.

[0014] In the AF-hi erar chi cal GVES, the structural module and the one or more assembly factor modules are operably linked by regulatory sequences allowing co-expression of the GV proteins with a Target Dosage Index (DI) ratio of structural-DI : AF1-DI : AF2-DI : AF3-DI. To mitigate cellular toxicity and metabolic bottlenecks during gas vesicle formation, the AF-hi erarchi cal GVES comprises an assembly factor stoichiometric configuration wherein the AF1-DI is greater than the AF2-DI, and the AF2-DI is greater than the AF3-DI (AF1-DI > AF2-DI > AF3-DI). By enforcing this specific descending hierarchy among the assembly factors, the system significantly increases the intracellular volumetric yield of assembled gas vesicles.

[0015] In preferred embodiments, this hierarchical assembly factor configuration operates in synergistic combination with a macro-stoichiometric configuration, wherein the structural-DI is configured to be greater than the assembly factor Dis. As a consequence of the maximized volumetric yield provided by this dual-level stoichiometric configuration, the target cells reach or surpass a threshold density of assembled gas vesicles. This allows the target cells to transition from an acoustically sub-threshold state to triggering a detectable nonlinear acoustic scattering signature that is statistically distinguishable from the environmental noise floor of the target site.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0016] n some embodiments of the AF -Hi erar chi cal GVES, the gvp gene modules are configured across a plurality of polynucleotide constructs or arranged along a single polycistronic construct utilizing tunable genetic elements (e.g., self-cleaving peptides, IRES, or ribosome binding sites) to enforce the descending Dosage Index (DI) hierarchy. This hierarchical configuration increases the gas vesicles' volumetric yield and improves cellular toxicity profiles by mitigating the accumulation of misfolded components, thereby reducing the metabolic burden on the host cell and enabling the system to achieve the density required for robust nonlinear acoustic detection..

[0017] In embodiments where the constructs are comprised within delivery vectors subject to physical packaging limits (e.g., viral vectors), the structural, AF1, AF2, and AF3 modules and related gvp genes are preferably configured across at least three polynucleotide constructs. This multi-construct partitioning further enhances the overall yield and transduction efficiency by bypassing the titer-capacity trade-off, ensuring the target cell receives the necessary genetic payload to assemble gas vesicles at a density sufficient for high-resolution acoustic mapping without inducing vector-mediated toxicity or genomic instability.

[0018] In addition or in the alternative, the present disclosure provides structural gas vesicle proteins configured to physically alter the mechanical stability of the gas vesicle shell, thereby providing engineered gas vesicles that exhibit distinct, measurable acoustic signatures. In a first configuration comprising wild-type structural proteins, the assembled gas vesicles exhibit a baseline acoustic collapse pressure profile. In a second configuration comprising engineered chimeric structural proteins, the resulting gas vesicles exhibit a right-shifted acoustic collapse pressure profile relative to the baseline configuration. This shifted collapse threshold provides a measurable parameter allowing the engineered gas vesicles to be acoustically distinguished from other gas vesicle populations within the same target environment.

[0019] In particular, according to a second aspect, the present disclosure provides a chimeric gas vesicle (GV) structural protein, related engineered polynucleotides, and engineered gas vesicles. The chimeric GV structural protein comprises an N-terminal core structural shell domain derived from a first gas vesicle structural protein (e.g., a GvpA or GvpB) of a first species, and a stabilizing C-terminal tail domain derived from a second gas vesicle structural protein (e.g., a GvpA or GvpB) of a second species, directly or indirectly fused to the core structural shell domain.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0020] The core structural shell domain of the chimeric GV structural protein is an N-terminal region of the chimeric GV structural protein, having a Core N-terminus and a Core C-terminus. The core structural shell domain comprises, in a Core N-terminus to Core C-terminus direction, an alphal-betal-beta2-alpha2 secondary structure topology. The core structural shell domain terminates at the C-terminal end of the alpha2 helix, which forms the Core C-terminus of the core structural shell domain.

[0021] The stabilizing tail domain of the chimeric GV structural protein is a C-terminal region of the chimeric GV structural protein, directly covalently attached to the Core C-terminus of the core structural shell domain. The stabilizing tail domain comprises an unstructured C-terminal region natively located downstream of the alpha2 helix of said second gas vesicle protein. Functionally, the stabilizing tail domain is configured to physically interact with at least one GvpU and / or GvpN assembly protein derived from the same second species. This interaction correlates with an increase in the assembly efficiency of the structural shell, providing an assembled gas vesicle at a detectable volumetric yield.

[0022] In the chimeric GV structural protein, the first species can be selected from species of GV-producing prokaryotes encoding a GvpA or GvpB having a core alphal-betal-beta2-alpha2 domain capable of forming a gas vesicle lattice or a derivative thereof. In particular, the first species can be selected from the group consisting of Anabaena flos-aquae, Halobacterium salinarum, Microcystis aeruginosa, Planktothrix rubescens, and Trichodesmium erythraeum.

[0023] In the chimeric GV structural protein, the second species can be selected from species of GV-producing prokaryotes encoding a GvpA or GvpB having a stabilizing C-terminal tail domain natively projecting from the structural lattice, or a derivative thereof. In particular, the second species can be selected from the group consisting of Bacillus megaterium, Serratia sp. 39006, Desulfovibrio vulgaris, and Streptomyces coelicolor.

[0024] A derivative of a chimeric GV structural protein or a domain thereof encompasses a derivative amino acid sequence having at least 70% overall sequence identity to the sequence of said chimeric GV structural protein or domain thereof. The derivative amino acid sequence can comprise substitutions, deletions, and / or insertions. In the derivative amino acid sequence, theTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT substitutions replace a base amino acid with a functionally equivalent amino acid possessing a side-chain van der Waals volume within + / - 30%, a net formal charge at pH 7.4 within + / - 1, and a Kyte-Doolittle hydropathy index within + / - 1.5 units relative to the corresponding residue of the reference sequence. In the derivative amino acid sequence, the deletions and insertions result in an amino acid sequence having substantially the same domain inclusive of secondary structure and tertiary structure, and substantially the same functionality as the base amino acid sequence.

[0025] In an exemplary representative embodiment, the first species is Anabaena flos-aquae and the second species is Bacillus megaterium, wherein the chimeric GV structural protein comprises an amino acid sequence having at least 70% overall sequence identity to the sequence: MAVEKTNSSSSLAEVIDRILDKGIVIDAWVRVSLVGIELLAIEARIVIASVETYLKYAEAV GLLRDDVEENGLPERSNSSEGQPRFSI (SEQ ID NO: 482).

[0026] Within this representative sequence, the core structural shell domain corresponds to amino acids 1 to 63 of SEQ ID NO: 482, and the stabilizing tail domain corresponds to amino acids 64 to 88 of SEQ ID NO: 482.

[0027] To maintain structural and functional integrity, sequence variance is configured by substituting functionally equivalent amino acids possessing a side-chain van der Waals volume within + / - 30%, a net formal charge at pH 7.4 within + / - 1, and a Kyte-Doolittle hydropathy index within + / - 1.5 units relative to the corresponding residue of the reference sequence.

[0028] Furthermore, sequence variance comprising insertions or deletions is configured such that the alpha 1 -beta l-beta2-alpha2 secondary structure topology of the core structural shell domain is maintained, and the structurally disordered conformation of the stabilizing tail domain is preserved. Specifically, insertions or deletions within the core domain are restricted to parameters that do not disrupt the beta-sheet pairing or the alpha-helical packing required for gas vesicle lattice formation, while insertions or deletions within the tail domain maintain the net negative charge profile required for homotypic repulsion and assembly factor interaction.

[0029] As provided herein, the chimeric GV structural protein can be utilized as an independent aspect to modulate the mechanical stability and acoustic collapse profile of a gas vesicle. Alternatively, the chimeric GV structural protein can be utilized in synergistic combination withTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT the assembly factor hierarchical configuration (Aspect 1), wherein the structural module of the AF -hierarchical GVES encodes the chimeric GV structural protein. In this combined configuration, the GVES yields gas vesicles characterized by both an order-of-magnitude increase in intracellular volumetric density and a right-shifted acoustic collapse threshold.

[0030] The corresponding engineered polynucleotide comprises a chimeric gvpAB gene encoding the aforementioned chimeric GV structural protein. In preferred embodiments, the chimeric gvpA / B gene comprises a fusion junction, lacking intervening linker sequence, between the nucleotide sequence encoding the core structural shell domain and the nucleotide sequence encoding the stabilizing tail domain. Furthermore, the disclosure provides a genetically engineered Gas Vesicle Expression System (GVES) comprising at least one structural gvp differentially expressed gene module comprising the chimeric gvpA / B gene under the control of a promoter and additional regulatory regions configured for expression of the chimeric GV structural protein in a target cell.

[0031] The engineered gas vesicle comprises a protein shell formed from a plurality of the chimeric GV structural proteins. Morphologically, when expressed in a target mammalian cell, the engineered gas vesicles exhibit a high-density clustering phenotype characterized by a multidirectional, interwoven bundling architecture. This architecture is structurally distinct from the parallel planar bundling observed in reference gas vesicles; specifically, adjacent engineered gas vesicles within a cluster intersect at variable non-parallel angles (e g., ranging from approximately 30 degrees to 90 degrees) to form dense, three-dimensional aggregates. The structural integration of the stabilizing tail domain physically reinforces the resulting engineered gas vesicle. Consequently, the engineered gas vesicle exhibits a right-shifted acoustic collapse pressure profile — demonstrating a higher median critical collapse pressure — relative to a reference gas vesicle consisting of a protein shell lacking the stabilizing tail domain. This mechanically robust, interwoven architecture enables distinct acoustic differentiation for multiplexed imaging applications and significantly enhanced ultrasound contrast signals due to the increased volumetric yield and localized spatial density of the assembled vesicles.

[0032] This mechanically robust, interwoven architecture enables distinct acoustic differentiation for multiplexed imaging applications and significantly enhanced ultrasound contrast signals dueTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT to the increased volumetric yield and localized spatial density of the assembled vesicles. Empirically, when subjected to a stepwise acoustic pressure ramp, the engineered gas vesicles exhibit a delayed, right-shifted acoustic collapse profile, sustaining structural integrity at acoustic pressures where reference gas vesicles have already collapsed. Furthermore, the chimeric structural shell remains fully compatible with outer scaffold proteins (e.g., GvpC or engineered variants thereof), allowing for compounding acoustic reinforcement. Based on this empirical acoustic scattering profile and biophysical modeling of the integrated stabilizing tail domain, a protein shell formed entirely from the chimeric GV structural proteins possesses an intrinsic median critical collapse pressure right-shifted by at least 150 kPa relative to a reference shell consisting solely of the first gas vesicle protein A, with theoretical buckling limits extending between approximately 700 kPa and 1.2 MPa.

[0033] The disclosure further provides an engineered polynucleotide comprising a chimeric gvp gene (e.g., a chimeric gvpA / B gene) encoding the aforementioned chimeric GV structural protein. In specific structural configurations, the chimeric gvp gene comprises a direct fusion junction lacking any intervening synthetic linker sequence between the nucleotide sequence encoding the core structural shell domain and the nucleotide sequence encoding the stabilizing tail domain. Furthermore, the disclosure provides a genetically engineered Gas Vesicle Expression System (GVES) comprising at least one structural gvp differentially expressed gene module comprising the chimeric gvp gene under the control of a promoter and additional regulatory regions configured for expression of the chimeric GV structural protein in a target cell.

[0034] The engineered gas vesicle comprises a protein shell formed from a plurality of the chimeric GV structural proteins. Morphologically, when expressed in a target cell, the engineered gas vesicles exhibit a measurable high-density clustering phenotype characterized by a multidirectional, interwoven bundling architecture. This architecture is structurally distinct from the parallel planar bundling observed in reference wild-type gas vesicles. Specifically, adjacent engineered gas vesicles within an intracellular cluster intersect at variable non-parallel angles, encompassing angles from approximately 30 degrees to 90 degrees, forming three-dimensional aggregates.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0035] The structural integration of the stabilizing tail domain physically alters the mechanical properties of the resulting engineered gas vesicle. Empirically, when subjected to a stepwise acoustic pressure ramp, the engineered gas vesicles exhibit a delayed, right-shifted acoustic collapse profile relative to a reference gas vesicle consisting of a protein shell lacking the stabilizing tail domain. This interwoven architecture and right-shifted collapse profile provides a distinct acoustic differentiation parameter for multiplexed imaging applications, while the high-density clustering correlates with an increase in localized spatial density of the assembled vesicles.

[0036] Specifically, a protein shell formed entirely from the chimeric GV structural proteins possesses an intrinsic median critical collapse pressure right-shifted by at least 150 kPa relative to a reference shell consisting solely of the first gas vesicle protein A. The structurally integrated tail domain provides the chimera with a theoretical buckling limit extending between approximately 700 kPa and 1.2 MPa. Furthermore, the chimeric structural shell maintains an exterior topology configured to physically interface with outer scaffold proteins (e g., GvpC or engineered variants thereof), allowing for compounding acoustic reinforcement through subsequent scaffold binding.

[0037] In addition or in the alternative, the disclosure provides yield-enhancing assembly complexes obtainable by the co-expression of a GvpU protein that physically interacts with and functionally stabilizes a GvpN assembly complex. This physical interaction correlates with a measurable increase in the intracellular volumetric yield of the assembled gas vesicles without detectably altering the mechanical properties of the structural shell.

[0038] In particular, according to a third aspect, the present disclosure provides an engineered gas vesicle (GV) assembly protein complex and a related genetically engineered Gas Vesicle Expression System (GVES). The engineered GV assembly protein complex comprises a GvpN assembly protein and a GvpU assembly protein. The related GVES comprises at least one assembly factor gvp differentially expressed gene module comprising a gvpU gene and a gvpN gene under the control of one or more promoters and additional regulatory regions configured to co-express the GvpU assembly protein and the GvpN assembly protein in a target cell.

[0039] To establish the requisite structural configuration for this co-expression, the gvpU gene and the gvpN gene can be operatively connected via a multi-cistronic linkage architectureTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT (comprising, for example, a P2A peptide, an Internal Ribosome Entry Site (IRES), or equivalent sequence), or they can be expressed as separate independent cassettes. Furthermore, the expression module is stoichiometrically configured to produce the GvpU assembly protein and the GvpN assembly protein at a functional relative ratio ranging from approximately 0.5:1 to 2.0:1.

[0040] To ensure functional compatibility and form the yield-enhancing assembly complex, the GvpU assembly protein and the GvpN assembly protein are derived from the same target species. Furthermore, this yield-enhancing assembly complex is configured to functionally operate in conjunction with an engineered gas vesicle structural protein comprising a C-terminal stabilizing tail domain derived from said same target species to provide an assembled engineered gas vesicle.

[0041] The integration and co-expression of the GvpU assembly protein functionally stabilizes the assembly complex, leading to a threshold yield improvement in gas vesicle production while substantially maintaining the intrinsic acoustic collapse threshold of the resulting engineered GVs. Because the GvpU assembly protein functions as an assembly chaperone rather than a structural shell component, the acoustic collapse threshold of the engineered GVs comprising the GvpU assembly protein remains within a variance of approximately + / - 10% (or within + / - 50 kPa) relative to the acoustic collapse threshold of a reference expression system lacking the gvpU gene.

[0042] Consequently, the formation of the species-matched GvpN-GvpU complex increases the intracellular volumetric yield of the assembled engineered gas vesicles by at least 5-fold, and preferably by at least an order of magnitude (e.g., at least 10-fold) relative to a baseline expression system lacking the matched GvpU protein.

[0043] In addition, or in the alternative, the disclosure provides an integrated acoustic reporter gene system that physically and functionally combines two or more of the individual gas vesicle engineering modules described herein. This integration provides compounding, measurable increases in the intracellular volumetric yield and acoustic distinctiveness of assembled gas vesicles within a target cell. Achievable via single-transcript or multi -transcript delivery architectures, combining a stoichiometrically optimized polycistronic architecture with a structurally reinforced chimeric shell and / or a yield-enhancing assembly complex provides a system exhibiting both expression efficiency and programmable acoustic performance.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0044] Accordingly, and according to a fourth aspect, a genetically engineered Gas Vesicle Expression System (GVES) and related methods of acoustic imaging are provided. The integrated GVES comprises a gene cluster of gvp genes encoding GV proteins capable of forming a gas vesicle, wherein the system integrates at least two of, and preferably all three of: (i) the assembly factor hierarchical configuration comprising the Target Dosage Index (DI) ratio according to the first aspect; (ii) the chimeric gas vesicle (GV) structural protein according to the second aspect; and (iii) the engineered gas vesicle (GV) assembly protein complex according to the third aspect.

[0045] The operative connection of these modular aspects provides a robust, highly sensitive platform for non-destructive, high-resolution singleplexed and / or, more preferably, multiplexed acoustic imaging. The specific imaging modality is dictated by the selected combinatorial configuration: configurations maximizing intracellular volumetric yield (e.g., integrating the stoichiometric optimization and / or the engineered assembly complex) provide enhanced acoustic contrast sensitivity optimal for singleplexed tracking. Conversely, configurations integrating the chimeric GV structural protein provide programmable acoustic distinctiveness (e.g., right-shifted acoustic collapse pressures) enabling spatiotemporal multiplexing. Ultimately, these combined architectures allow for the precise real-time tracking of cellular therapies, molecular biosensors, and dynamic gene expression circuits deep within living tissues.

[0046] In preferred combined configurations comprising the chimeric GV structural protein and the engineered GV assembly protein complex operating within the hierarchical framework, the integrated GVES comprises a modified Target Dosage Index (DI) ratio relative to a baseline wildtype system. Because the yield-enhancing assembly complex increases the total physical volume of the structural lattice formed, the stoichiometric demand for late-stage capping or accessory assembly factors is correspondingly altered.

[0047] For example, to prevent assembly bottlenecking, the modified Target Dosage Index (DI) ratio can comprise an AF3 Dosage Index (DI) (comprising, e.g., the gvpK gene) that is quantitatively higher than the AF3 Dosage Index (DI) required for reference wild-type systems. While the overall integrated GVES remains functional without this adjustment, configuring this modified DI ratio maximizes the terminal assembly efficiency of the volumetrically enhanced vesicle pool.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0048] According to a fifth aspect, a genetically engineered Gas Vesicle Reporting molecular component (GVRMC) is described, configured as an integrated molecular system for the nonlinear acoustic detection of a target cell. The GVRMC comprises at least one of the Gas Vesicle Expression Systems (GVES) and / or the set of Gas Vesicle polynucleotide constructs (GVPCs) described herein (integrating, for example, the stoichiometrically optimized architecture, the structurally reinforced chimeric shell, and / or the yield-enhancing assembly complex). The regulatory regions of the GVRMC comprise a gas vesicle reporting (GVR) target region configured to be activated and / or inhibited by a molecular component of a genetic circuit.

[0049] In the GVRMC, the gvp genes and regulatory regions are in a configuration allowing expression of the GV proteins encoded by the gvp genes through activation and / or inhibition of the GVR target region, when the genetic circuit operates according to a circuit design in the target cell. In preferred embodiments, the target cell is a mammalian cell and / or the target site is a physiological environment.

[0050] According to a sixth aspect, a genetically engineered gas vesicle reporting (GVR) genetic circuit (GVRGC) configured for expression in a target cell within a target site is described. In the GVR genetic circuit, molecular components are connected one to another in a target cell in accordance with a circuit design by activating, inhibiting, binding, or converting reactions to form a fully connected network of interacting components.

[0051] The GVR genetic circuit comprises a GVRMC described herein in a configuration in which GV proteins encoded by the gvp genes of the GVRMC are expressed — yielding a programmable acoustic distinctiveness or target volumetric yield — when the genetic circuit operates according to the circuit design. In preferred embodiments, the target cell is a mammalian cell and / or the target site is a physiological environment.

[0052] According to a seventh aspect, a method to express gas vesicles in a mammalian cell is described. The method comprises introducing into the target cell within a target site a genetically engineered Gas Vesicle expression system (GVES) herein described for a time and under conditions to allow expression of GV proteins encoded by the gvp genes of the GVES andTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT production of the gas vesicle type in the target cell. In preferred embodiments, the target cell is a mammalian cell and / or the target site is a physiological environment.

[0053] According to an eighth aspect, a genetically engineered target cell is described comprising the Gas Vesicle expression system (GVES) and / or Gas Vesicle Polynucleotide Construct (GVPC) herein described, configured for expression in the genetically engineered target cell.

[0054] According to a ninth aspect, a method to provide a gas vesicle in a host is described. The method comprises introducing into a target cell of the host the genetically engineered Gas Vesicle expression system (GVES) herein described, the introducing performed for a time and under conditions to allow expression of the GV proteins encoded by the gvp genes of the GVES and the production of the gas vesicle type in the target cell. In preferred embodiments, the target cell is a mammalian cell and the host is a mammalian host.

[0055] According to a tenth aspect, a genetically engineered non-human host is described comprising the Gas Vesicle Expression System (GVES), the set of Gas Vesicle Polynucleotide Constructs (GVPCs), the Gas Vesicle Reporting Molecular Component (GVRMC), or the Gas Vesicle Reporting (GVR) genetic circuit (GVRGC) described herein. The non-human host is configured for expression of the GV proteins encoded by the gvp genes and the production of a Gas Vesicle (GV) type in the genetically engineered non-human host.

[0056] According to an eleventh aspect, a method to provide a genetically engineered target cell comprising a GVR genetic circuit (GVRGC) is described. The method comprises genetically engineering the target cell to introduce into the target cell one or more genetically engineered Gas Vesicle Reporting Molecular Components (GVRMC) described herein to provide the GVRGC.

[0057] According to a twelfth aspect, a method is described to image a biochemical event in a target cell comprised in an imaging target site. The method comprises introducing into the target cell a Gas Vesicle Reporting Molecular Component (GVRMC) described herein to provide a GVR genetic circuit in which expression of GV proteins encoded by the gvp genes of the GVRMC and production of the GV type, or an intracellular spatial translocation of the GV type, occurs when the GVR genetic circuit operates according to the circuit design in response to the biochemical event. The introducing is performed for a time and under conditions allowing expression of theTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT GV proteins and production of the GV type, or an intracellular spatial translocation of the GV type, in response to the biochemical event. The method further comprises imaging the target site comprising the target cells by applying a magnetic field and / or ultrasound to obtain an MRI and / or an ultrasound image of the target site. A system is also provided comprising the genetically engineered Gas Vesicle Expression System (GVES), Gas Vesicle Polynucleotide Construct (GVPC), Gas Vesicle Reporting Molecular Components (GVRMC), and / or GVR genetic circuits (GVRGC), related components and / or target cells in a combination for simultaneous combined or sequential use in the imaging methods herein described.

[0058] According to a thirteenth aspect, a method is described to label a target cell. The method comprises introducing into the target cell a GVRMC herein described to provide a GVR genetic circuit in which expression of GV proteins encoded by the gvp genes of the GVRMC and production of the GV type, or an intracellular spatial translocation of the GV type, occurs when the GVR genetic circuit operates according to the circuit design in response to a trigger molecular component. The introducing is performed under conditions resulting in the presence of the trigger molecular component in the target cell. In some embodiments, the method further comprises imaging the target site comprising the target cell by applying a magnetic field and / or ultrasound to obtain an MRI and / or an ultrasound image of the target site. A system is also provided comprising the genetically engineered GVES, GVPC, related polynucleotide constructs, GVR genetic circuits, related components and / or target cells in a combination for simultaneous combined or sequential use in the imaging methods described herein.

[0059] According to a fourteenth aspect, a composition for the stoichiometric expression and nonlinear detection of gas vesicles is described. The composition comprises at least one of a genetically engineered GVES, a set of GVPCs, a GVRMC, and / or a GVRGC described herein, optionally comprised within one or more vectors (e.g., a single-transcript polycistronic lentiviral vector), and / or genetically engineered cells described herein, together with a suitable vehicle. The vehicle is configured for the delivery of the GVES or related components to a target site in a host.

[0060] According to a fifteenth aspect, a method for volumetric nonlinear acoustic detection of acoustic reporter genes using an ultrasound imaging system is described. The method comprises acquiring a temporal sequence of ultrasound volumes of a target region to form a 4DTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT spatiotemporal data stack. The method further comprises calculating a voxel-wise correlation coefficient by comparing a temporal intensity profile of said voxel to a temporal template vector representing a programmable mechanical collapse event of a gas vesicle (e.g., the right-shifted acoustic collapse threshold of the chimeric GV structural protein). A 3D signal map is generated by applying a statistical indicator function to the calculated correlation coefficients to achieve a specific background rejection rate.

[0061] According to a sixteenth aspect, a system for activity-dependent acoustic imaging and non-invasive monitoring of deep-site dynamics of a target cell within a target site is described. The system comprises at least one of the Gas Vesicle Expression System (GVES), the set of Gas Vesicle polynucleotide constructs, or the activity-dependent genetic circuit described herein. The system further comprises an ultrasound controller and matrix array transducer configured to apply differential acoustic pressures and acquire a volumetric temporal stack, and a processor configured to reconstruct a 3D activity map by executing the voxel-wise statistical correlation algorithm.

[0062] The genetically engineered Gas Vesicle Expression Systems (GVES), the chimeric gas vesicle structural proteins, the engineered gas vesicle assembly protein complexes, the set of Gas Vesicle polynucleotide constructs (GVPCs), the Gas Vesicle Reporting Molecular Components (GVRMC), the GVR genetic circuits (GVRGC), and the related vectors, genetically engineered cells, non-human hosts, compositions, imaging methods, and hardware systems described herein can be utilized in a modular "toolbox" configuration. By allowing a person skilled in the art to selectively deploy and combine the assembly factor stoichiometric optimization, the structurally reinforced chimeric shell, and the yield-enhancing assembly complex, the genetically engineered systems overcome the rigid, single-phenotype limitations of conventional reporters.

[0063] Accordingly, the genetically engineered Gas Vesicle Expression Systems (GVES), the chimeric gas vesicle structural proteins, the engineered gas vesicle assembly protein complexes, the set of Gas Vesicle polynucleotide constructs (GVPCs), the Gas Vesicle Reporting Molecular Components (GVRMC), the GVR genetic circuits (GVRGC), and the related vectors, genetically engineered cells, non-human hosts, compositions, imaging methods, and hardware systems described herein provide an engineered, configurable Gas Vesicle Architecture, providingTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT increased Gas Vesicle (GV) yield and target detectability with respect to existing GV architecture counterparts.

[0064] When configured for maximized expression, the engineered configurable Gas Vesicle Architecture of the present disclosure results in a threshold-triggered increase in nonlinear acoustic signal magnitude of at least 10-fold relative to a baseline reference system lacking the modified Target Dosage Index (DI) ratio and the yield-enhancing assembly protein complex. These systems achieve improved delivery efficiency enabled by the single-transcript polycistronic architecture. Furthermore, the systems are characterized by an optimized stoichiometric hierarchy and the integration of the engineered GV assembly protein complex, offering predictably higher ultrasound sensitivity and broader applicability across mammalian cell types, including primary cells.

[0065] In particular, the engineered configurable Gas Vesicle Architecture of the present disclosure can be used to report the location of cells with target spatial sensitivity, quantifiable by achieving a BURST Signal -to-Background Ratio (SBR) greater than 100 (e.g., as observed in mammalian cells in vitro). This enhanced sensitivity results from the structural and functional integration of the stoichiometric optimization and the GvpN-GvpU stabilization complex, which enables high transduction and expression via a single polycistronic vector without requiring multivector co-transfection. This architecture achieves high functional expression while substantially maintaining cell viability, demonstrating a Toxicity / Viability Index statistically equivalent to an unengineered control cell population under Standard Conditions, and preserving primary cell effector functions such as target T-cell cytotoxicity.

[0066] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in several embodiments to allow high-resolution, multiplexed acoustic imaging. The structural integration of the chimeric GvpA / B shell introduces a predictably right-shifted acoustic collapse pressure profile. This structural control serves as a result-effective variable, allowing the engineered gas vesicles to be statistically and acoustically distinguished from distinct gas vesicle populations (e.g., reference wild-type or differently engineered GVs) co-expressed in the same target site or organism. This provides a functional dynamic range for non-destructive, multi-Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT population spatiotemporal mapping, characterized by a median critical collapse pressure right-shifted by at least 150 kPa relative to a reference wild-type gas vesicle.

[0067] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in several embodiments to detect events such as dynamic gene expression, wherein the enhanced GV yield correlates with a high Assembly Efficiency (AE) of functional particles. This high-yield system is achieved through the predefined polycistronic stoichiometric optimization, specifically the Assembly factor microoptimization (wherein AF1-DI > AF2-DI > AF3-DI) operating in conjunction with the structural macrooptimization. This dual-level tuning enables the system to reliably reach a nonlinear visibility threshold, improving the temporal sensitivity of detection for transient biochemical events.

[0068] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in several embodiments to report biochemical events with sensitivity enhanced by the system's combinatorial architecture. This architecture provides high functional output and efficient delivery to primary cells (e.g., primary human T cells or neurons), establishing a wide functional dynamic range. Furthermore, the genetically engineered systems can be used to produce dynamic contrast reflecting cell presence and / or activity. The stoichiometric optimization of the system — characterized by DI ratios achieving macrooptimization (e.g., Structural-DI higher than the total AF-DI) and microoptimization — is specifically configured to facilitate robust quantitative measurements and signal correlations even in high-noise environments.

[0069] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in several embodiments to provide ultrasound imaging, allowing for the detection of gas vesicle-expressing cells at highly sensitive volumetric concentrations in vitro and in vivo. These systems provide high-resolution ultrasound imaging which, in several embodiments, allows the detection of cellular activity in hard-to-transduce primary cells by reaching a required volumetric density of assembled gas vesicles. Once this target density is reached, the system crosses the nonlinear visibility threshold, providing a distinct increase in nonlinear signal magnitude.

[0070] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in several embodiments to track the presence, location, and / or expansion of cells and areTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT particularly suitable for tracking therapeutic cells, such as primary T-cells or chimeric antigen receptor (CAR) T-cells. This is due to the efficient delivery enabled by the single-transcript polycistronic architecture, which circumvents the packaging and co-transduction limitations of multi-construct systems. These system features are characterized by a high Transduction-Expression Index (TEI) and robust acoustic signal over reference controls.

[0071] The engineered configurable Gas Vesicle Architecture of the present disclosure can be used in connection with various applications wherein reporting, labeling, and / or tracking is desired with high sensitivity and efficiency. In these embodiments, the modular GVES architecture and the integration of the yield-enhancing GvpN-GvpU complex enable the non-invasive tracking of cellular homing and expansion in deep tissue. For example, the systems can be used for the visualization of biological events, including the monitoring of cell-based therapies trafficking to tumors. The optimized stoichiometric configuration and structurally robust chimeric shell maximize the nonlinear acoustic properties of the gas vesicles, facilitating tracking in high-noise environments where linear correlations were previously insufficient to distinguish target activity from static physiological clutter.

[0072] The engineered configurable Gas Vesicle Architecture of the present disclosure can also be used in developmental biology, stem cell research, and induced pluripotent stem cell (iPSC)-derived applications, offering improved suitability due to the enhanced GV yield and minimized cellular toxicity. Furthermore, the system provides efficient single-transcript delivery capabilities and functional expression in primary cells without significantly impairing critical physiological functions.

[0073] Additional exemplary applications include uses of the combinatorial GVES, related polynucleotide constructs, GVR genetic circuits, vectors, genetically engineered cells, and genetically engineered non-human mammals in fields including basic biology research, applied biology, bio-engineering, bio-energy, medical research, medical diagnostics, and therapeutics. In particular, the integration of activity -dependent genetic circuits and computational spatiotemporal demixing algorithms enables these systems to be used as biological sensors and actuators. These systems are further applicable for the non-invasive, high-resolution mapping of transient biological events, cellular activity, and neuromodulation in high-noise environments, such as the mammalianTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT brain, and in additional fields identifiable by a skilled person upon reading of the present disclosure.

[0074] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.

[0001] Figure 1 shows an exemplary Clustal omega alignment of amino acid sequences of selected exemplary gvpA and gvpB proteins (SEQ ID NO: 7-10 and 457-472).

[0076] Figure 2 shows exemplary phylogenetic relationships of the gvpA protein sequences from the indicated prokaryotic species . [1]

[0077] Figure 3 shows exemplary phylogenetic relationships of the gvpF and gvpL protein sequences from the indicated prokaryotic species. [1]

[0078] Figure 4 shows exemplary phylogenetic relationships of the gvpN protein sequences from the indicated prokaryotic species. [1]

[0079] Figure 5 shows diagrams illustrating the organization of exemplary gas vesicle gene clusters. Gas vesicle gene clusters from the indicated organisms are shown, with genes shown as block-shaped arrows, and genes of predicted similar function indicated in the same shade of grey. The direction of the transcription of genes within a gene cluster is indicated by the direction of the block-shaped arrows, and genes grouped together having block arrows pointed in the same direction are typically organized in the same operon. The scale bar indicates 1 kb. [1]Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0080] Figure 6 shows diagrams illustrating organization of exemplary gvp gene clusters, wherein each letter indicates a gvp gene, and an arrow beneath a group of letters indicates an operon, with the direction of the arrow indicating the direction of transcription. [2]

[0081] Figure 7A shows a graph illustrating the stoichiometry optimization of GvpK and GvpJ in an Anabaena flos-aquae genetic background, depicting the relative BURST ultrasound contrast level measured in mammalian cells transiently transfected with plasmids encoding GvpA and other gas vesicle proteins at a set DNA molar ratio with variable amounts of plasmids encoding GvpJ and / or GvpK.

[0082] Figure 7B shows a graph illustrating a similar titration and stoichiometry optimization of GvpF, GvpG, and GvpL in a Bacillus megaterium genetic background, where GvpB is titrated while other chaperones are held constant except for specific doubled components.

[0083] Figure 7C shows a schematic of an original mammalian polycistronic construct alongside a newly rearranged construct designed for achieving optimal stoichiometry through the strategic placement of self-cleaving linkers, accompanied by a graph comparing the acoustic performance of the new arrangement with the original.

[0084] Figure 8A shows an image of an overlay of Anabaena flos-aquae GvpA over a CryoEM density of Bacillus megaterium GvpB, with arrows highlighting extra density present in GvpB gas vesicles that is not present in GvpA.

[0085] Figure 8B shows a schematic and sequence alignment detailing the rationale for the engineered GvpA / B hybrid protein, indicating the specific residue differences between GvpB, the engineered GvpA / B hybrid, and GvpA.

[0086] Figure 8C shows a schematic of an original acoustic reporter gene expression system (mARG 2.1) utilizing a wild-type Anabaena GvpA structural protein and Anabaena GvpN.

[0087] Figure 8D shows a schematic of a new, right-shifted acoustic reporter gene expression system (mARG 2.2) utilizing the engineered GvpA / B hybrid structural protein and Bacillus megaterium GvpN.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0088] Figure 8E shows a graph characterizing the acoustic collapse and contrast profiles of the expression systems from Figures 8C and 8D using cross-amplitude modulation (xAM) ramped across a stepwise voltage range.

[0089] Figure 9A shows a computational structural prediction of Bacillus megaterium GvpN and GvpU hexamers, illustrating the GvpU homohexamer interacting with the beta-barrel hexamerization domain of GvpN.

[0090] Figure 9B shows a graph characterizing the acoustic collapse and contrast profile of an updated acoustic reporter gene system incorporating GvpU, demonstrating its performance compared with previous systems using cross-amplitude modulation ramped across a stepwise voltage range.

[0091] Figure 9C shows confocal reflectance microscopy images of gas vesicles over cell autofluorescence, demonstrating the dramatic improvement in expression upon the addition of GvpU to the system.

[0092] Figure 9D shows transmission electron microscopy images of buoyancy purified gas vesicles from cell lysates, confirming the morphology and improved expression of the systems corresponding to Figure 9C.

[0093] Figure 10 shows an example diagram of a 3D imaging system as described herein illustrating the workflow for volumetric nonlinear acoustic detection, including spatiotemporal data stack construction, voxel-wise correlation analysis, and statistical signal rejection.

[0094] Figure 11A is a representative Transmission Electron Microscopy (TEM) micrograph illustrating the planar, parallel bundling architecture of reference mARGana gas vesicles expressed in target cells

[0095] Figure 11B is a representative TEM micrograph illustrating the high-density, multidirectional interwoven bundling architecture of engineered mARG 3.0 gas vesicles comprising the chimeric GvpA / B structural protein expressed in target cells.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0096] Figure 12A: Graphs illustrating the acoustic collapse profiles of the reference mARG 2.1 system co-expressed with wild-type GvpC (left) and engineered URoC (right).

[0097] Figure 12B: Graphs illustrating the right-shifted acoustic collapse profiles of the mARG 2.2 system co-expressed with wild-type GvpC (left) and engineered URoC (right).

[0098] Figure 12C: Graphs illustrating the high-yield acoustic collapse profiles of the fully integrated mARG 3.0 system co-expressed with wild-type GvpC (left) and engineered URoC (right).

[0099] Figure. 13A is a graph illustrating the acoustic collapse pressure profile, measured as cross-amplitude modulation (xAM) signal per cell over time, of a reference mARG 2.1 expression system co-expressed with varying stoichiometric levels of wild-type GvpC (Hi C, Med C, Low C, and AC).

[0100] Figure 13B is a graph illustrating the right-shifted acoustic collapse pressure profile of a mARG 2.2 expression system, comprising the chimeric GvpA / B structural protein and Bacillus megaterium GvpN, co-expressed with varying levels of wild-type GvpC.

[0101] Figure 13C is a graph illustrating the increased intracellular volumetric yield and right-shifted acoustic collapse pressure profile of a mARG 3.0 expression system, comprising the chimeric GvpA / B structural protein and the engineered GvpN-GvpU assembly protein complex, co-expressed with varying levels of wild-type GvpC.DETAILED DESCRIPTION

[0102] Provided herein are genetically engineered gas vesicle expression systems (GVES) and related polynucleotide constructs, including optimized polycistronic architectures and chimeric structural proteins, configured for the efficient expression of a gas vesicle (GV) type in a target cell, such as a mammalian cell. Also provided are related engineered gas vesicle gene clusters, gas vesicle shells, assembly protein complexes, genetic circuits, vectors, genetically engineered target cells, hosts, compositions, methods, and systems.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0103] In particular, the GVES and related components of the present disclosure are configured as enhanced nonlinear contrast and high-resolution nonlinear detection systems. This is achieved through a multifaceted architectural approach that integrates: (i) specific polycistronic configurations designed to enforce a predefined stoichiometric hierarchy (Target Dosage Index ratio) among structural and assembly factor gene modules; (ii) structurally reinforced chimeric gas vesicle shells engineered to provide distinct acoustic collapse signatures; and / or (iii) specific yieldenhancing assembly protein complexes. Together, these systems are configured to significantly increase overall expression yield, enabling the system to efficiently achieve and surpass a threshold density of assembled gas vesicles within a target cell at a target site, thereby triggering a high-magnitude, detectable nonlinear acoustic scattering signature obtained through gas vesicles of the disclosure.

[0104] The wordings “gas vesicles”, GV”, “gas vesicles protein structure”, or “GVPS”, refer to a gas-filled protein structure natively intracellularly expressed by certain bacteria or archaea as a mechanism to regulate cellular buoyancy in aqueous environments [4], In particular, gas vesicles are protein structures natively expressed almost exclusively in microorganisms from aquatic habitats, to provide buoyancy by lowering the density of the cells [4], GVs have been found in over 150 species of prokaryotes, comprising cyanobacteria and bacteria other than cyanobacteria [5, 6], from at least 5 of the 11 phyla of bacteria and 2 of the phyla of archaea described by Woese (1987) [7], Exemplary microorganisms expressing or carrying gas vesicle protein structures and / or related genes include cyanobacteria such as Microcystis aeruginosa Aphanizomenon flos aquae Oscillatoria agardhii, Anabaena, Microchaete diplosiphon and Nostoc, phototropic bacteria such as Amoebobacter, T hiodiclyon, Pelodiclyon, and Ancalochloris. non phototropic bacteria such as Microcyclus aquaticus, Gram-positive bacteria such as Bacillus megaterium Gram-negative bacteria such as Serratia, and archaea such as Haloferax mediterranei, Methanosarcina barkeri, and Halobacteria salinarium, as well as additional microorganisms identifiable by a skilled person.

[0105] In particular, a GV in the sense of the disclosure is an intracellularly expressed structure forming a hollow structure wherein a gas is enclosed by a protein shell, which is a shell substantially made of protein (at least 95% protein). In gas vesicles in the sense of the disclosure,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT the protein shell is formed by a plurality of proteins herein also indicated as GV proteins or gvps, which form in the cytoplasm a gas permeable and liquid impermeable protein shell configuration encircling gas. Accordingly, a protein shell of a GV is permeable to gas but not to surrounding liquid such as water. In particular, GV protein shells exclude water but permit gas to freely diffuse in and out from the surrounding media [8] making them physically stable despite their usual nanometer size, unlike microbubbles, which trap pre-loaded gas in an unstable configuration.

[0106] GV structures are typically nanostructures with widths and lengths of nanometer dimensions (in particular with widths of 45 - 250 nm and lengths of 100 - 800 nm) but can have lengths up to 2 pm in prokaryotes but can have larger dimensions such as up to 8- 10pm as will be understood by a skilled person upon reading of the present disclosure. In certain embodiments, the gas vesicles protein structure have average dimensions of 1000 nm or less, such as 900 nm or less, including 800 nm or less, or 700 nm or less, or 600 nm or less, or 500 nm or less, or 400 nm or less, or 300 nm or less, or 250 nm or less, or 200 nm or less, or 150 nm or less, or lOOnm or less, or 75 nm or less, or 50nm or less, or 25 nm or less, or 10 nm or less. For example, the average diameter of the gas vesicles may range from 10 nm to 1000 nm, such as 25 nm to 500 nm, including 50 nm to 250 nm, or 100 nm to 250 nm. By “average” is meant the arithmetic mean.[00107J GVs in the sense of the disclosure have different shapes depending on their genetic origins [8], For example, GVs in the sense of the disclosure can be substantially spherical, ellipsoid, cylindrical, or have other shapes such as football shape or cylindrical with cone shaped end portions depending on the type of bacteria providing the gas vesicles.

[0108] Representative examples of endogenously expressed GVs native to bacterial or archaeal species are the gas vesicle protein structure produced by the Cyanobacterium Anabaena flos-aquae (Ana GVs) [4], and the Halobacterium Halobacterium salinarum (Halo GVs) [9], In particular, Ana GVs are cone-tipped cylindrical structures with a diameter of approximately 140 nm and length of up to 2 pm and in particular 200-800 nm or longer. Halo GVs are typically spindle-like structures with a maximal diameter of approximately 250 nm and length of 250-600 nm.

[0109] In bacteria or archaea expressing GVs, the genes (herein also gvp genes) encoding for the proteins forming the GVs (herein also GV proteins), are organized in a gas vesicle gene cluster ofTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 8 to 14 different genes depending on the host bacteria or archaea, as will be understood by a skilled person.

[0110] The term “Gas Vesicle Genes Cluster” or “GVGC” as described herein indicates a gene cluster encoding a set of GV proteins capable of providing a GV upon expression within a bacterial or archaeal cell Since the ability of expressed GV proteins to assemble in a GV depends on the cell environment where GV proteins are expressed and a same group of gvp genes may or may not form a GV upon expression in a cell, gvp genes provide GVGCs in a cell dependent manner as will be understood by a skilled person (see on point US application 15 / 663,635 published as US 2018 / 0030501).

[0111] The term “gene cluster” as used herein means a group of two or more genes found within an organism's DNA that encode two or more polypeptides or proteins, which collectively share a generalized function or are genetically regulated together to produce a cellular structure and are often located within a few thousand base pairs of each other. The size of gene clusters can vary significantly, from a few genes to several hundred genes

[0010] , Portions of the DNA sequence of each gene within a gene cluster are sometimes found to be similar or identical; however, the resulting protein of each gene is distinctive from the resulting protein of another gene within the cluster. Genes found in a gene cluster can be observed near one another on the same chromosome or native plasmid DNA, or on different, but homologous chromosomes. An example of a gene cluster is the Hox gene, which is made up of eight genes and is part of the Homeobox gene family. In the sense of the disclosure, gene clusters as described herein also comprise gas vesicle gene clusters, wherein the expressed proteins thereof together are able to form gas vesicles.

[0112] The term “gene” as used herein indicates a polynucleotide encoding for a protein that in some instances can take the form of a unit of genomic DNA within a bacteria, plant, or other organism. The term gene as used herein incudes naturally occurring polynucleotide encoding for a protein as well as engineered polynucleotide whose sequences have been modified from the original sequence for example to optimize expression, e.g. through codon changes (see Examples section) and / or through introduction of modified N-and / or C-terminal modifications, while still maintaining the ability to encode for the protein encoded by the naturally occurring polynucleotide or a or a functional variant thereof.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0113] The term “polynucleotide” as used herein indicates an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers respectively to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or a with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, and in particular DNA RNA analogs and fragments thereof.

[0114] The term “protein” as used herein indicates a polypeptide with a particular secondary and tertiary structure that can interact with another molecule and in particular, with other biomolecules including other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and / or small molecules. The term “polypeptide” as used herein indicates an organic linear, circular, or branched polymer composed of two or more amino acid monomers and / or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full-length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer, peptide, or oligopeptide. In particular, the terms “peptide” and “oligopeptide” usually indicate a polypeptide with less than 100 amino acid monomers. In particular, in a protein, the polypeptide provides the primary structure of the protein, wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds, and additional bonds identifiable by a skilled person. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of alpha-amino acid residues covalently linked by peptide bond or a synthetic covalent linkage. The two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NPb-group), whichTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT is the end where the amino group is not involved in a peptide bond to the C-terminal end (-COOH group) which is the end where a COOH group is not involved in a peptide bond. Proteins and polypeptides can be identified by x-ray crystallography, direct sequencing, immunoprecipitation, and a variety of other methods as understood by a person skilled in the art. Proteins can be provided in vitro or in vivo by several methods identifiable by a skilled person. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and / or analogs thereof are joined through chemically-mediated condensation of an organic acid (-COOH) and an amine (-NH2) to form an amide bond or a “peptide” bond.

[0115] As used herein the term “amino acid”, “amino acid monomer”, or “amino acid residue” refers to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid. In particular, alpha- or a- amino acid refers to organic compounds composed of amine (-NH2) and carboxylic acid (-COOH), and a side-chain specific to each amino acid connected to an alpha carbon. Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity, and pKa. Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the amine group of a first amino acid and the carboxylic acid group of a second amino acid. Amino acid in the sense of the disclosure refers to any of the twenty naturally occurring amino acids, non-natural amino acids, and includes both D an L optical isomers.

[0116] In embodiments herein described identification of a gene cluster encoding GV proteins naturally expressed in bacteria or archaea as described herein can be performed for example by isolating the GVs from the bacteria or archaea, isolating the protein for the protein shell of the GV and deriving the related amino acidic sequence with methods and techniques identifiable by a skilled person (see e.g. procedures described in

[0011]

[0012] ). The sequence of the genes encoding for the GV proteins can then be identified by methods and techniques identifiable by a skilled person. For example, gas vesicle gene clusters can also be identified by persons skilled in the art by performing gene sequencing or partial- or whole-genome sequencing of organisms using wet lab and in silica molecular biology techniques known to those skilled in the art. As understood by those skilled in the art, gas vesicle gene clusters can be located on the chromosomal DNA or native plasmid DNA of microorganisms. After performing DNA or cDNA isolation from aTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT microorganism, the polynucleotide sequences or fragments thereof or PCR-amplified fragments thereof can be sequenced using DNA sequencing methods such as Sanger sequencing, DNASeq, RNASeq, whole genome sequencing, and other methods known in the art using commercially available DNA sequencing reagents and equipment, and then the DNA sequences analyzed using computer programs for DNA sequence analysis known to skilled persons.

[0117] In some embodiments, identification of a gene cluster encoding for GV proteins [9, 13, 14] can also be performed by screening DNA sequence databases such as GenBank, EMBL, DNA Data Bank of Japan, and others. Gas vesicle gene cluster gene sequences in databases such as those above can be searched using tools such as NCBI Nucleotide BLAST and the like, for gas vesicle gene sequences and homologs thereof, using gene sequence query methods known to those skilled in the art. For example, genes of the gene cluster for the exemplary haloarchael GVs (which have the largest number of different gvp genes) and their predicted function and features are illustrated in Example 26 of related U.S. Application No. 15 / 613,104, filed on June 2, 2017, which is incorporated herein by reference in its entirety. GV gene clusters can also be identified using a combination of genomic vicinity (e.g. antiSMASH), protein homology and prior GV gene annotation as will be understood by a skilled person.

[0118] A GV gene cluster encoding for GV proteins typically comprises Gas Vesicle Assembly (GV A) genes and Gas Vesicle Structural (GVS) genes.

[0119] The term Gas Vesicle Structural (GVS) proteins as used herein indicates proteins forming part of a gas-filled protein structure intracellularly expressed by certain bacteria or archaea and can be used as a mechanism to regulate cellular buoyancy in aqueous environments [8], In particular, GVS shell comprises a GVS identified as gvpA or gvpB (herein also referred to as gvpA / B) and optionally also a GVS identified as gvpC.

[0120] In particular, gvpB gene is a gene encoding for gas vesicle structural protein B. gvpB genes is highly homologous to gvpA gene encoding for gas vesicle structural protein A. A gvp A / B is a protein of the GV shell that has a higher than 60% and possibly higher than 70% identity to the following consensus sequence: SSSLAEVLDRILDKGXVIDAWARVSLVGIEILTIEARVVIASVDTYLR (SEQ ID NO: 3)Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT wherein X can be any amino acid. In particular in a gvpA / B of prokaryotes, the consensus sequence of SEQ ID NO: 3 typically forms a conserved secondary structure having an alpha-beta- beta-alpha structural motif formed by portions of the consensus sequence comprising the amino acids LDRILD (SEQ ID NO:4) having an alpha helical structure, RILDKGXVIDAWARVS (SEQ ID NO:5) wherein X can be any amino acid, having a beta strand, beta strand structure, and DTYLR (SEQ ID NO: 6) having an alpha helical structure, as will be understood by a skilled person.

[0121] As used herein, “homology”, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.

[0122] A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.

[0123] A person skilled in the art would understand that similarity between sequences is typically measured by a process that comprises the steps of aligning the two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, i.e. characters similar or identical between the two aligned sequences, and calculating the total numberTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

[0124] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0125] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length protein or protein fragment. A reference sequence can comprise, for example, a sequence identifiable a database such as GenBank and UniProt and others identifiable to those skilled in the art.

[0126] As understood by those skilled in the art, determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller

[0015] , the local homology algorithm of Smith etal.

[0016] ; the homology alignment algorithm of Needleman and Wunsch

[0017] ; the search-for-similarity-method of Pearson and Lipman

[0018] ; the algorithm of Karlin and Altschul

[0019] , modified as in Karlin and Altschul

[0020] , Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTF1T, BLAST, FASTA

[0018] , and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0127] Further examples of modern implementations to determine sequence identity include CLUSTAL [1] [[2] [3]); and T-Coffee ([4] [4]) for multiple sequence alignments. Additional high-throughput and cloud-based implementations useful for large-scale or metagenomic sequence comparison include NCBI BLAST+ [5]), Diamond ([6]), MMseqs2[7], and DeepAlign [Ref. Wang et al., Bioinformatics 29:2640-2648, 2013), which employ GPU acceleration or machinelearning-based scoring functions to enhance alignment speed and accuracy. Alignments using these programs can be performed using the default parameters or parameters optimized according to the needs of the user, as understood by those skilled in the art. Alignments using these programs can be performed using the default parameters optimized according to the needs of the user, as understood by those skilled in the art.

[0128] Thus, a gvpA / B protein in a prokaryote of interest can be identified for example by isolating GVs from a prokaryote of interest, isolating the protein from the protein shell of the GV and obtaining the amino acid sequence of the isolated protein. In addition or in the alternative to isolating the GVs and isolating the protein, the method can include obtaining amino acidic sequences of the shell proteins of the GV of the prokaryote of interest from available database. The method further comprises performing a sequence alignment of the obtained amino acidic sequences against the gvpA / B protein consensus sequence of SEQ ID NO:3.

[0129] In particular the isolating GVs from a prokaryote of interest can be performed following methods to isolate gas vesicles as described in U.S. Application No. 15 / 613,104, filed on June 2, 2017. The isolating the protein for the protein shell of the GV and obtaining the related amino acidic sequence can be performed with tandem liquid chromatography mass-spectrometry alone or in combination with obtaining amino acid sequences of the isolated protein with wet lab techniques or from available databases comprising the sequences of the prokaryote of interest as well as additional techniques and approaches identifiable by a skilled person. Obtaining amino acid sequences of GV shell proteins of the prokaryote of interest can be performed by screening available databases of gene and protein sequences identifiable by a skilled person. Performing a sequence alignment of the sequences of the isolated GV proteins or proteins encoded in the genome of a prokaryote of interest can be performed (using Protein BLAST or other alignment algorithms known in the art) against the gvpA / B protein consensus sequence of SEQ ID NO:3. In particular,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT a sequence alignment can be performed using gvpA / B protein sequences from the closest phylogenetic relative to the prokaryote of interest. Reference is made to Example 1 showing exemplary phylogenetic relationships between gvpA / B proteins of exemplary prokaryotic species.

[0130] The optional gvpC gene encodes for a gvpC protein which is a hydrophilic protein of a GV shell, including repetitions of one repeat region flanked by an N-terminal region and a C terminal region. The term “repeat region” or “repeat” as used herein with reference to a protein refers to the minimum sequence that is present within the protein in multiple repetitions along the protein sequence without any gaps. Accordingly, in a gvpC multiple repetitions of a same repeat is flanked by an N-terminal region and a C-terminal region. In a same gvpC, repetitions of a same repeat in the gvpC protein can have different lengths and different sequence identity one with respect to another.

[0131] Repeat regions within any given gvpC sequence 'X' from organism 'Y' can be identified by comparing the related sequence with the sequence of a known gvpC (herein e.g. reference gvpC sequence “Z”). In particular, the comparing can be performed by aligning sequence 'X' to the reference gvpC sequence 'Z' using a sequence alignment tools such as BLASTP or other sequence alignment tools identifiable by a skilled person at the date of filing of the application upon reading of the present disclosure. In particular, a reference sequence 'Z' is chosen from a host that is the closest phylogenetic relative of ‘ Y’, from a list of Anabaena flos-aquae, Halobacterium salinarum, Haloferax mediditerranei, Microchaetae diplosiphon and Nostoc sp. The sequence alignment of 'X' and 'Z' (e.g. a BLASTP) is performed by performing a first alignment of sequence X and sequence Z to identify a beginning and an end of a repeat in 'X as well as a number of repetition of the identified repeat, in accordance with the known repeats in 'Z'. The first alignment results in at least one first aligned portion of X with respect to reference sequence Z. The aligning can also comprises performing a second alignment between the at least one first aligned portion of X identified following the first alignment and additional portions of X to identify at least one repeat 'RT in X. Other repeats in 'X' (i.e. R2, R3, R4...) can subsequently be identified with respecttoRl. In performing alignment steps sequence are identified as repeat when the sequence shows at least 3 or more of the characteristics described in US application 15 / 663,635 published as USTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 2018 / 0030501 (incorporated herein by reference in its entirety) which also include additional features of gvpC proteins and the related identification.

[0132] In a GVGC, the GVS genes are comprised with Gas Vesicle Assembly genes. The Gas Vesicle Assembly genes are genes encoding for GVA proteins. GVA proteins comprise proteins with various putative functions such as nucleators and / or chaperons as well as proteins with an unknown specific function related to the assembly of the GV.

[0133] In a prokaryotic cell GVA genes are all the genes within one or more operons comprising at least one of a gvpN and a gvpF excluding any gvpA / B and gvpC gene possibly present within said one or more operons. Therefore GVA genes can be identified by identifying an operon in a prokaryote including at least one of a gvpN and a gvpF excluding any gvpA / B and gvpC gene.

[0134] Preferably the one or more operons comprising all the GVA genes of a prokaryote can be identified and detected by detecting a gvpN gene encoding for a GV protein consensus sequence RALXYLQAGYXVHXRGPAGTGKTTLAMHLAXXLXRPVMLIXGDDEFXTSDLIGSESGY XXKKVVDNYIHSVVKVEDELRQNWVDNRLTXACREGFTLVYDEFNRSRPEXNNVLLS VLEEKILXLP (SEQ ID NO: 1) wherein X indicates any amino acid or a sequence of any length having at least 50%, and more preferably 60% or higher, most preferably from 50% to 83% identity.

[0135] gvpN genes of various microorganisms have a sequence encoding for a gvpN protein within the consensus SEQ ID NO: 1. In particular, gvpN gene in the sense of the disclosure can be a gene encoding for sequence MTVLTDKRKKGSGAFIQDDETKEVLSRALSYLKSGYSIHFTGPAGGGKTSLARALAKKR KRPVMLMHGNHELNNKDLIGDFTGYTSKKVIDQYVRSVYKKDEQVSENWQDGRLLEA VKNGYTLIYDEFTRSKPATNNIFLSILEEGVLPLYGVKMTDPFVRVHPDFRVIFTSNPAEY AGVYDTQDALLDRLITMFIDYKDIDRETAILTEKTDVEEDEARTIVTLVANVRNRSGDEN SSGLSLRASLMIATLATQQDIPIDGSDEDFQTLCIDILHHPLTKCLDEENAKSKAEKIILEE CKNIDTEEK (SEQ ID NO: 11) or a sequence of any length having at least 30% sequence identity with respect to SEQ ID NO: 11, preferably at least 50%, and more preferably 60% or higher, and gvpF gene in the sense of the disclosure can be a gene encoding for sequenceTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT MSETNETGIYIFSAIQTDKDEEFGAVEVEGTKAETFLIRYKDAAMVAAEVPMKIYHPNR QNLLMHQNAVAAIMDKNDTVIPISFGNVFKSKEDVKVLLENLYPQFEKLFPAIKGKIEVG LKVIGKKEWLEKKVNENPELEKVSASVKGKSEAAGYYERIQLGGMAQKMFTSLQKEV KTDVFSPLEEAAEAAKANEPTGETMLLNASFLINREDEAKFDEKVNEAHENWKDKADF HYSGPWPAYNFVNIRLKVEEK (SEQ ID NO: 12) or a sequence of any length having at least 20% sequence identity with respect to SEQ ID NO: 12, preferably at least 50%, more preferably 60%, and at least 70% or higher.

[0136] The term “operon” as described herein indicates a group of genes arranged in tandem in a prokaryotic genome as will be understood by a skilled person. Operons typically encode proteins participating in a common pathway are organized together as understood by those skilled in the art. Typically, genes of an operon are transcribed together into a single mRNA molecule referred to as polycistronic mRNA. Polycistronic mRNA comprises several open reading frames (ORFs), each of which is translated into a polypeptide. These polypeptides usually have a related function and their coding sequence is grouped and regulated together in a regulatory region, containing a promoter and an operator. Typically, repressor proteins bound to the operator sequence can physically obstruct the RNA polymerase enzyme from binding the promoter, preventing transcription. An example of a prokaryotic operon is the lac operon, which natively regulates transport and metabolism of lactose in E. coli and many other enteric bacteria.

[0137] In contrast, eukaryotic and mammalian cells generally do not possess native operons, as transcriptional units are typically monoci stronic, resulting in one mRNA per gene. However, operon-like expression strategies can be engineered for use in mammalian systems through the incorporation of self-cleaving peptide sequences (such as 2A peptides derived from picornaviruses), internal ribosome entry sites (IRES elements), or other multi ci stronic expression constructs. These synthetic or chimeric operons permit coordinated expression of multiple open reading frames from a single promoter while enabling translation of distinct polypeptides in mammalian cells. Accordingly, operon-based or operon-inspired constructs as described herein may be adapted for use in prokaryotic or eukaryotic host cells, including mammalian systems.

[0138] In an operon, each ORF typically has its own ribosome binding site (RBS) so that ribosomes simultaneously translate ORFs on the same mRNA. Some operons also exhibitTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT translational coupling, where the translation rates of multiple ORFs within an operon are linked. This can occur when the ribosome remains attached at the end of an ORF and translocates along to the next ORF without the need for a new RBS. Translational coupling is also observed when translation of an ORF affects the accessibility of the next RBS through changes in RNA secondary structure.

[0139] In engineered mammalian expression systems, analogous coupling effects can be achieved through use of viral IRES elements or ribosomal skipping sequences (for example, Thosea asigna virus 2A or porcine teschovirus 2A peptides), which mediate efficient multi ci str onic translation from a single transcript. Such constructs enable the coordinated expression of multiple proteins in mammalian cells in a manner functionally comparable to natural prokaryotic operons, while maintaining proper folding, localization, and stoichiometric balance among the expressed polypeptides.

[0140] In some embodiments, a GV cluster comprises one of gvpN or gvpF. In several embodiments GV clusters include both gvpN and gvpF as will be understood by a skilled person. In this connection, reference is made to Example 12 and Figures 20 and 21 of related application US application 15 / 663,635 published as US 2018 / 0030501 incorporated herein by reference in its entirety, showing exemplary gas vesicle gene clusters operons [1, 2] comprising GVS and GV A genes and related exemplary configuration. In particular, as shown in Example 12 of related applicationUS application 15 / 663,635 published as US 2018 / 0030501, typically a native GV gene cluster has GVA genes comprising both gvpN and gvpF genes, even if native GV gene clusters are known having a gvpN gene or a gvpF gene, as understood by skilled persons.

[0141] Accordingly, for a certain prokaryote, GVA genes in the sense of the disclosure indicate all the genes that are comprised in the one or more operons having at least one of a gvpN and / or a gvpF herein described and excluding any Gas Vesicle Structural (GVS) genes of the prokaryotes possibly comprised within the one or more operons.

[0142] Thus, GVA genes comprised in a gas vesicle gene cluster in a prokaryote can be identified for example by obtaining genome sequence of the prokaryote of interest and performing a sequenceTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT alignment of the protein sequences encoded in the genome of the prokaryote of interest against a gvpN protein sequence and / or a gvpF protein sequence.

[0143] In particular, obtaining the genome sequence of the prokaryote of interest, can be performed either using wet lab techniques identifiable by a skilled person upon reading of the present disclosure, or obtained from databases of gene and protein sequences also identifiable by a skilled person upon reading of the present disclosure. Performing a sequence alignment of the protein sequences encoded in the genome of the prokaryote of interest can per performed using Protein BLAST or other alignment algorithms identifiable by a skilled person. Exemplary gvpN protein sequence and / or a gvpF protein sequence, that can be used in performing the alignment are sequences SEQ ID NO: 11 and / or SEQ ID NO: 12. In particular, a sequence alignment can be performed using gvpN and / or gvpF protein sequences from the closest phylogenetic relative to the prokaryote of interest. Reference is made to Example 2 showing exemplary phylogenetic relationships between gvpF and gvpN proteins of exemplary prokaryotic species. Accordingly, one or more operons that comprise the gvpN and / or gvpF genes can be identified, and any other gvps within the one or more operons can also be identified, wherein the other gvps are comprised in ORFs within the one or more operons, excluding any ORFs encoding gvpA / B or gvpC genes comprised in the one or more operons of the GV gene cluster.

[0144] Accordingly, GVA genes can also be identified based on the configuration of operon and Gene Clusters identified through homology (see e g. Example 1), phylogenesis (see e.g. Example 2) also using the the gvpA / B, gvpN and / or gvpF consensus of SEQ ID Nos: 1, 3, and 11-12 herein provided, preferably gvpA / B consensus of SEQ ID NO: 3 and gvpN consensus of SEQ ID NO: 1. Reference is also made in this connection to the indication of Example 3 reporting exemplary GVGC configurations of naturally occurring Gas Vesicle gene clusters identified with method herein described and additional methods identifiable by a skilled person.

[0145] GVS genes of a GVGC of the disclosure, identified with methods herein indicated, typically comprise gvpA or gvpB which have similar sequences and are equivalent in their purpose and optionally gvpC. Exemplary sequences for gvpA and gvpB genes of GV gene clusters in the sense of the disclosure, which can also be used to identify additional GVS and GVGC throughTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT homology and alignment in addition to the use of the consensus sequence SEQ ID NO: 3, are reported in Example 4.

[0146] GVA genes of a GVGC of the disclosure, identified with methods herein indicated, typically comprise proteins identified as gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and gvpU. GVA genes and proteins can also comprise gvpR and gvpT (see e.g. B.megaterium GVA) gvpV, gvpW (se Anaboena flos aque and Serratia GVA) and / or gvp X, gvp Y and gvp Z (see e.g. Serratiai GVA. Preferably GVGC of the disclosure further comprise gvpN which result in a more robust detection with many detection methods herein described. Exemplary sequences for GVA genes of GV gene clusters in the sense of the disclosure which can also be used to identify additional GVAs and GVGC through homology and alignment are reported in Example 4.

[0147] In GVGC herein described co-expression of the GVS genes and the GVA genes in connection with regulatory sequence capable of operating in a host cell are configured to provide a GV type, with a different GVGC typically resulting in a different GV type.

[0148] The wording “GV type” in the sense of the disclosure indicates a gas vesicle having dimensions and shape resulting in distinctive mechanical, acoustic, surface and / or magnetic properties as will be understood by a skilled person upon reading of the present disclosure. In particular, a skilled person will understand that different shapes and dimensions will result in different properties in view of the indications in provided in US Applicant S / N 15 / 613,104 published as US2018 / 0028693 and US S / N 15 / 663,600 published as US2018 / 0038922 and additional indications identifiable by a skilled person Typically, larger volume results in stronger per-particle scattering, smaller diameter generally results in higher collapse pressure after removal of gvpC, and different dimensions result in different ratios of T2 / T2* relaxivity per volume-averaged magnetic susceptibility (

[0021] ).

[0149] In addition, a “GV type” can further encompass variations determined by the structure, truncation, or modification of the outer scaffold protein GvpC, as will be understood by a skilled person. In particular, GV types can comprise truncated or partially deleted GvpC (for example, AN-, AC-, or AN&C-terminus variants) and GV types lacking GvpC entirely (AGvpC) which can exhibit reduced collapse pressures and enhanced nonlinear acoustic responses relative to wild-typeTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT GVs [Ref. patent US 10,493,172], Similarly, GVs with weakened or cleaved GvpC coatings facilitate acoustic collapse and cavitation under therapeutic ultrasound conditions, representing a distinct functional GV type [Ref. patent US 11,446,523] and mammalian-expressible GV types can include full-length, truncated, or omitted GvpC forms, each conferring specific acoustic and magnetic profiles in eukaryotic expression environments [Ref patent US 11,761,008], Additionally, modification of the gvpC can also result in the protease-sensitive GVPC constructs which can be included in GV type bearing cleavable sequences, wherein protease-mediated cleavage dynamically alters the mechanical reinforcement of the GV wall and thereby changes the collapse pressure and nonlinear imaging response [Ref. U.S. Appl. Serial No.17 / 006,591],

[0150] Accordingly, the “GV type” in the sense of the present disclosure includes any gas vesicle structure whose mechanical or acoustic characteristics are determined not only by vesicle size and geometry but also by the configuration of GvpC — whether full-length, truncated, omitted, or engineered to be protease-cleavable — and combinations thereof, as will be understood by a skilled person upon reading of the present disclosure.

[0151] In addition, the GV type encompasses vesicles whose structural or functional properties are modified post-translationally or post-assembly, including chemical or enzymatic alteration of GvpC or other shell proteins. Such modifications can include stripping or partial removal of GvpC from the vesicle surface, chemical crosslinking, PEGylation, acylation, or conjugation of small molecules, peptides, or polymers to the GV shell. These treatments, as described for example in U.S. Pat. Nos. 10,493,172 B2, 11,446,523 B2, and 11,761,008 B2, can be used to modulate GV stability, surface charge, hydrophobicity, or acoustic non-linearity, thereby providing additional GV types distinguished by their post-translational or chemical modification state. Accordingly, GV type in the sense of the disclosure encompasses native, genetically engineered, and chemically or biochemically modified gas vesicles whose collapse pressure, resonance behavior, or magnetic or optical response is determined by both protein composition and post-synthetic modification of the GV shell as will be understood by a skilled person.

[0152] Accordingly, in embodiments herein described, GVGC can be selected based on desired properties of the corresponding GV type. In particular, to this extent, a skilled person can useTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT naturally occurring GVGC, can provide engineered GVGC wherein some of the naturally occurring gvp genes are omitted, and / or can provide hybrid GVGC in which GVAs and GVS genes of naturally occurring GVGCs are combined to provide GV types having the shape and dimensions resulting in the desired properties.

[0153] The term “hybrid gene cluster” or “hybrid cluster” as used herein indicates a cluster comprising at least two genes native to different species and resulting in a cluster not natively in any organisms. Typically, a hybrid gene cluster comprises a subset of gas vesicle genes native to a first bacterial species and another subsets of gas vesicle genes native to one or more bacterial species, with at least one of the one or more bacterial species different from the first bacterial specie Accordingly, a hybrid GV gene clusters includes a combination of GV genes which is not native in any naturally occurring prokaryotes.

[0154] In particular, identification of a desired GVGC for a target cell and therefore of the ability of the corresponding gvp genes combination to result in production of functional GV proteins capable of assembling in a GV thus providing a corresponding detectable GV type can be performed through a testing method also directed to verify detectability of the GV by a detection method of choice. The testing method can be performed in the target cell where detection of the GV type is desired or in testing cells having a cell environment equivalent to the cell environment of the target cell in terms of expression of GV genes and GV formation and thus provide a model to verify ability of the gvp genes to provide a GVGC for the target cells. In the method to identify a desired GVGC the introducing can be performed using engineered polynucleotide constructs contacted with the target cell or testing cell for a time and under conditions to allow expression of the GVGC and formation of the GV type (e.g. using the methods described in US application 15 / 663,635 published as US 2018 / 0030501 incorporated herein by reference in its entirety) . The method further comprises detecting formation of a gas vesicle in the target cell or testing cell following the introducing with a pre-set method of detection. Preset methods of detection can be directed to detect acoustic and / or magnetic properties that are of interest in desired applications of the corresponding GV type. Preferably the testing can be performed in a target cell or testing cell, that have been modified, either chemically or genetically, to have the same cellular turgor pressure as mammalian cells according to methods identifiable by a skilled person.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0155] Experiments performed with GVGC herein described (e.g., in the Examples section) provide proof of principle that a permissive mammalian cell line (e.g., HEK293T) known to be highly transducible is an effective initial model for assessing the ability of a GVGC to correctly assemble and produce a signal in other, less-permissive or hard-to-transduce mammalian cells (e.g., primary T cells). Accordingly, detecting expression and ultrasound signal from a candidate GVGC in a model mammalian cell line using a pre-set method is indicative of the ability of the corresponding GV proteins to form a functional GV type in other mammalian cells, including less permissive or hard to transduce types like clinically relevant primary T cells. Therefore, a method for optimizing gas vesicle expression in a target mammalian cell type comprises: (a) providing the multi-vector gas vesicle expression system components; (b) transducing aliquots of the target cells with the components using systematically varied total viral titers and systematically varied relative ratios of the vector components; and (c) measuring gas vesicle expression levels and / or functional ultrasound signal under standard conditions for each aliquot to identify the titer and ratio combination that yields maximal functional output in the target cell type.

[0156] In exemplary embodiments where a GV type is to be used in differential ultrasound imaging or image-subtracted ultrasound, the pre-set method of detection can comprise imaging with ultrasound a target site comprising the cell following the introduction of the GVGC, applying acoustic pressure to the target site at a pressure expected to collapse the GVs and then imaging the target site with ultrasound again, and the difference of the images (before and after collapse) shows if collapsing GVs (having a collapse threshold below the acoustic pressure) were present at the target site.

[0157] In exemplary embodiments where a GV type is to be used in MRI (magnetic resonance imaging), imaging, the pre-set method of detection can comprise imaging with MRI a target site comprising the cell following the introduction of the GVGC, applying hydrostatic pressure to the target site at a pressure expected to collapse the GVs. The target site is then imaged with MRI again, and the difference of the images (before and after collapse) shows if collapsing GVs (having a collapse threshold below the hydrostatic pressure) were present at the target site. In further embodiments, detection of the GV type can also include measurement of an increase in magnetic susceptibility or non-linearity of the signal, as the collapse or structural modulation of the GVTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT shell, particularly in variants having truncated or cleavable GvpC as described in U.S. Pat. No.10,493,172 B2 and U.S. Pat. No. 11,761,008 B2, produces a quantifiable change in MRI contrast. Multiplexed detection of multiple GV types, distinguished by their differing collapse pressures or relaxation behavior, can further be employed to enable multi-parametric or multi-target MRI imaging of distinct GV populations within the same field of view.

[0158] In exemplary embodiments where a GV type is to be used in BURST (burst ultrasound reconstruction with signal templates) imaging described herein and in US application S / N 16 / 736,581 filed on January 7, 2020 and herein incorporated by reference in its entirety, the preset method of detection can comprise imaging with ultrasound a target site comprising the cell following the introduction of the GVGC, over successive frames, at a peak positive pressure (PPP) well below the expected collapse threshold pressure for the GVs. While the frames are being taken, increasing the PPP step-wise to a value over the expected collapse threshold pressure for at least 9 half-cycles. Frames from before, during, and after the application of the increased pressure undergo template mixing to detect a BURST signal from the collapsing GVs, if present. Additionally, GV detection can include monitoring increases in acoustic non-linearity, such as harmonic or subharmonic signal generation, characteristic of GVs with truncated, weakened, or protease-cleavable GvpC coatings as disclosed in U.S. Pat. Nos. 10,493,172 B2, 11,446,523 B2, and 11,761,008 B2. These nonlinear responses can be analyzed to discriminate GV types according to their distinct collapse thresholds, enabling multiplexed ultrasound imaging in which multiple GV populations are simultaneously resolved by their unique nonlinear pressure-response profiles

[0159] Additional methods of detection such as Transmission Electron Microscopy (TEM) and optical scattering, optical phase detection, and xenon hyperCEST MRI can be used. Further exemplary embodiments can employ detection of GV types based on acoustic or magnetic nonlinearity, for instance by analyzing harmonic, subharmonic, or higher-order components of the ultrasound or magnetic resonance signal as described in U.S. Pat. Nos. 10,493,172 B2, 11,446,523 B2, and 11,761,008 B2. Such nonlinear detection enables identification of GVs that differ by GvpC truncation, deletion, or protease sensitivity, which manifest distinct collapse thresholds or harmonic response profiles. In some embodiments, multiplexed detection of multiple GV types can be achieved by combining signals from GVs having different nonlinear characteristics orTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT collapse pressures, allowing simultaneous imaging or sensing of multiple biological events or reporter populations within a single target site. Additional optical and magnetic resonance approaches, including phase-contrast microscopy, dynamic light scattering, and susceptibility-weighted MRI, can likewise be used to distinguish GV types based on changes in scattering intensity, relaxation rates, or susceptibility following GV collapse or modification.

[0160] An exemplary method of detection of a functional GVGC in the sense of the disclosure performed in E.Coli is reported in Example 5 of the present disclosure. Additional methods to be performed other prokaryotic cells and / or mammalian cells using the engineered configurable Gas Vesicle Architecture of the disclosure can be identified by a skilled person upon reading of the present disclosure.

[0161] Several detectable GVGC with one or more detection methods of interests have been identified and can be used for production of GV types in various cells through various genetically engineered constructs as will be understood by a skilled person upon reading of the present disclosure and US application 15 / 663,635 published as US 2018 / 0030501 herein incorporated by reference in its entirety.

[0162] Exemplary GVGCs comprise native or engineered clusters that give rise to gas vesicles exhibiting distinct collapse pressures, acoustic non-linearities, or magnetic susceptibilities, thereby providing characteristic signatures under ultrasound or MRI detection as described in U.S. Pat. Nos. 10,493,172 B2, 11,446,523 B2, and 11,761,008 B2. In particular, GVGCs comprising gvpC variants that are truncated, partially deleted, or engineered to include cleavable or proteasesensitive domains yield GV types with modulated mechanical stability and enhanced nonlinear acoustic responses, enabling multiplexed imaging or sensing of different GV populations within a single specimen. Such GVGCs can be incorporated into expression systems suitable for prokaryotic, eukaryotic, or mammalian hosts, under the control of appropriate promoters and regulatory elements, to produce reporter cells or organisms optimized for one or more of the aforementioned detection modalities.

[0163] In some embodiments described herein GVGC, the instant disclosure can be naturally occurring combination of gvp genes which can have a naturally occurring sequence or a sequenceTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT modified to optimize the expression in the cell where detection is to be performed. For example GVGC clusters of the instant disclosure comprise a GVGC of B. megaterium formed by the gvpA or gvpB genes, gvpR, gvpN gvpF, gvpG, gvpL gvpS, gvpK, gvpj, gvpT, gvpU of B. megaterium, or the GVGC of Anaboena Flos Aquae formed by the gvpA or gvpB genes of Anaboena Flos Aquae (see e.g. the sequences in Table 6 of Example 4) and the GVA gvpC, gvpN, gvpJ, gvpK, gvpF, gvpG, gvpV, gvpW of Anaboena Flos Aquae (see e.g. sequences in Table 10 of Example 4).

[0164] The gvp genes in one or more genes of the GVGC cluster of the present disclosure can have a naturally occurring sequence or a sequence modified to optimize the expression in the cell where detection is to be performed. For example, a B. megaterium GVGC can have a gvpA or gvpB genes having the sequences in Table 6 of Example 4, and / or any one of the gvpR, gvpN gvpF, gvpG, gvpL gvpS, gvpK, gvpj, gvpT, gvpU genes having the sequences in Table 8 of Example 4. Similarly, an Anaboena Flos Aquae GVGC can have the gvpA or gvpB genes having the sequences reported in Table 6 of Example 4 and / or any one of the gvpC, gvpN, gvpj, gvpK, gvpF, gvpG, gvpV, gvpW having the. sequences reported in Table 10 of Example 4.

[0165] In some embodiments, described herein, GVGC of the instant disclosure can be modified version of naturally occurring GV gene clusters. An example is provided by the GVGC of B. megaterium comprising gvpB, gvpR, gvpN gvpF, gvpG, gvpL gvpS, gvpK, gvp J, gvpT, gvpU wherein the gvpR and gvpT genes of the naturally occurring GVGC from B. megaterium have been omitted (see e.g. the sequences reported in Example 6 and Table 9 of the instant disclosure). Another example is provided by GV gene clusters comprising gvpA, Ana-gvpC gvpN, gpvJ, gvpK, gvpF, gvpG, gvpW, and gvpV from Anabaena flos-aquae or GV gene clusters comprising gvpA+ gvpN, gpvJ, gvpK, gvpF, gvpG, gvpW, gvpV from Anabaena flos-aquae (see Anabaena flos-aquae genes in Table 4 and Table 10 of Example 4 of the present disclosure).

[0166] In other embodiments described herein, GVGC of the instant disclosure can be a hybrid GV gene cluster in a Gas Vesicle expression system of the disclosure, can comprise a combination of genes from A. flos-aquae (herein also Ana-gvp) and genes from B. megaterium (herein also Mega-gvp). In particular, in exemplary embodiments, the hybrid GV gene cluster can comprise B. megaterium GVA assembly genes gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpj, gvpT andTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT gvpU and further comprise structural gvpA gene from Anabaena flos-aquae. In some of those embodiments, the hybrid GV gene cluster can comprise gvpA, gvpC from Anabaena flos-aquae and GVA genes from B. megaterium possibly excluding gvpR and / or gvpT. In some of those embodiments, the hybrid GV gene cluster can comprise Ana-gvpA and mega GVA genes possibly excluding gvpR and / or gvpT. In some embodiments GVGC of the instant disclosure can include gvpA, gvpC, gvpN from Anabaena flos-aquae and GVA genes from B. megaterium, as well as other combinations identifiable by a skilled person upon reading of the present disclosure.

[0167] In some embodiments herein described, a GVGC comprising gvp genes A / B, C and N (gvpA / B, gvpC, gvpN genes) from a same or different prokaryote. Preferably the GVGC comprises a gvpN gene as presence of gvpN protein results in an increased detectability of the related GV type.

[0168] For example, in one exemplary embodiment, all the gvp genes B, N, F, G, L, S, K, J and U are from B. megaterium. GVs from B. megaterium are typically cone-tipped cylindrical structures with a diameter of approximately 73 nm and length of 100-600 nm, encoded by a cluster of eleven or fourteen different genes, including the primary structural protein, gvpB, and several putative minor components and putative chaperones [22, 23] as would be understood by a person skilled in the art.

[0169] In some embodiments, some of the set of nine gvp genes can be from Bacillus megaterium and the rest genes are from Anabaena flos-aquae such as the GVGC comprising Ana-A, Ana-C, Ana-N, mega: gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and gvpU with / without gvpR and gvpT, and additional examples identifiable by a skilled person upon reading of the present disclosure (see Example 4 and Example 5 of the present disclosure).

[0170] In embodiments herein described, the sequences of at least one gvp gene can be modified with respect to the natural occurring sequence to improve the related expression (e.g. to be codon optimized) and / or the inclusion in the engineered configurable Gas Vesicle Architectureof the disclosure (e.g. by modification of the N- and / or C- terminal portions to allow the use of linker or other elements to be included in a cassette or construct of the disclosure).Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0171] In some embodiments, the GVGC can comprise Serratia gvp genes as Serratia GVs can express functional GV proteins in E. coli, as reported in literature (

[0024]

[0025] ).

[0172] GVES and related constructs have been herein provided based on the surprising finding that a naturally occurring, or engineered functional GVGC can be transfected and expressed in target cells with a construct including differentially expressed gene modules stoichiometrically configured to allow expression in the target cell of GV proteins encoded by the GVGC, resulting in formation of a corresponding GV type in the target cell with a significantly increased yield in GV type obtained that provides an enhanced, detectable acoustic signature upon expression in cells

[0173] As used herein, the term “ cell” indicates the basic structural and functional units of life. Cells in the sense of the disclosure comprise prokaryotic cells, which include the domains of Bacteria and Archaea. Prokaryotic target cells and in particular engineered probiotic bacteria (e.g., Escherichia, Bacillus, Lactobacillus). Prokaryotic cells can frequently utilized as living diagnostics or therapeutics configured to localize within specific target sites such as the gastrointestinal tract, the respiratory tract, or the interstitial spaces of a tumor microenvironment. In these contexts, the prokaryotic cell serves as a self-replicating vehicle for the gas vesicle expression system, providing a nonlinear acoustic readout of its spatial distribution or metabolic activity within the host. Cells in the sense of the disclosure also comprise eukaryotic cells, which encompass a broad range of cells from multi-cellular and single-cellular organisms including mammalian, vertebrate, avian, insect, yeast, and plant cells. Within the eukaryotic category, the mammalian cell represents a preferred subgenus for high-resolution deep-tissue imaging and clinical intervention. The term “mammalian cell” refers to cells from a mammal tissue comprising cell within a mammal host and cell isolated from and expanded in culture for use as therapeutic and research tools. Exemplary mammalian cells that can express GVES of the disclosure are primary cells (cells that are directly harvested from an animal and genetically engineered with GVs. Exemplary mammalian cell culture that can be genetically engineered with GV constructs described herein configured to allow expression of GVs comprise HEK 293T, CHO-K1 cells, HEK293, CHO-K1, N2A cells, HeLa, Jurkat, NIH3T3, and other identifiable by those skilled in the art.

[0174] Additional examples of mammalian cells suitable to be used in connection with Stoichiometric GVES of the disclosure include induced pluripotent stem cells (iPSCs), embryonicTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT stem cells, mesenchymal stem cells, neuronal and glial cells, hepatocytes, cardiomyocytes, fibroblasts, macrophages, T lymphocytes, and natural killer (NK) cells, as well as immortalized derivatives thereof. Mammalian cells can be further classified into immortalized cell lines, such as HEK 293T, CHO, HeLa, Jurkat, and NIH3T3, and primary cells directly isolated from mammalian tissue. Primary cells retain the specialized differentiated characteristics of their tissue of origin and include, without limitation, neurons, astrocytes, microglia, hepatocytes, cardiomyocytes, skeletal muscle cells, and fibroblasts.

[0175] In the context of the present disclosure, mammalian cells expressing GV expression systems (GVES) or related constructs (GVPC, GVRMC, GVRGC) may provide GVs exhibiting cell-type-specific assembly or post-translational processing of GV proteins, including glycosylation, phosphorylation, proteolytic cleavage, or partial stripping of GvpC or related structural proteins, as described for example in U.S. Pat. Nos. 10,955,496 B2 and 11,761,008 B2. These cell-specific biochemical environments can modulate GV stability, collapse pressure, and acoustic non-linearity, thereby giving rise to distinct GV types within mammalian expression systems.

[0176] Accordingly, “mammalian cell” in the sense of the present disclosure includes any genetically engineered cell derived from a mammalian lineage that is capable of expressing, assembling, modifying, or maintaining GVs or GV-like nanostructures as described herein, whether in vitro, in vivo, or ex vivo.

[0177] As used herein, the term “differentially expressed gene module” refers to a set or subset of two or more coding sequences within a single gene expression cassette — such as a polycistronic or multicistronic construct — that are expressed at distinct relative levels or stoichiometric ratios as a result of one or more internal regulatory elements. A differentially expressed gene module thus enables coordinated yet non-equivalent expression of multiple proteins from a common transcript or promoter while maintaining functional assembly of the encoded complex or structure.

[0178] Differential expression within the module may be governed by transcriptional, translational, or post-translational regulatory features, including but not limited to: (i) promoters or sub-promoters of differing strengths; (ii) internal ribosome entry sites (IRES) with variableTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT initiation efficiency; (iii) ribosome-binding sites or Kozak sequences of differing translation initiation potential; (iv) separation elements such as viral 2A peptides or protease-cleavable linkers with differential cleavage kinetics; (v) RNA structural elements or codon-usage biases affecting mRNA stability or translation rate; and (vi) post-translational processing motifs or degradation tags altering protein half-life.

[0179] In certain embodiments, each differentially expressed gene module comprises a subset of gvp genes — for example, gvpA / B, gvpC, and one or more accessory gvp genes — whose relative expression ratios are optimized to yield a gas vesicle type with defined size, shell composition, and acoustic response. The internal regulatory architecture of the module can thus be designed to ensure that stoichiometric balance among Gvp proteins is maintained within a single polycistronic transcript, while still permitting differential expression amplitudes suitable for proper vesicle assembly.

[0180] In exemplary embodiments, differentially expressed gene modules are designed using computational modeling and synthetic-biology design tools (for example, in silico prediction of translation efficiency or ribosome profiling data) to achieve target expression ratios and dynamic ranges. These ratios can be predetermined experimentally or through predictive modeling to optimize vesicle morphology, collapse threshold, or nonlinear acoustic contrast.

[0181] Differentially expressed gene modules can be used individually or in combination within a multicassette or multigenic construct, enabling hierarchical or multi-layer control of expression. For instance, one module may encode structural GV proteins expressed at high levels, while a second module encodes regulatory or accessory GV proteins expressed at reduced or inducible levels. Such modular design allows precise tuning of vesicle assembly kinetics, mechanical integrity, and imaging properties in mammalian, prokaryotic, or cell-free expression systems.

[0182] Accordingly, the term “differentially expressed gene module” encompasses any operably linked group of genes within a cassette whose expression levels are intentionally varied relative to one another by engineered regulatory architecture. These modules permit rational control of protein stoichiometry and post-transcriptional dynamics, thereby enabling predictable customization of GV types and acoustic or magnetic phenotypes across host systems.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0183] In some embodiments, a differentially expressed gene module can be included as part of a monocistronic or a polycistronic gene expression cassete, as will be understood by a skilled person.

[0184] The term “gene cassette” as used herein indicated a mobile genetic element that contains at least one gene and a recombination site. Accordingly, a gene cassette can contain a single gene (herein a "monocistronic cassette") or multiple genes (herein a "polycistronic cassette"), which may be organized in an operon structure. A gene cassette can be transferred from one DNA sequence (usually on a vector) to another by 'cutting' the fragment out using restriction enzymes or transposase, CRISPR, viral and / or recombinase enzymes and other nucleases and 'pasting' it back into the new context or other molecular biology and cloning techniques (e.g. per, CRISPR, TALENs, ZFN). Gene cassetes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer.

[0185] A ‘ ‘gene expression cassette” is a gene cassette comprising regulatory sequence to be expressed by a transfected cell. Following transformation, the expression cassette directs the cell's machinery to make RNA and proteins. Some expression cassettes are designed for modular cloning of protein-encoding sequences so that the same cassette can easily be altered to make different proteins. An expression cassete is composed of one or more genes and the sequences controlling their expression. An expression cassette typically comprises at least three components: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. An expression cassette can be formed by manipulable fragment of DNA carrying, and capable of expressing, one or more genes of interest optionally located between one or more sets of restriction sites Gene expression cassetes as used herein typically comprise further regulatory sequences additional to the prompter to regulated the expression of the gene or genes within the open reading frame herein also indicated as coding region of the cassette. As used herein, an expression cassette comprising a single gene or open reading frame is a "monocistronic expression cassette," and an expression cassette comprising two or more genes or open reading frames (e g., linked by 2A peptides or IRES elements) is a "polycistronic expression cassette."

[0186] In particular, in embodiments of the GVES herein described, the gene expression cassettes of the system comprise one or more gvp genes under control of regulatory sequenceTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT capable of operating in the mammalian host and are thus configured to provide a GV type in the mammalian cell.

[0187] The term “regulatory sequence” or “regulatory regions” as described herein indicate a segment of a nucleic acid molecule which is capable of increasing or decreasing transcription or translation of a gene within an organism either in vitro or in vivo. In particular, coding regions of the GV genes herein described comprise one or more protein coding regions which when transcribed and translated produce a polypeptide.

[0188] Accordingly the wording regulatory region refers to a nucleotide sequence that influences the transcription, translation, mRNA stability, or localization of an operably linked gene module. Regulatory regions include, but are not limited to, promoters, enhancers, silencers, insulators, 5’ and 3’ untranslated regions (UTRs), polyadenylation signals, and separation elements (e.g., IRES or 2 A peptides).

[0189] Regulatory regions of a gene herein described comprise promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein-protein binding domains, RNA binding domains, DNA binding domains, silencers, insulators, 5’ and 3’ untranslated regions (UTRs), polyadenylation signals, and separation elements (e.g., IRES or 2A peptides)and additional regulatory regions that can alter gene expression in response to developmental and / or external stimuli as will be recognized by a person skilled in the art.

[0190] The term “promoter” refers to a DNA sequence recognized by the synthetic or cellular machinery required to initiate transcription of a gene module. Promoters suitable for the present disclosure comprise constitutive promoters (e.g., CMV, EFl a CAG, or PGK) providing steadystate expression across various target cells, Cell-type or tissue-specific promoters (e.g., Synapsin for neurons, Lek or CD4 for T-cells) providing expression localized to a specific target site; and additional promoters identifiable by a skilled person. Promoters initiate transcription of a gene in operative connection with transcription factors regulatory genes and additional components depending on the transcription system.

[0191] The term “operative connection” as used herein indicate an arrangement of elements in a combination enabling production of an appropriate effect. With respect to genes and regulatoryTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT sequences an operative connection indicates a configuration of the genes with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease transcription or translation of the genes.

[0192] Regulatory sequences used in gene expression cassettes herein described identified herein also as mammalian regulatory regions are configured to operate in a mammalian cell.

[0193] Exemplary regulatory regions capable of operating in mammalian cells comprise promoters, enhancers, silencers, terminators, regulators, operators, ribosome binding / entry sites, and riboswitches, among others known in the art. Regulatory regions capable of operating in a mammalian host can be selected by a skilled person following selection of the mammalian host of interest. Exemplary constitutive and inducible mammalian promoters and operators suitable for regulating expression of GVs in a mammalian host comprise and others identifiable by those skilled in the art and described herein.

[0194] Mammalian regulatory regions comprised in a gene expression cassette herein described, typically comprise a mammalian promoter, 5’UTR regions, 3’UTR regions, and a terminator as will be understood by a skilled person. Additional regulatory features, such as upstream open reading frames (uORFs), post-transcriptional response elements, or RNA-stabilizing motifs, may be included to modulate expression kinetics or mRNA stability.

[0195] A “mammalian promoter” in the sense of the disclosure suitable for gene expression in a mammalian cell is a region of DNA that leads to initiation of transcription of a particular gene. Exemplary are typically located on a same strand and upstream on a DNA sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the genes whose transcription they initiate. In mammalian cells organisms, promoters typically comprise the eukaryotic TATA (SEQ ID NO: 13) box. Promoters are located near the transcription start sites of genes, upstream on the DNA. Promoters can typically be about 100-1000 base pairs long. In particular promoters that can be used in gene expression cassettes herein described can be a constitutive promoter or a conditional promoter.

[0196] The term “conditional promoter” refers to a promoter with activity regulatable or controlled by endogenous transcription factors or exogenous inputs such as chemical, or thermalTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT inducers or optical induction. Examples of mammalian constitutive promoters include inducible promoters based on exogenous agents such as TET (tetracycline-response elements, TET-ON / TET-OFF), Lac, dCas-transactivator, Zinc-finger- TF, TALENs-ZF Gal4-uas, synNotch and inducible promoters based on endogenous signals TNF-alpha, cFOS and others identifiable to a skilled person. Conditional promoters enable spatial and temporal control of GV expression and can be used to link vesicle formation to specific biological events or therapeutic triggers.

[0197] The term “constitutive promoter” refers to an unregulated promoter that allows for continual transcription of its associated genes. Exemplary mammalian constitutive promoters that can be used for expression in mammalian cell include CMV from human cytomegalovirus, EFla from human elongation factor 1 alpha, SV40 from the simian vacuolating virus 40, PGK1 from phosphoglycerate kinase gene, Ubc from human ubiquitin C gene, human beta actin, CAAG, Synl and others identifiable to those skilled in the art. Promoter strength and expression kinetics can be modulated by incorporating promoter variants, tandem promoter arrays, or synthetic hybrid promoters.

[0198] Similarly, the term “inducible promoter” refers to synthetic or exogenous systems that respond to a chemical trigger. Exemplary systems include ligand-responsive systems such as the Tetracycline-Response Element (TRE) operably linked to a reverse Tetracycline-controlled Transactivator (rtTA). In these systems, transcription is initiated only upon the administration of a specific inducer (e.g., Doxycycline), allowing for precise control over the timing of gas vesicle production within a target site.

[0199] The wording “5’UTR region” refers to the region upstream from the initiation codon as will be understood by a person of ordinary skill in the art and is therefore outside the coding region of the cassette. The 5’UTR region can contain a Kozak sequence. The Kozak sequence used herein refers to a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts as will be understood by a person skilled in the art. The Kozak sequence locates approximately 6 nucleotide sequence upstream of the ATG start codon. Exemplary Kozak sequence include GCCACCATG (SEQ ID NO: 475), TTCACCATG (SEQ ID NO: 476), (CCC)TTCACCATG (SEQ ID NO: 477) consensus sequence XXX[A / G]XXATGTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT (SEQ ID NO: 478) wherein X indicates any nucleotide, and additional sequences identifiable by a skilled person.

[0200] The “3’UTR region” refers to an untranslated region that immediately follows the translation termination codon and is therefore outside the coding region of the cassette. 3’UTR region often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3’UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA as will be understood by a person skilled in the art. In some embodiments, the 3 ’UTR contains silencer regions which are configured to bind to repressor proteins and inhibit the expression of the mRNA. The 5 UTR can further contain regulatory motifs such as upstream ORFs, stem -loop structures, or aptamers that modulate translation efficiency and responsiveness to cellular signals. Engineering of the 3 'UTR region can be used to fine-tune transcript half-life, enabling modulation of GV protein levels in response to environmental or developmental cues.

[0201] A “terminator” as used herein indicates a sequence-based element that defines the end of a transcriptional unit and initiates the process of releasing the synthesized mRNA. Exemplary mammalian terminators include polyadenylation sites. A “polyadenylation site” indicates an element target by the polyadenylation enzymes such as CPSF and typically comprises the sequence AAUAAA (SEQ ID NO: 14) on the RNA. Polyadenylation sites will result in cleavage of the construct 10-30 nucleotides downstream the site, and addition of a poly(A) tail located at the end of 3’UTR as will be understood by a person skilled in the art. In gene expression cassette the poly(A) site can include SV40 polyadenylation element, hGH poly(A) signal, and other poly(A) signal that have the canonical AAUAAA (SEQ ID NO: 14) region as will be understood by a skilled person.

[0202] In some embodiments, a gene expression cassette can include additional mammalian regulatory regions configured to increase or decrease the expression of the GV coding regions of the cassette, as will also be understood by a skilled person.

[0203] Exemplary mammalian regulatory sequences increasing transcription of the operatively linked gene comprise enhancers that can be located more distally from the transcription start siteTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT compared to promoters, and either upstream or downstream from the regulated genes, as understood by those skilled in the art. Enhancers are typically short (50-1500 bp) regions of DNA that can be bound by transcriptional activators to increase transcription of a particular gene. Typically, enhancers can be located up to 1 Mbp away from the gene, upstream or downstream from the start site. An exemplary additional mammalian regulatory regions directed to enhance the expression levels of the GV genes, include Woodchuck Hepatitis Virus Posttranscri phonal Regulatory Element (WPRE) placed downstream of the genes between GV gene and the poly(A) tail. The WPRE and WPRE-like (e.g. RE of Hepatitis B virus (HPRE)) element is known to increase transgene expression from a variety of viral vectors. Additional examples include viral enhancers (SV40, CMV), synthetic enhancer arrays, and post-transcriptional regulatory elements such as the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or its analogs (e.g., HPRE), which stabilize transcripts and increase transgene expression in mammalian hosts.

[0204] Exemplary mammalian regulatory sequences decreasing transcription of the operatively linked gene comprise RNAi / miRNA / shRNA sites that can be located upstream or downstream of the GV genes to control mRNA translation or degradation. For example, by binding to specific sites within the 3’UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript.

[0205] Additional mammalian regulatory sequences that can be included in a gene expression cassette include post transcriptional regulatory sequences such as riboswitches typically present in eukaryotic untranslated regions (UTRs) of encoded RNAs. These sequences are configured to switch between alternative secondary structures in the RNA depending on the concentration of key metabolites. The secondary structures then either block or reveal other regulatory sequence regions such as RNA binding proteins. Further examples of additional post transcriptional regulatory sequences regulatory sequences comprise aptazymes fusions composed of an aptamer domain and a self-cleaving ribozyme which can be used for conditional gene expression to control mRNA levels with small molecules (e.g. tetracycline).

[0206] In general, selection of promoter and other regulatory sequences to be included in expression polynucleotidic constructs comprised in GVES of the present disclosure can beTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT performed by one or more of the following: detecting functionality of a promoter and / or additional regulatory sequence in the host cells, selecting promoters and / or additional regulatory sequences known to be functional in the host cells; detecting the strength of the promoters and / or additional regulatory sequences in connection with protein production and / or selecting promoter and / or additional regulatory sequences of known strength; and selecting inducible promoters and / or additional regulatory sequence to control GV expression.

[0207] Mammalian regulatory sequences can be provided in any configuration which is directed to provide a desired expression of the GV protein in the coding regions. For example, a gene expression cassette can an end of UTR with polyA site only, or can be with WPRE and poly A site, or it can be with WPRE only. A combination of WPRE and polyA tail is expected to result in highest expression (highest copy of translated protein). Additional configuration can be identified by a skilled person.

[0208] In embodiments of the GVES herein described GV genes other than gvpA / B can be provided in a single gene expression cassette in various combinations and in any order to the extent that when the cassette comprises two or more gvp genes other than gvpA / B, the two or more gvp genes are configured to have each GV gene linked to another by a separation element.

[0209] A “separation element” as used herein refers to an element that can be placed between two adjacent coding genes allowing for a separate transcription or translation of the two adjacent coding genes. Separation elements are used in the present disclosure to enable coordinated expression of multiple gas vesicle (GV) proteins from a single transcript while ensuring that each protein is produced as a discrete polypeptide capable of proper folding and assembly into a functional GV type.

[0210] In some embodiments, a separation element can be an internal ribosome entry site (“IRES”). An internal ribosome entry site (IRES) used herein refers to an element that allows for translation initiation in a cap-independent manner. In some embodiments herein described, an IRES element is placed between two coding genes to allow for initiation of translation from an internal region of the mRNA. It allows the coordinated expression of two genes using the same promoter in a single gene cassette as will be understood by a person skilled in the art. Thus, theTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT genes separated by IRES can be expressed from a bicistronic mRNA without requiring either cleavage of a polyprotein or generation of a monocistronic mRNA. In GV constructs, the use of IRES elements facilitates expression of downstream gvp genes, such as gvpC, gvpF, or gvpN, from a shared promoter in mammalian cells, ensuring balanced stoichiometry of GV structural and assembly proteins while preserving the integrity of each polypeptide product

[0211] Internal ribosome entry sites are approximately 450 nucleotides in length and are characterized by moderate conservation of primary sequence and strong conservation of secondary structure. The most significant primary sequence feature of the IRES is a pyrimidine-rich site whose start is located approximately 25 nucleotides upstream of the 3' end of the IRES. Detailed information on IRES can be found in Jackson, et al., Trends Biochem. Sci., vol. 15, No. 12, pp.477-483, 1990. Variants of IRES sequences and synthetic IRES mimetics can also be used, including those optimized for mammalian codon usage and RNA folding stability, as described in U.S. Pat. No. 11,761,008 B2.

[0212] Examples of IRES known in the art include IRES obtainable from picomavirus and IRES obtainable from viral or cellular mRNA sources such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huezetal. (1998) Mol. Cell. Biol.18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4, the encephelomycarditis virus (EMCV) which is commercially available from Novagen (Duke et al. (1992) J. Virol 66(3): 1602-9) and the VEGFIRES (Huez et al. (1998) Mol Cell Biol 18(11):6178-90). IRES have also been reported in different viruses such as cardiovirus, rhinovirus, aphthovirus, HCV. Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). As used herein, IRES encompasses functional variations of IRES sequences as long as the variation is able to promote direct internal ribosome entry to the initiation codon of a cistron.

[0213] In some embodiments, a separation element is a post-translation cleavage element comprising a cleavage site sequence. A post-translation cleavage element is typically placed between two adjacent coding genes. In the context of the present disclosure, IRES elements can thus be used to produce bicistronic or multi ci str onic GV expression systems (GVES) that yieldTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT correctly folded GV proteins within mammalian cells, contributing to the formation of distinct GV types based on transcriptional or translational stoichiometry.

[0214] In some embodiments, the post-translation cleavage element comprises a 2A element. The term “2A element” or “2A sequence” refers to a post-translational or co-translational processing cleavage site sequence. The 2A sequence can be a DNA sequence or the peptide expression produce of the DNA sequence. The latter is referred to as the 2A peptide. The 2A peptides are known to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream. The cleavage occurs between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline. The 2A elements used herein are placed between two adjacent GV coding genes. Exemplary 2A peptides are listed in Table 1 below:Table 1: Exemplary 2A peptide sequencesP2A ATNFSLLKQAGDVEENPGP (SEQ ID NO: 15)T2A EGRGSLLTCGDVEENPGP (SEQ ID NO: 16)E2A QCTNYALLKLAGDVESNPGP (SEQ ID NO: 17)F2A VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 18)BmCPV DVFRSNYDLLKLCGDIESNPGP (SEQ ID NO: 19)BmIFV TLTRAKIEDELIRAGIESNPGP (SEQ ID NO: 20)

[0215] In Table 1, the bold residues are the consensus residues among each type of 2A element (P2A, T2A, E2A or F2A). In each 2A element of Table 1, the cleavage occurs between the last G / P. In some embodiments, a linker sequence such as GAPGSG linker (SEQ ID NO: 21) is placed between a GV coding gene and the 2A sequence optionally using a linker, wherein any linker sequences such as GSG, GSGSG (SEQ ID NO: 2), SGS, and other linkers identifiable by a skilled person can be used. For example, a polynucleotide construct can comprise from 5’ to 3’ GV gene 1 -GAPGSG -2 A sequence-GV gene 2.

[0216] In some embodiments, the post-translation cleavage element comprises a cleavage recognition site that can be targeted and subsequently cleaved by protease enzymes. ExemplaryTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT protease enzymes include TEV, HCV NS3 / 5 protease, HIV protease, CMV protease, and HSV protease.

[0217] The term “protease cleavage site” in the sense of the disclosure indicates target sites for proteolytic cleavage by enzymes such as peptidases, proteases or proteolytic cleavage enzymes which break peptide bond between amino acids in proteins. The general nomenclature of cleavage site positions of the substrate were formulated by Schechter and Berger, 1967

[0026] and Schechter and Berger, 1968

[0027] Accordingly, the cleavage site is designated between Pl -Pl', incrementing the numbering in the N-terminal direction of the cleaved peptide bond (P2, P3, P4, etc.). On the carboxyl side of the cleavage site the numbering is incremented in the same way (PT, P2', P3' etc.).

[0218] Protease cleavage sites that can be inserted in engineered microcompartment proteins of the disclosure comprise regions up to 25 residues. In particular, protease cleavage sites are inserted in a configuration which makes them surface accessible. In some embodiments protease cleavage site are included in an unstructured segment or within an alpha helical or beta sheet secondary structured segment. Exemplary protease cleavage sites that can be inserted in engineered microcompartment proteins herein described comprise TEV protease cleavage sites with sequence ENLYFQG, (SEQ ID NO: 25) which is unstructured and others identifiable by a skilled person upon reading of the present disclosure (see Table 2).

[0219] Recognition sequences and cleavage sites of exemplary proteases are shown in Table 2.Forward slash ( / ) indicates where protease cleaves the protein sequence.Table 2: Recognition sequences and cleavage sites of exemplary proteases Enzyme Name Sequence and Cleavage SEQ ID NO Human Rhinovirus (HRV) 3C Protease LEVLFQ / GP 22 Enterokinase DDDDK / 23Factor Xa IEGR / 24Tobacco etch virus protease (TEV protease) ENLYFQ / G 25Thrombin LVPR / GS 26NS3 / 4A DLEVVT / STWV 27NS4A / 4B DEMEEC / ASHL 28Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT Table 2: Recognition sequences and cleavage sites of exemplary proteases Enzyme Name Sequence and Cleavage SEQ ID NO NS4B / 5A DCSTPC / SGSW 29NS5A / 5B EDVVCC / SMSY 30NS4A / 4B DEMEEC / SQH 31

[0220] In some embodiments, the cleavage recognition site comprises a TEV protease cleavable sequence that can be placed between two GV coding genes when the TEV enzymes are coexpressed. The TEV peptide can be cleaved to release the two GV proteins.

[0221] In some embodiments, the cleavage recognition site comprises a recognition sequence targeted by one or more non- structural protein NS3, NS4A, NS4B and NS5 sequence.

[0222] In some embodiments herein described, post-translation cleavage element comprises an intein or hedgehog family auto-processing domains or variants therefore, inserted in an open reading frame between multiple coding genes. The term “intein” refers to the protein equivalent of gene introns which facilitate protein splicing. The intein element contains the necessary components needed to catalyze protein slicing and often contains an endonuclease domain that participates in intein mobility (Perler, F. B., et al., Nucleic Acids Research 1994, 22, 1127).

[0223] The Hedgehog family auto-processing domains used herein comprise the hedgehog protein carboxy-terminal autocatalytic domain HhC. As a person skilled in the art will understand, the hedgehog (“Hh”) proteins are composed of two domains, an amino-terminal domain HhN, which has the biological signal activity, and a carboxy-terminal autocatalytic domain HhC, a carboxy-terminal autocatalytic domain HhC which cleaves Hh into two parts in an intramolecular reaction and adds a cholesterol moiety to the HhN. HhC has sequence similarity to the self-splicing inteins, the shared region is termed Hint. New classes of proteins containing the Hint domain have been discovered in bacteria and eukaryotes.

[0224] ] In some embodiments, stoichiometric Gas Vesicle Expression Systems (GVES), polynucleotide constructs, and related methods provided herein are configured to modulate the yield of functional gas vesicles and cellular toxicity parameters in a target cell by configuring the relative expression levels of the constituent GV assembly factor proteins. This stoichiometricTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT control provides a measurable increase in gas vesicle expression relative to a baseline configuration wherein gas vesicle gene cassettes are expressed at a 1 : 1 ratio.

[0225] An operational principle of the GVES described herein involves establishing a defined, multi-tier stoichiometric hierarchy among the assembly factors themselves. In some embodiments, the system is configured to express specific early-stage assembly factors at a higher level relative to late-stage or accessory chaperones. In a baseline, non-hierarchical configuration, equal expression of these assembly factors correlates with the accumulation of misfolded intermediates and measurable cellular toxicity. In the hierarchical configuration, this toxicity is detectably reduced and the intracellular yield of assembled gas vesicles is increased.

[0226] In specific configurations, the GVES is partitioned into a multi-modular architecture comprising structurally distinct differentially expressed gene modules. To enforce the assembly factor hierarchical configuration, the GVES comprises an AF1 module encoding a first subset of highly expressed assembly factors (comprising at least a gvpG gene and a gvpF gene), an AF2 module encoding a moderately expressed second subset of assembly factors (comprising at least a gvpN gene, and optionally a gvpW gene), and an AF3 module encoding a limited third subset of assembly factors (comprising at least a gvpK gene, and optionally a gvpJ gene).

[0227] To quantify and control this stoichiometric relationship, a "Dosage Index" (DI) is utilized. As used herein, the term "Dosage Index" or "DI" refers to a normalized quantitative parameter representing the relative stoichiometry of gene expression output within a defined gene system, operon, or expression cassette set. The DI reflects the ratio between the vector-encoded dosage of individual genes or gene cassettes and the corresponding levels of their expressed products (e.g., messenger RNA or protein), measured under Standard Conditions. The DI is determined by the regulatory architecture of the cassette (e.g., promoter strength, UTRs, enhancers, mRNA stability elements, translation control elements), rather than merely its physical copy number.

[0228] Accordingly, as used herein, the wording "Dosage Index" refers to a normalized quantitative measure of the expression output of a gene cassette relative to a designated reference cassette, measured under standard conditions. This DI is determined by the regulatory architecture (e g., promoter strength, UTRs, enhancers, mRNA stability elements) of the cassette, not by itsTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT physical copy number. For example, a GVES configured with a DI ratio of 4:1:1 expresses the first component at a 4-fold higher level than the second and third components, even when all three cassettes are present at a 1 : 1 copy number.

[0229] In embodiments, herein described the Dosage Index for a given gene i within a system comprising n genes is determined according to the following relationship:DIi =Ei / Gi (1)

[0230] where Et represents the relative abundance of the expressed product of gene z, and Gi represents the relative dosage of the same gene, expressed as copy number, promoter strength, or molar fraction within the construct. The DI for a system can be reported as a vector of individual Dli values or as a composite normalized average reflecting the overall stoichiometric balance of the system. When all constituent genes exhibit DI values approaching 1.0 under Standard Conditions, the system is deemed stoichiometrically balanced. Deviations from this equilibrium indicate disproportionate expression, which may adversely affect assembly or functionality of the encoded structure.

[0231] In accordance with the present disclosure, the DI serves as a verifiable variable for configuring the stoichiometric balance.

[0232] Utilizing this metric, the AF-Hierarchical GVES is configured such that the DI of the first assembly factor module (AF1-DI) is greater than the DI of the second assembly factor module (AF2-DI), and the AF2-DI is greater than the DI of the third assembly factor module (AF3-DI), thereby establishing a descending expression hierarchy (AF1-DI > AF2-DI > AF3-DI).

[0233] In certain configurations, the AF1 module (comprising gvpF and gvpG) is maintained at a target Dosage Index (AF1-DI) of at least 2.0, or at a molar ratio of at least 2:1 relative to the AF2 module. This assembly factor hierarchy functions as an independent modular mechanism capable of modulating yield. In a first structural configuration maintaining a 1:1 macro ratio between the primary structural protein and the assembly factors, an equivalent AF-DI hierarchy results in a baseline yield level. In a second configuration, selectively configuring the AF1-DI to at least a 2-Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT fold excess over the AF2-DI, while maintaining the same 1:1 structural macro ratio, yields a rescued gas vesicle expression that achieves a detectable intracellular acoustic density.

[0234] In further configurations, this AF-hierarchical configuration operates alongside a foundational macro-stoichiometric configuration. This macro-level principle configures the system to provide a relative excess of a primary structural protein module (encoding, e g., GvpA or a chimeric GvpA / B) as compared to the total pool of assembly factor proteins.

[0235] In baseline macro-stoichiometric configurations, a DI ratio of the structural protein cassette to the combined assembly factor cassettes of at least 2:1 (e.g., 2:1:1 or 4:1:1) correlates with an increase in functional GV yield compared to a 1:1:1 baseline. For example, baseline embodiments configured with a 4:1:1 DI ratio produced a BURST Signal-to-Background Ratio (SBR) of approximately 118, while a 2:1:1 ratio produced an SBR of approximately 85. In comparison, the non-macro-stoichiometric baseline configuration of 1:1:1 produced an SBR of approximately 10.

[0236] In some combined configurations, the GVES utilizes both the macro-stoichiometric baseline and the assembly factor hierarchical configuration simultaneously. To provide functional integration across different target cell types and expression platforms, the synergistic system can be configured to operate within a broadly effective DI ratio range of approximately 2 to 12 for the structural module, 2 to 8 for the AF1 module, 0.5 to 2 for the AF2 module, and 0.125 to 1 for the AF3 module (i.e., a ratio range of approximately (2-12) : (2-8) : (0.5-2) : (0.125-1)).

[0237] In further defined parameter ranges, the system is characterized by an overall Target Dosage Index (DI) ratio of Structural : AF1 : AF2 : AF3 wherein the Structural DI is at least 2-fold higher than any individual AF DI, and the AF Dis follow the descending hierarchy. Within this framework, specific verifiable target DI ratios comprise approximately 4:2: 1:0.5, or approximately 6:2:1:0.25.

[0238] lln preferred embodiments utilizing the 6:2:1:0.25 optimized expression ratio, the engineered mARG 3.0 system yields a significantly enhanced Signal-to-Background Ratio (SBR) under BURST ultrasound imaging of approximately 4.5-fold, or within a functional variance of + / - 25% of said valueTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0239] The requisite stoichiometric ratios can be configured through approaches comprising the selection of specific regulatory sequences, transcription factors, and / or post-translational regulatory elements. In certain embodiments, computational modeling and synthetic biology design tools are used to predict regulatory strength and dynamic range to configure the Target DI. Such computational approaches can include in silico simulation of transcriptional and translational processes, thermodynamic modeling of promoter-transcription-factor interactions, codon-usage optimization, mRNA secondary-structure prediction, and kinetic modeling of protein expression and folding. Gene regulatory networks can be modeled using ordinary -differential-equation (ODE) or stochastic frameworks to determine parameters suitable for balancing the target DI hierarchy.

[0240] In some embodiments, synthetic-biology design platforms and computational modeling suites — including, but not limited to, SBOL-compliant DNA-assembly software, BioStudio™, Benchling®, Geneious®, or MATLAB® SimBiology® — can be used to construct and simulate the GVES cassettes prior to experimental implementation. Computational pipelines may integrate experimentally derived data (for example, RNA-seq or proteomics) with machine-learning models trained to predict transcriptional strength, translation rate, or post-translational modification patterns in a given host cell. By utilizing these or equivalent modeling platforms, a person skilled in the art can identify the specific regulatory architectures required to achieve the target Dosage Index stoichiometry and ensure stable vesicle formation without undue experimentation.

[0241] In addition to, or in the alternative to, the assembly factor hierarchical configurations described herein, the genetically engineered Gas Vesicle Expression System (GVES) provides structural gas vesicle proteins configured to physically alter the mechanical stability of the gas vesicle shell.

[0242] As used herein, the term "operative connection" or "configuration" indicates an arrangement of elements in a combination enabling production of an appropriate effect. With respect to genes and regulatory sequences, an operative connection indicates a configuration of the genes with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease transcription or translation of the genes. With respect to structuralTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT proteins, a configuration indicates the specific spatial arrangement and covalent linkage of distinct polypeptide domains to provide a target secondary or tertiary structure.

[0243] In these configurations, the structural gvp differentially expressed gene module comprises a chimeric gvp gene (e.g., a chimeric gvpA / B gene) encoding a chimeric GV structural protein. The chimeric GV structural protein comprises a core structural shell domain derived from a first gas vesicle structural protein directly or indirectly fused to a stabilizing tail domain derived from a second gas vesicle structural protein.

[0244] As previously described herein with reference to the native gas vesicle gene clusters, the term "gas vesicle structural protein" refers to the highly hydrophobic proteins that polymerize to form the crystalline ribs and structural lattice of the gas vesicle shell. While representative configurations herein describe these components utilizing the conventional nomenclature of "GvpA" (typically denoting a core-only structural protein) or "GvpB" (typically denoting a structural protein natively comprising a stabilizing C-terminal tail domain), it is understood by a person skilled in the art that genomic annotation and nomenclature vary across prokaryotic species. Accordingly, the genus of gas vesicle structural proteins encompasses any homologous latticeforming protein — regardless of whether it is annotated in a database as GvpA, GvpAl, GvpA2, GvpB, or a functionally equivalent structural homolog — provided the protein comprises the requisite alphal-betal-beta2-alpha2 secondary structure topology capable of assembling into a gas vesicle shell.

[0245] ] As used herein, the term “lattice” or “gas vesicle lattice” refers to the highly ordered, crystalline protein shell structure formed by the multimeric assembly and polymerization of gas vesicle structural proteins (e g., GvpA, GvpB, or chimeric derivatives thereof). Morphologically, the lattice comprises a repeating series of interlocking structural ribs that enclose a hollow interior volume, forming a semi-permeable shell that excludes liquid water while permitting the free diffusion of gas. The structural integrity and formation of the lattice are fundamentally dependent on the conserved alpha 1 -beta l-beta2-alpha2 secondary structure topology of the monomeric structural subunits.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0246] The successful of a functional gas vesicle lattice can be objectively detected and verified by a person skilled in the art using morphological, physical, and / or acoustic assays. Morphological detection of the lattice is performed using Transmission Electron Microscopy (TEM) or CryoElectron Microscopy (Cryo-EM) to visually identify the repeating, striated crystalline rib structures and the characteristic cylindrical or spindle-shaped overall nanostructure. Physical detection of the lattice is performed via centrifugally-driven buoyancy purification; because a properly assembled, gas-filled lattice imparts physical buoyancy, functional gas vesicles can be detected by their ability to partition to the meniscus (float) when centrifuged in a cellular lysate or culture medium. Acoustic detection of the lattice is performed by subjecting the target cells or purified structures to an ultrasound imaging sequence (such as cross-amplitude modulation (xAM) or BURST) and measuring the resulting nonlinear acoustic scattering or characteristic acoustic collapse signature. The presence of these nonlinear acoustic transients uniquely verifies the successful formation of a rigid, gas-filled protein lattice.

[0247] The two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond to the C-terminal end (-COOH group) which is the end where a COOH group is not involved in a peptide bond. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and / or analogs thereof are joined through chemically-mediated condensation of an organic acid (-COOH) and an amine (-NH2) to form an amide bond or a “peptide” bond.

[0248] The term "attach" or "attached" as used herein, refers to connecting or uniting by a bond, link, force, or tie in order to keep two or more components together, which encompasses either direct or indirect attachment. For example, "direct attachment" refers to a first molecule directly bound to a second molecule or material, while "indirect attachment" in refers to one or more intermediate molecules being disposed between the first molecule and the second molecule or material. In the context of the chimeric structural proteins herein described, "directly covalentlyTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT attached" refers to a first domain directly bound to a second domain through a covalent peptide bond, lacking any intervening synthetic spacer or linker sequence.

[0249] Structurally, the core structural shell domain of the chimeric GV structural protein comprises an alphal-betal-beta2-alpha2 secondary structure topology. The core domain terminates at the C-terminal end of the alpha2 helix. To establish this boundary across uncharacterized gas vesicle-producing species, a person having ordinary skill in the art can identify the C-terminal termination point of the alpha2 helix utilizing standard secondary structure prediction algorithms, including but not limited to AlphaFold, JPred, or Phyre2.

[0250] The stabilizing tail domain is a C-terminal region of the chimeric GV structural protein directly covalently attached to said C-terminal end of the alpha2 helix. The stabilizing tail domain comprises a structurally disordered (unstructured) C-terminal region natively located subsequent to the alpha2 helix of said second gas vesicle protein. Functionally, the stabilizing tail domain is configured to physically interact with at least one GvpU and / or GvpN assembly protein derived from the same second species. In a first configuration lacking the tail domain, interaction with the assembly proteins yields a baseline assembly efficiency. In a second configuration comprising the integrated tail domain, this physical interaction correlates with an increase in assembly efficiency, providing an assembled gas vesicle at a detectable volumetric yield.

[0251] To identify, isolate, and extract the core structural shell domain and the stabilizing tail domain from uncharacterized or newly sequenced prokaryotic species, a person skilled in the art can execute a defined bioinformatic protocol. First, a sequence homology search (utilizing algorithms comprising BLAST, PSI-BLAST, or HMMER) is performed against prokaryotic genomic databases to identify native GvpA or GvpB structural protein candidates.

[0252] Second, secondary structure prediction algorithms (comprising AlphaFold, JPred, or Phyre2) are applied to the identified candidates to map the conserved alphal-betal-beta2-alpha2 topology. The core domain boundary is strictly defined as the terminal amino acid residue of the alpha2 helix.

[0253] Third, candidates are screened for the presence of a natively unstructured C-terminal extension. The stabilizing tail domain is structurally defined as the contiguous amino acidTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT sequence extending from the residue immediately subsequent to the alpha2 helix termination point to the native C-terminus of the protein. By filtering for tail domains characterized by structural disorder and a net negative formal charge, functional cross-species chimeric pairs can be systematically identified and genetically fused without undue experimentation.

[0254] Accordingly, in the chimeric GV structural protein, the first species can be selected from species of GV-producing prokaryotes encoding a GvpA or GvpB having a core alphal-betal-beta2-alpha2 domain capable of forming a gas vesicle lattice or a derivative thereof.

[0255] In the chimeric GV structural protein, the second species can be selected from species of GV-producing prokaryotes encoding a GvpA or GvpB having a stabilizing C-terminal tail domain natively projecting from the structural lattice, or a derivative thereof.

[0256] In particular, the first species can be independently selected from the group consisting of Anabaena flos-aquae, Halobacterium salinarum. Microcystis aeruginosa, Planktothrix rubescens, and Trichodesmium erythraeum. and the second species can be independently selected from the group consisting of Bacillus megaterium, S rratia sp. ATCC 39006, Desulfovibrio vulgaris, and Streptomyces coelicolor.

[0257] The chimeric protein comprises sequences from the base gene or a derivative thereof. A derivative of a chimeric GV structural protein or a domain thereof encompasses a derivative amino acid sequence having at least 70% overall sequence identity to the sequence of said chimeric GV structural protein or domain thereof. The derivative amino acid sequence can comprise substitutions, deletions, and / or insertions. In the derivative amino acid sequence, the substitutions replace a base amino acid with a functionally equivalent amino acid possessing a side-chain van der Waals volume within + / - 30%, a net formal charge at pH 7.4 within + / - 1, and a Kyte-Doolittle hydropathy index within + / - 1.5 units relative to the corresponding residue of the reference sequence. In the derivative amino acid sequence, the deletions and insertions result in an amino acid sequence having substantially the same domain inclusive of secondary structure and tertiary structure, ability to form quaternary structure, and substantially the same functionality as the base amino acid sequence.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0258] The term “domain” as related to the protein indicates any continuous part of a protein sequence from a single amino acid up to the full protein associated to an identifiable structure within the protein. An “identifiable structure” in the sense of the disclosure indicates a spatial arrangement of the primary structure or portions thereof which can be detected by techniques such as crystallography, hydrophobicity analysis or additional techniques known by a skilled person. In many instances, a protein domain comprises one or more secondary structures of the protein, which together form a tertiary or quaternary structure of a protein.

[0259] The “secondary structure” of a protein refers to local sub-structures with a repeating geometry identifiable within the crystal structure of the protein, by circular dichroism, or by additional techniques identifiable by a skilled person. In some instances, a secondary structure of a protein can be identified by the patterns of hydrogen bonds between backbone amino and carboxyl groups. Secondary structures can also be defined based on a regular, repeating geometry, being constrained to approximate values of the dihedral angles y and cp of the amino acids in the secondary structure unit on the Ramachandran plot. Two main types of secondary structure are the alpha helix and the beta strand or beta sheets as will be identifiable by a skilled person. Both the alpha helix and the beta sheet represent a way of establishing non-covalent hydrogen bonds between constituents of the peptide backbone, thus forming secondary structural features. Secondary structure formation can be promoted by formation of hydrogen bonds between backbone atoms. Amino acids that can minimize formation of a secondary structure by destabilizing the structure of the hydrogen bonding interactions are referred to as secondary structure breakers. Amino acids that can promote formation of a secondary structure by stabilizing formation of hydrogen bonding interactions are referred to as structure makers.

[0260] The term “tertiary structure” refers to the three-dimensional structure of a protein, stabilized by non-covalent interactions among non-adjacent segments of the protein and optionally by one or more additional compounds or ions interacting through covalent or non-covalent interactions with one or more segments of the proteins. Exemplary non-covalent interactions stabilizing the three-dimensional structure of the proteins comprise non-specific hydrophobic interactions, burial of hydrophobic residues from water, specific tertiary interactions, such as salt bridges, hydrogen bonds, the tight packing of side chains, chelation and disulfide bonds andTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT additional interactions identifiable by a skilled person. Exemplary covalent interactions among compounds or ions and segments of the protein comprise N-linked glycosylation, cytochrome C haem attachment and additional interactions identifiable by a skilled person.

[0261] The term “quaternary structure” when referred to a complex refers to the three-dimensional structure of a protein complex, also called a multimer, stabilized by non-transitory interactions between the two or more proteins forming the complex. Accordingly, the quaternary structure can be stabilized by some of the same types of non-covalent and covalent interactions as the tertiary structure as will be understood by a skilled person. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-", for example, a heterotetramer, such as the two alpha and two beta chains of hemoglobin. “Non-transitory interactions” as used herein indicates interactions between proteins or related segments that are detectable by laboratory techniques such as immunoprecipitation, crosslinking and Forster Resonance Energy Transfer (FRET) measurements, crystallography, Nuclear Magnetic Resonance (NMR) and additional techniques identifiable by a skilled person.

[0262] Detection of three-dimensional secondary, tertiary, or quaternary protein structure can be performed using techniques such as x-ray crystallography, NMR spectroscopy, and dual polarization interferometry among others known to a skilled person. Using such techniques, the position of structural features comprising alpha-helices, beta strands, turns, beta bridges, bends, loops, coils, coiled coils, and others identifiable by a skilled person within the N-terminal to C-terminal primary sequence of a protein can be detected.

[0263] In particular, detection of repeated motifs in a protein domain in the sense of the disclosure can be performed using structure prediction servers COILS, Paircoil2, LOGICOIL and JPred. Structure of polypeptides and proteins can also be obtained from publicly available sources such as the Protein Data Bank and others known to a skilled person.

[0264] As used herein, a derivative having "substantially the same secondary structure and tertiary structure" indicates that the derivative sequence rigorously maintains the local spatial conformation (secondary structure) necessary to preserve the alpha 1 -beta l-beta2-alpha2 topologyTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT of the core domain, and the structurally disordered conformation of the stabilizing tail domain. Furthermore, the three-dimensional folding and spatial packing (tertiary structure) of the monomeric protein chain is maintained such that the stabilizing tail domain correctly projects outward from the core domain. When assembled, a derivative possessing substantially the same secondary and tertiary structure will maintain the high-density clustering phenotype, exhibiting a multi-directional interwoven bundling architecture with intersection angles between adjacent vesicles within a variance of + / - 15 degrees relative to the 30 to 90 degree non-parallel angles observed in the reference chimeric gas vesicles.

[0265] As used herein, a derivative having "substantially the same functionality" indicates that the derivative core domain retains the physical ability to polymerize into a functional gas vesicle lattice, and the derivative stabilizing tail domain retains the ability to physically interact with assembly factors, such as GvpN and GvpU, to provide mechanical reinforcement. The maintained functionality of the derivative can be objectively verified by a person skilled in the art. Morphological functionality (lattice assembly and intersection angle variance) is verified by expressing the derivative in a target cell and observing the high-density gas vesicle assembly via Transmission Electron Microscopy (TEM). Acoustic functionality (mechanical reinforcement) is verified by co-expressing the derivative with the cognate GvpN and GvpU assembly proteins, and measuring the resulting acoustic collapse pressure profile using a cross-amplitude modulation (xAM) or BURST ultrasound imaging sequence. A functional derivative will yield an assembled engineered gas vesicle possessing an intrinsic median critical collapse pressure right-shifted by at least +50 kPa relative to a reference gas vesicle lacking the stabilizing tail domain. To ensure the preservation of said structure and functionality, in defined parameter ranges, the sequence variance comprising deletions and insertions within the derivative amino acid sequence is restricted. Specifically, the derivative amino acid sequence comprises no more than 8 consecutive amino acid insertions or deletions within the core structural shell domain, and no more than 8 consecutive amino acid insertions or deletions within the stabilizing tail domain.

[0266] In a representative embodiment, the core structural shell domain is derived from an Anabaena flos-aquae GvpA protein, and the stabilizing tail domain is derived from a BacillusTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT megaterium GvpB protein. In this specific configuration, the chimeric GV structural protein comprises an amino acid sequence having at least 70% overall sequence variation to the sequence:MAVEKTNSSSSLAEVIDRILDKGIVIDAWVRVSLVGIELLAIEARIVIASVETYLKYAEAV GLLRDDVEENGLPERSNSSEGQPRFSI (SEQ ID NO: 482).

[0267] Within this reference sequence, the core structural shell domain corresponds to amino acids 1 to 63 of SEQ ID NO: 482, and the stabilizing tail domain corresponds to amino acids 64 to 88 of SEQ ID NO: 482.[00268J The term "percent variation" or "percentage variation" as used herein means the difference between two amino acid residue sequences, expressed as a percentage, wherein the difference between two amino acid sequences is measured by a process that comprises the steps of aligning the two amino acid sequences, then detecting one or more differences between the aligned sequences, and calculating the total number of differences divided by the total number of aligned amino acids in each amino acid sequence, including gaps with the result expressed as a percentage.

[0269] The term "alignment" as used herein means aligning the positions of structural features of statistically significant structural similarity between two amino acid sequences, where "statistically significant structural similarity" means greater than 95% probability that two structural features are structurally homologous, for example, alpha-helix. The term 'difference" indicates mismatches in the position of structural features in the position of structural features in the two amino acid sequences, whereby each amino acid that comprises part of a mismatched structural feature is counted as one difference between the two aligned amino acid sequences. Mismatches between aligned sequences can comprise an insertion, a deletion, and / or a replacement of one or more structural features in one amino acid sequence compared to the other aligned amino acid sequence as would be understood by a skilled person. Several publicly available online servers can be used to detect protein structure alignment and calculate percent variation, such as FATCAT, SuperPose, iPBA, MAPSCI, and others identifiable by a person skilled in the art.

[0270] To maintain structural and functional integrity of the protein shell within this percent variation, allowable sequence variance within the chimeric GV structural protein are conservative substitutions.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0271] As used herein, a "functionally equivalent" conservative substitution refers to a modification that does not detectably disrupt the required alphal-betal-beta2-alpha2 secondary structure topology of the core domain, nor abolish the ability of the chimeric protein to polymerize into an assembled gas vesicle shell. Specifically, a functionally equivalent substitution is one wherein the substituted amino acid possesses a side-chain van der Waals volume within + / - 30%, a net formal charge at a physiological pH of 7.4 within + / - 1, and a Kyte-Doolittle hydropathy index within + / - 1.5 units relative to the corresponding residue of the reference sequence.

[0272] The biophysical parameters utilized to define a functionally equivalent conservative substitution are anchored to standard biochemical metrics. As used herein, the "side-chain van der Waals volume" refers to the spatial volume occupied by the unperturbed amino acid side chain. To maintain the steric packing of the gas vesicle shell, allowable substitutions in defined configurations possess a van der Waals volume within + / - 30%, within + / - 20%, or strictly within + / - 10% of the volume of the corresponding residue in the reference sequence.

[0273] As used herein, the "net formal charge" refers to the predicted electrostatic charge of the amino acid side chain calculated at a physiological target environment pH of 7.4. To maintain electrostatic interactions and homotypic repulsion, allowable substitutions in defined configurations possess a net formal charge within + / - 1 unit, or are identically charge-matched (a difference of 0) relative to the net formal charge of the corresponding residue in the reference sequence.

[0274] As used herein, the "Kyte-Doolittle hydropathy index" refers to the standard numerical scale representing the relative hydrophobic or hydrophilic character of an amino acid side chain. To maintain the amphipathic boundaries of the shell architecture, allowable substitutions in defined configurations possess a Kyte-Doolittle hydropathy index within + / - 1.5 units, within + / -1.0 units, or strictly within + / - 0.5 units relative to the corresponding residue in the reference sequence.

[0275] This architecture is structurally distinct from the parallel planar bundling observed in reference gas vesicles. Specifically, adjacent engineered gas vesicles within a cluster intersect at variable non-parallel angles. In broad parameter ranges, this intersection angle extends fromTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT approximately 15 degrees to 90 degrees. In further defined parameter ranges, the intersection angle is from approximately 30 degrees to 90 degrees, or strictly between 45 degrees and 75 degrees. The combination of these variable non-parallel intersection angles and the interwoven architecture correlates with a known or expected increase in localized intracellular packing density, yielding higher spatial acoustic contrast relative to wild-type parallel arrays

[0276] In specific configurations, the sequence variance is distributed asymmetrically across the chimeric GV structural protein. The core structural shell domain independently comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence variation to the reference core domain (e.g., amino acids 1 to 63 of SEQ ID NO: 482). Because the stabilizing tail domain is structurally disordered, it tolerates a higher degree of sequence variance. Accordingly, the stabilizing tail domain independently comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence variation to the reference tail domain (e.g., amino acids 64 to 88 of SEQ ID NO: 482), provided the net negative formal charge at pH 7.4 is maintained.

[0277] Within the core structural shell domain, the sequence variance can be further differentially allocated to preserve the lattice-forming mechanics. In defined configurations, the discrete segments corresponding to the alpha!, betal, beta2, and alpha2 secondary structures possess a high sequence variation of at least 85%, at least 90%, at least 95%, or at least 99% relative to the corresponding segments of the reference sequence. In contrast, the intervening unstructured loop regions connecting said secondary structures independently possess a lower baseline sequence variation of at least 40%, at least 50%, at least 60%, or at least 70% relative to the reference sequence, provided the steric beta-sheet pairing and alpha-helical packing are maintained.

[0278] To maintain the physical buckling limits and interaction kinetics of the engineered gas vesicle, the absolute lengths of the domains are structurally bounded. In functional configurations, the core structural shell domain consists of an absolute length from 50 to 80 amino acids, preferably 60 to 70 amino acids. The directly covalently attached stabilizing tail domain consists of an absolute length from 10 to 60 amino acids, preferably 15 to 35 amino acids. Substitutions, insertions, or deletions that extend or compress the domains beyond these absolute parameters correlate with a detectable loss of volumetric yield or acoustic right-shifting.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0279] Furthermore, sequence variance comprising insertions or deletions is configured such that the alpha 1 -beta l-beta2-alpha2 secondary structure topology of the core structural shell domain is maintained, and the structurally disordered conformation of the stabilizing tail domain is preserved.

[0280] In specific alternative configurations, the stabilizing tail domain of the chimeric GV structural protein can be truncated to modulate the interaction affinity with the assembly proteins. In specific parameter ranges, the stabilizing tail domain comprises a truncation of at least 5, at least 10, or at least 15 amino acids from the extreme C-terminus relative to the wild-type second gas vesicle protein, provided the net negative charge profile is maintained.

[0281] While the stabilizing tail domain is natively and preferably located at the C-terminus of the core structural shell domain, in alternative structural configurations, an equivalent unstructured, charged stabilizing domain derived from a gas vesicle protein is directly covalently attached to the Core N-terminus (upstream of the alphal helix). This N-terminal configuration is structurally tolerated provided the fusion does not sterically hinder the beta-sheet pairing required for shell polymerization.

[0282] The right-shifted acoustic profile and high-density bundling phenotype demonstrated by the representative Anabaena-Bacillus chimera are structurally driven by the integration of the charged, unstructured tail domain, rather than the specific primary amino acid sequence of the core domain. Because the alphal-betal-beta2-alpha2 secondary structure topology is evolutionarily conserved with high structural homology across gas vesicle-producing prokaryotes, the core structural shell domain functions as a universal modular scaffold. The stabilizing tail domain operates via localized steric and electrostatic interactions that are functionally independent of the specific core sequence, provided the direct fusion junction at the C-terminus of the alpha2 helix is maintained. Consequently, empirical data demonstrating the mechanical reinforcement and increased volumetric yield of the representative Anabaena-Bacillus chimera provides verifiable written description and predictive enablement for cross-species chimeras.

[0283] Because this structural alteration is driven by conserved biophysical parameters, the components of the chimeric GV structural protein can be derived from other gas vesicle-producingTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT species. In one alternative configuration, the chimeric GV structural protein comprises a core structural shell domain derived from Microcystis aeruginosa and a stabilizing tail domain derived from Bacillus megaterium, comprising an amino acid sequence having at least 70% overall sequence variation relative to the based sequence of Microcystis aeruginosa and Bacillus megaterium.

[0284] In another alternative configuration, the chimeric GV structural protein comprises a core structural shell domain derived from Anabaena flos-aquae and a stabilizing tail domain derived from Serratia sp. ATCC 39006, comprising an amino acid sequence having at least 70% overall sequence variation relative to the based sequence of Anabaena flos-aquae and Serratia sp. ATCC 39006.

[0285] In further configurations, the chimeric GV structural protein further comprises a heterologous cargo moiety fused to the N-terminus of the core domain or the C-terminus of the stabilizing tail domain. The cargo moiety is selected from a fluorescent protein, an enzymatic domain, or a targeting ligand, wherein the fusion does not disrupt the alphal-betal-beta2-alpha2 topology required for shell assembly.

[0286] To encode these structures, an isolated engineered polynucleotide comprises a chimeric gvp gene encoding the chimeric GV structural protein. In specific configurations, the chimeric gvp gene comprises a direct fusion junction lacking any intervening synthetic linker sequence between the nucleotide sequence encoding the core structural shell domain and the nucleotide sequence encoding the stabilizing tail domain. A genetically engineered Gas Vesicle Expression System (GVES) can comprise a structurally reinforced chimeric gas vesicle shell by utilizing at least one structural gvp differentially expressed gene module comprising the chimeric gvp gene. The chimeric gvp gene is under the control of a promoter and additional regulatory regions configured for expression of the chimeric GV structural protein in a target cell.

[0287] When a plurality of these chimeric GV structural proteins assemble to form an engineered gas vesicle in a target cell, the resulting protein shell exhibits measurable phenotypic and mechanical alterations relative to a wild-type reference gas vesicle. Morphologically, the engineered gas vesicles exhibit a high-density clustering phenotype characterized by a multi-Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT directional, interwoven bundling architecture. This architecture is structurally distinct from the parallel planar bundling observed in reference gas vesicles; specifically, adjacent engineered gas vesicles within a cluster intersect at variable non-parallel angles ranging from approximately 30 degrees to 90 degrees to form three-dimensional aggregates.

[0288] The structural integration of the stabilizing tail domain physically alters the mechanical compliance of the resulting protein shell. Consequently, the engineered gas vesicle exhibits a right-shifted acoustic collapse pressure profile relative to a reference gas vesicle consisting of a protein shell lacking the stabilizing tail domain. Based on empirical acoustic scattering profiles, this architecture provides an intrinsic median critical collapse pressure right-shifted by at least 150 kPa relative to a wild-type Anabaena flos-aquae reference.

[0289] Furthermore, the aforementioned structural parameters — domain length, regional sequence variance, and tail truncation — can be deployed in specific combinations to precisely configure the expected acoustic output. In a defined combined configuration, an engineered gas vesicle comprising a core domain absolute length of 60 to 70 amino acids and a tail domain absolute length of 15 to 35 amino acids, wherein the core domain sequence identity is at least 85% and the tail domain sequence identity is at least 70%, is known or expected to correlate with a median critical collapse pressure right-shifted by at least 150 kPa.

[0290] In an alternative combined configuration, variating the structural parameters by utilizing a truncated stabilizing tail domain (e.g., lacking 5 to 15 C-terminal amino acids) combined with an identical core domain length, is known or expected to modulate the assembly protein interaction kinetics. This combination correlates with an expected fine-tuned right-shift of between 50 kPa and 100 kPa, demonstrating that the structural parameters can be combined and variated to yield predictably distinct acoustic sub-populations.

[0291] The magnitude of the right-shifted acoustic collapse pressure profile is a configurable parameter directly correlated with the structural configuration of the stabilizing tail domain. Specifically, altering the absolute length or the net formal charge of the stabilizing tail domain (e.g., via truncations or species-specific derivation) modulates the interaction efficiency with the assembly proteins, thereby modulating the mechanical compliance of the shell.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0292] In defined parameter ranges, the engineered gas vesicle possesses an intrinsic median critical collapse pressure right-shifted by at least 50 kPa, at least 100 kPa, at least 150 kPa, at least 200 kPa, or at least 250 kPa relative to a reference gas vesicle lacking the stabilizing tail domain.

[0293] Correspondingly, the structural buckling limit of the engineered protein shell is a configurable parameter extending between approximately 600 kPa and 1.5 MPa. In further defined parameter ranges, depending on the specific tail domain length and cross-species assembly interaction, the structural buckling limit extends between 700 kPa and 1.2 MPa, or strictly between 800 kPa and 1.0 MPa.

[0294] In certain configurations, multiscale modeling is employed to connect molecular-level structural parameters to the macroscopic acoustic properties of the engineered gas vesicles. Finite-element simulations and molecular-dynamics calculations are utilized to estimate GV shell stiffness, resonance frequency, and theoretical buckling limits as a function of the stabilizing tail domain's length, charge, and structural disorder. Utilizing these models, the structural buckling limit of the protein shell extends between approximately 700 kPa and 1.2 MPa. By utilizing these predictive models, a person skilled in the art can iteratively refine the fusion junction and tail sequence to achieve a target right-shifted acoustic signature without undue experimentation.

[0295] This mechanically robust architecture enables distinct acoustic differentiation for multiplexed imaging applications, allowing the chimeric GVs to be acoustically distinguished from sub-threshold gas vesicle populations in the same environment by applying differential acoustic pressures.

[0296] Furthermore, the GVES can incorporate specific yield-enhancing assembly complexes to increase the intracellular volumetric yield of the assembled gas vesicles. In these configurations, at least one assembly factor gvp differentially expressed gene module comprises a gvpU gene and a gvpN gene under the control of one or more regulatory regions configured to co-express a GvpU assembly protein and a GvpN assembly protein.

[0297] As previously described herein with reference to the native gas vesicle gene clusters, the "GvpN assembly protein" refers to a highly conserved endogenous gas vesicle assembly factor functionally characterized as an ATPase / NTPase, which typically comprises a beta-barrelTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT hexamerization domain and conserved Walker A and Walker B nucleotide-binding motifs. Similarly, the "GvpU assembly protein" refers to an accessory assembly factor or chaperone protein natively encoded within certain yield-optimized prokaryotic gas vesicle operons.

[0298] Building upon the general identification of gas vesicle gene clusters described above, a person skilled in the art can identify, isolate, and extract functional GvpN and GvpU assembly proteins from uncharacterized or newly sequenced prokaryotic species utilizing a targeted bioinformatic protocol. Utilizing sequence homology searches (e.g., BLAST, PSLBLAST, or HMMER), identified native gvp operons are specifically screened for the co-localization of a gvpN gene and a gvpU gene. Because the GvpN and GvpU proteins are defined by their functional domains and operon co-localization rather than strict primary sequence identity alone, a person skilled in the art can systematically identify homologous yield-enhancing pairs across varying species by referencing the cluster architecture without undue experimentation.

[0299] To provide a target assembly stabilization, the GvpU assembly protein and the GvpN assembly protein are derived from a target species selected from the group consisting of Bacillus megaterium, Anabaena flos-aquae, Halobacterium salinarum, Microcystis aeruginosa, and Serratia sp. ATCC 39006, or a functionally equivalent gas vesicle-producing species.

[0300] As used herein, the phrase "functionally equivalent gas vesicle-producing species" is distinct from the concept of a functionally equivalent amino acid residue. A functionally equivalent gas vesicle-producing species is defined biologically and bioinformatically as a prokaryotic organism that natively encodes a complex gvp gene cluster comprising not only the core structural lattice genes (e.g., gvpA or gvpB homologs), but also the accessory assembly chaperone network specifically including homologous gvpN and gvpU sequences. To determine whether a target species is a functionally equivalent gas vesicle-producing species, a defined method is executed: (1) querying the genomic sequence of the target species to identify the presence of a gvp operon; (2) verifying the operon encodes a structural protein capable of forming a gas vesicle lattice; and (3) bioinformatically confirming the co-localization within or adjacent to said operon of a gvpN ortholog (encoding an ATPase / NTPase domain) and a gvpU ortholog (encoding a chaperone capable of stabilizing the assembly complex). By way of example, functionally equivalent gas vesicle-producing species comprise Bacillus megaterium and Serratia sp. ATCC 39006. ByTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT contrast, a non-functionally equivalent species is one that either lacks a gvp gene cluster entirely, or a prokaryote that possesses an evolutionarily minimal gvp operon natively devoid of a gvpU homologous chaperone sequence.

[0301] While GvpN and GvpU assembly proteins are identifiable via operon co-localization, their specific amino acid sequences can variate while maintaining the functional capacity to stabilize the assembly complex. To provide functional flexibility, allowable sequence variance within the GvpN and GvpU assembly proteins is restricted to the functionally equivalent conservative substitutions (governed by the side-chain van der Waals volume, net formal charge, and Kyte-Doolittle hydropathy index parameters) previously defined herein. Accordingly, the GvpN assembly protein independently comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence variation relative to a wildtype reference GvpN assembly protein, provided the sequence variance maintains the conserved Walker A and Walker B nucleotide-binding motifs and the beta-barrel hexamerization domain required for ATPase / NTPase activity. Similarly, the GvpU assembly protein independently comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence variation relative to a wild-type reference GvpU assembly protein. Because GvpU functions as an accessory chaperone rather than a structural lattice component, it tolerates a higher baseline degree of sequence variance, provided the structurally variated sequence maintains the physical interaction capacity required to chaperone the GvpN complex without causing assembly failure.

[0302] The co-expressed GvpU and GvpN proteins are configured to physically and functionally interact, forming a stabilized GvpN-GvpU assembly complex. While computational modeling indicates the GvpU homohexamer may structurally interface with the beta-barrel hexamerization domain of the GvpN assembly protein, the integration and co-expression of the GvpU assembly protein functionally stabilizes the assembly complex as a chaperone. Because GvpU acts as a functional chaperone rather than a structural lattice component of the gas vesicle shell, its integration does not detectably alter the morphological assembly or the mechanical properties of the resulting gas vesicle shell.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0303] Specifically, the intrinsic acoustic collapse pressure of the engineered gas vesicles produced by the system comprising the GvpU assembly protein remains substantially constant relative to the critical collapse pressure of a reference expression system lacking the gvpU gene. In defined parameter ranges, this mechanical stability is characterized by an acoustic collapse pressure variance strictly within + / - 15%, within + / - 10%, or within + / - 5% relative to the baseline system, or measured as an absolute variance within + / - 75 kPa, within + / - 50 kPa, or within + / - 25 kPa.

[0304] Because this stabilization is driven by functional chaperone interactions rather than strict species-specific lock-and-key mechanisms with the core structural shell, the GvpN-GvpU assembly complex functions as an independent modular assembly module that can operate to enhance yield across varying structural contexts. Specifically, the co-expressed GvpU and GvpN assembly proteins are configured to provide an expression enhancement for gas vesicle structural proteins (e.g., the chimeric GV structural proteins) that possess a charged, unstructured stabilizing C-terminal tail domain, regardless of the species of the core structural shell domain.

[0305] To ensure structural compatibility across these modular components — analogous to fitting a specific cap to a complementary pen body, or an engine to a chassis — a person skilled in the art can verify compatibility prior to expression. In preferred configurations, the GvpU assembly protein and the GvpN assembly protein are derived from the same target species as each other (species-matched), and are further matched to the species from which the stabilizing C-terminal tail domain of the structural protein is derived. However, in cross-species modular configurations, a GvpU and GvpN complex from a first species can be combined with a structural C-terminal tail domain from a second species. A person skilled in the art can evaluate and verify this cross-species compatibility by bioinformatically or experimentally confirming (e g., via AlphaFold-Multimer modeling or in vitro binding assays) that the electrostatic charge profile and steric volume of the structural protein's C-terminal tail domain physically interfaces with the chaperone binding pocket of the cross-species GvpU / N complex. If the interface is conserved, the modular components are functionally compatible.

[0306] To achieve a target increase in intracellular volumetric yield, the GvpU and GvpN assembly proteins are co-expressed within a defined stoichiometric window. Expression of GvpUTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT relative to GvpN at a molar ratio of 5:1 or higher (GvpU:GvpN) correlates with a measurable reduction or complete cessation of functional gas vesicle formation (assembly failure). Therefore, the GVES is stoichiometrically configured to co-express the GvpU and GvpN assembly proteins at a functional molar ratio ranging from approximately 0.5:1 to 3.0:1. In further defined parameter ranges, the functional molar ratio is configured from 0.8:1 to 2.0:1. In preferred configurations, the GvpU and GvpN assembly proteins are co-expressed at a substantially equal molar ratio of approximately 1:1, thereby increasing intracellular yield while maintaining the integrity of the gas vesicle assembly pathway.

[0307] To configure these molar ratios, the gvpU gene and the gvpN gene can be operatively connected via a multi-cistronic linkage architecture, comprising a P2A peptide, an Internal Ribosome Entry Site (IRES), or an equivalent sequence, ensuring proportional co-translation. Alternatively, they may be expressed as separate independent cassettes utilizing promoters of calibrated relative strengths.

[0308] The aforementioned structural and stoichiometric parameters can be deployed in specific combinations to precisely configure the expected volumetric yield output. In a defined combined configuration, co-expressing a GvpN-GvpU assembly complex (whether species-matched or verified as cross-species compatible) at a functional molar ratio of approximately 1:1, alongside a structural protein comprising a stabilizing tail domain, correlates with a measurable increase in the intracellular yield of assembled gas vesicles.

[0309] This volumetric yield increase is measurable via established acoustic and optical metrics, comprising cross-amplitude modulation (xAM) signal amplitude, BURST Signal-to-Background Ratio (SBR), or Transmission Electron Microscopy (TEM) volumetric density. In defined parameter ranges, the formation of the stabilized GvpN-GvpU complex increases the measured intracellular yield metric by at least 3-fold, at least 5-fold, or at least 8-fold relative to a reference expression system lacking the gvpU gene. In optimal combined configurations, the intracellular yield is increased by at least an order of magnitude (e.g., at least 10-fold) while maintaining the baseline acoustic collapse pressure.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0310] As introduced in the initial descriptions of the genetically engineered Gas Vesicle Expression System (GVES), the distinct structural and genetic innovations detailed herein operate collectively as a modular synthetic biology toolbox. Specifically, the assembly factor stoichiometric optimization (Module 1), the structurally reinforced chimeric GV structural protein (Module 2), and the yield-enhancing GvpN-GvpU assembly complex (Module 3) are independently deployable modules. They can be utilized alone or structurally and functionally integrated in combinatorial permutations to precisely control two primary system outcomes: intracellular volumetric yield and acoustic detectability.

[0311] The selection and internal configuration of a specific module, or a combination of modules, is dictated by the physical constraints of the target cells, the acoustic attenuation of the target site (e.g., tissue depth), and the required imaging modality. To systematically configure the optimal GVES architecture, a person skilled in the art can apply a defined, step-wise configuration matrix utilizing the specific parameters described herein:

[0312] Step 1: Determine the Detectability Outcome (Singleplex vs. Multiplex) via Module 2. First, the required acoustic contrast modality is evaluated. If the application requires multiplexed detectability (e.g., differentiating engineered target cells from native background vesicles), the system is configured to include Module 2 (the chimeric GV structural protein). To tune this outcome, a person skilled in the art can variate specific structural parameters: altering the absolute length of the stabilizing tail domain (e.g., 15 to 35 amino acids) or utilizing truncations (e.g., removing 5 to 15 residues) predictably modulates the acoustic collapse pressure shift. Selecting these defined parameter ranges allows the user to program the resulting engineered gas vesicles with a tailored structural buckling limit (extending between approximately 600 kPa and 1.5 MPa) and a right-shifted acoustic detectability profile (e.g., shifted by +50 kPa to +250 kPa).

[0313] Step 2: Evaluate Target Cell Constraints and Baseline Yield via Module 1. Second, the biological tolerance of the host system is assessed to ensure target expression levels do not induce cytotoxicity, which would fundamentally abrogate detectability. If the target cell is a mammalian host subject to strict payload limits, the system is configured to include Module 1 (the assembly factor stoichiometric optimization). To tune this outcome, the user configures the Target Dosage Index (DI) ratios (e.g., Structural-DI > AF1-DI > AF2-DI > AF3-DI). By selecting specificTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT defined DI sub-ranges (for example, restricting AF2 or AF3 to a DI of 0.1 to 0.5 relative to the structural proteins) and utilizing specific polycistronic linkages, the user maximizes the metabolic efficiency of the host's translation machinery, thereby ensuring a baseline volumetric yield without cellular toxicity.

[0314] Step 3: Assess Target Site Attenuation and Maximize Yield via Module 3. Third, the physical environment of the target site is analyzed. If the target site is located deep within living tissue where acoustic attenuation is severe, the system requires maximized intracellular yield to achieve a detectable cross-amplitude modulation (xAM) or BURST signal. In such cases, the system is configured to include Module 3 (the yield-enhancing GvpN-GvpU assembly complex). To optimize this volumetric yield, the user configures the stoichiometric parameter of the GvpU:GvpN ratio. By avoiding the assembly failure threshold (ratios of 5: 1 or higher) and tuning the complex to the preferred molar ratio parameter (approximately 1:1), the user can achieve an order-of-magnitude increase (e g., at least 5-fold to 10-fold) in measurable volumetric yield and target detectability. Furthermore, the user can reference the cross-species compatibility protocols to mix and match GvpU / N species with differing structural tails.

[0315] By executing this step-wise decision matrix and variating the integrated structural and stoichiometric parameters, a person skilled in the art can systematically select and combine these individual functional modules. When a target application simultaneously requires programmable multiplexing detectability (Step 1), mammalian toxicity mitigation (Step 2), and maximal deeptissue volumetric yield (Step 3), the modular matrix explicitly guides the user to the fully integrated, combinatorial expression system.

[0316] In advanced configurations, a genetically engineered combinatorial Gas Vesicle Expression System (GVES) is provided that physically and functionally integrates multiple structural and genetic modules to achieve compounding, measurable improvements in both expression efficiency and programmable acoustic performance. The combinatorial GVES integrates at least two, and preferably all three, of the following functional aspects: (i) the assembly factor hierarchical configuration comprising the Target Dosage Index (DI) ratio; (ii) the structurally reinforced chimeric gas vesicle (GV) structural protein; and (iii) the yield-enhancing engineered GvpN-GvpU assembly protein complex.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0317] A person skilled in the art can selectively deploy specific combinations of these modular aspects to achieve target imaging modalities — specifically, singleplexed or multiplexed acoustic imaging. To configure a system for singleplexed acoustic imaging requiring maximized contrast sensitivity, the GVES is configured to combine the dual-level stoichiometric optimization (Aspect i) with the yield-enhancing GvpN-GvpU assembly complex (Aspect iii). This specific combination drives high-efficiency translation while chaperoning assembly, yielding a structurally validated increase in intracellular volumetric yield of at least 5-fold to 10-fold relative to a baseline system, without substantially altering the baseline acoustic collapse pressure.

[0318] Alternatively, to configure a system for multiplexed acoustic imaging, the GVES integrates the structurally reinforced chimeric GV structural protein (Aspect ii) in combination with the stoichiometric optimization (Aspect i) and / or the assembly complex (Aspect iii). By integrating the chimeric structural shell (comprising the specific core and stabilizing tail domains), the resulting combinatorial system possesses the aforementioned right-shifted acoustic collapse pressure profile (e g., right-shifted by at least 150 kPa). This mechanically robust combinatorial architecture allows the engineered gas vesicles to be acoustically distinguished from sub-threshold gas vesicle populations in the same environment by applying differential acoustic pressures, allowing for the precise spatiotemporal tracking of cellular therapies or biosensors.

[0319] In a fully integrated "omnibus" embodiment (e.g., the mARG 3.0 system), the genetically engineered GVES comprises all three modular aspects. Specifically, the combinatorial GVES comprises: (i) the assembly factor stoichiometric optimization configured to provide an expression hierarchy wherein AF1-DI > AF2-DI > AF3-DI; (ii) a structural gvp differentially expressed gene module encoding the structurally reinforced chimeric GV structural protein; and (iii) the yieldenhancing assembly complex comprising co-expressed, species-matched GvpU and GvpN assembly proteins.

[0320] The integration of these three features produces a compounding functional outcome. Specifically, the assembly factor stoichiometric optimization and the yield-enhancing GvpN-GvpU complex interact with the chimeric GV structural protein to support the folding, assembly, and accumulation of the structurally reinforced gas vesicles without inducing target cell toxicity.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0321] To ensure proper physical assembly when utilizing the fully integrated omnibus configuration, the hierarchical stoichiometric ratios can be predictively adjusted. Because the yield-enhancing assembly complex and the chimeric structural proteins increase the total physical volume of the structural lattice formed within the cell, the stoichiometric demand for late-stage capping or accessory assembly factors is correspondingly altered. To prevent morphological bottlenecking, the modified Target Dosage Index (DI) ratio can comprise an AF3 Dosage Index (DI) (comprising, e.g., the gvpK gene) that is quantitatively higher than the AF3 Dosage Index (DI) optimized for reference wild-type systems. This stoichiometric adjustment maximizes the terminal assembly efficiency of the volumetrically enhanced vesicle pool.

[0322] Furthermore, the combinatorial GVES is physically configured to maximize delivery efficiency. The differentially expressed gene modules comprising the structural, stoichiometric, and yield-enhancing components can be arranged sequentially on a single polycistronic transcript. This single-transcript configuration is physically sized to fit within the payload capacity limits of standard single-vector delivery systems (comprising, for example, lentiviral vectors, which typically possess a functional packaging limit of approximately 8.0 to 10.0 kilobases), thereby bypassing the necessity and inefficiency of multi-vector co-transfections.

[0323] In some embodiments, the assembly factor stoichiometric optimization (the AF GVES), the structurally reinforced chimeric gas vesicle structural proteins (the A / B chimera), the yieldenhancing gas vesicle assembly protein complexes (the U / N assembly complex), and their combinatorial permutations — collectively define an engineered configurable Gas Vesicle Architecture.

[0324] A person skilled in the art can physically configure this architecture by selectively integrating the aforementioned modules to predictably increase intracellular volumetric yield, target acoustic detectability, or a programmed combination of both, in accordance with the physiological constraints of the target site and host cell.

[0325] In particular in some exemplary embodiments, when the engineered configurable Gas Vesicle Architecture is configured to maximize expression efficiency and structural accumulation (for example, by integrating the stoichiometric assembly factor optimization and the U / N assemblyTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT complex), the architecture provides a quantified increase in intracellular volumetric yield. Specifically, this configured architecture produces an intracellular yield of assembled gas vesicles that is increased by at least 3-fold, at least 5-fold, or optimally at least 10-fold relative to a baseline reference expression system lacking these modifications. This order-of-magnitude increase is structurally measurable via objective acoustic and optical metrics, comprising cross-amplitude modulation (xAM) signal amplitude, BURST Signal-to-Background Ratio (SBR), or cellular Transmission Electron Microscopy (TEM) volumetric density.

[0326] Alternatively or concurrently, in some exemplary embodiments when the engineered configurable Gas Vesicle Architecture is configured to maximize target acoustic detectability (for example, by integrating the chimeric GV structural protein alongside the optimized assembly factors), the architecture provides a predictably right-shifted acoustic collapse pressure profile. This structural control allows the architecture to provide an acoustic distinctiveness characterized by a median critical collapse pressure right-shifted by at least +50 kPa, at least +100 kPa, or optimally at least +150 kPa relative to reference wild-type gas vesicles. This configured shift serves as a quantifiable result-effective variable, directly enabling the spatial multiplexing of distinct gas vesicle populations within a single target environment.

[0327] In embodiments herein described to physically realize these quantified functional outcomes, the engineered configurable Gas Vesicle Architecture can be provided in various specific genetic layouts, transcript arrangements, and vector configurations. Exemplary specific polynucleotide architectures and physical delivery frameworks are detailed below.

[0328] In some embodiments, the genetic constructs encoding the GVES can be provided in various architectures, such as being comprised in one, two, three, or more separate polynucleotides, vectors, or cassettes.

[0329] In a particularly preferred embodiment, all components of the GVES are provided on a single polynucleotide or vector configured to execute both the macrooptimization and the Assembly factor microoptimization. In one such single-vector configuration, all components of the GVES are provided within a single, complex polycistronic cassette under the control of a single promoter. To achieve the multi-tier stoichiometric hierarchy, the genetic modules are spatiallyTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT ordered from 5' to 3' to match the desired Target Dosage Index ratio, utilizing separation elements to link the cistrons. For example, the construct may be arranged to sequentially encode the structural module (e.g., gvpA / B), followed by the highly expressed AF1 module (e.g., gvpF, gvpG, gvpW), the moderately expressed AF2 module (e.g., gvpN, gvpJ), and the strictly limited AF3 module (e.g., gvpK).

[0330] In this single-transcript embodiment, the desired stoichiometric DI ratios are achieved not by separate promoters or varying viral titers, but by the physical 5'-to-3' spatial ordering of the genes and the differential translational efficiency of the regulatory linkers (e.g., IRES elements, 2A peptides) and other post-transcriptional elements. Because downstream cistrons in extended polycistronic configurations often experience sequential reductions in translational efficiency, placing the genes in the exact sequence of the required stoichiometric hierarchy natively governs the final protein-level dosage of each component from a single transcript. In some embodiments, configuring the single-transcript embodiment to integrate the structural macrooptimization, the assembly factor microoptimization, and the engineered GvpN-GvpU assembly complex produces the aforementioned volumetric yield increase of at least 10-fold relative to a baseline system.

[0331] In another single-vector embodiment, the single vector comprises multiple distinct gene expression cassettes. In this configuration, the single vector comprises, for example: a first expression cassette (e.g., [Promoter 1] - [Structural module] - [Terminator 1]); a second polycistronic expression cassette (e.g., [Promoter 2] - [AF1 module] - [Terminator 2]); and a third expression cassette encoding the lower-tier assembly factors (e.g., [Promoter 3] - [AF2 / AF3 modules] - [Terminator 3]). The stoichiometric macrooptimization and microoptimization are achieved in this embodiment by selecting regulatory elements (e.g., Promoter 1, Promoter 2, Promoter 3) that inherently provide the desired relative Dosage Index ratios upon expression.

[0332] In some embodiments, the highly compressed combinatorial GVES described herein is physically sized to fit within standard single-vector packaging limits (e.g., the -8-10 kb capacity of lentiviral vectors). In other embodiments, to improve delivery efficiency for vectors subject to physical packaging constraints, the system is provided as a multi-vector architecture, such as a two-cassette or three-cassette system partitioned across separate vectors (e.g., separate lentiviral vectors). In a three-vector system, a first vector encodes the structural module, a second vectorTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT encodes the AF 1 module, and a third vector encodes the AF2 and / or AF3 modules. In a two-vector system, a first vector encodes the structural module, while a second vector encodes the full set of assembly factors divided into differentially expressed modules operatively linked by self-cleaving peptides or IRES elements. In such multi-vector embodiments, the desired macrooptimization and / or Assembly factor microoptimization is achieved by delivering the vectors to the host cell at a corresponding particle ratio (e g., co-transducing at a viral ratio of 4: 1 : 1), thereby achieving the target DI ratio.

[0333] In embodiments here described the macrooptimized and / or microoptimized configurations can be comprised within a monocistronic constructs and / or polycistronic constructs.

[0334] The term “polycistronic construct” as used herein refers to a construct capable of simultaneously translating multiple genes from a single transcript as will be understood by a person skilled in the art, within a single cassette or in different cassettes on the construct if the cassettes are separated by an internal ribosome entry site.

[0335] In some embodiments, the polycistronic construct can be a biocistronic construct which comprises two genes separated by an Internal Ribosome Entry Site (IRES) element which allows for initiation of translation from an internal region of the mRNA. In some embodiments, the use of an IRES allows for the upstream protein to be translated independently without a terminal peptide appendage, and the downstream protein concurrently acquires a minor peptide addition to its N-terminus. The second protein may be expressed at a lower level compared with the first protein since the ribosome entry site is less efficient than the 5’cap / UTR as will be understood by a skilled person.

[0336] In some embodiments, some of the gene of a GVGC are expressed at a lower level compared to other gvp genes of the GV gene cluster when expressed under a same promoter and regulatory regions in those embodiments, the stoichiometry of the expression of the specific cassette can be increased to provide an optimal functionality of the GVES in the target cell.

[0337] . In some embodiments, specific highly expressed modules within the engineered configurable Gas Vesicle Architecture (such as the structural macrooptimization module or theTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 'booster' AF1 module) can be comprised on one or more gene cassettes, wherein the physical stability of the transcript is leveraged to tune the stoichiometry of the translated proteins. Regulatory elements that stabilize mRNAs (for example Poly A, WPRE) can be used on the booster constructs. For genes that need to be expressed at lower relative stoichiometries, these stability elements can be removed, or can be conditionally removed using siRNAs / shRNAs / aptazymes / cas9 and etc. While the other GV cassette can include these mRNA stability elements. In some embodiments, the other GV cassettes can selectively include or exclude these mRNA stability elements to achieve the desired Dosage Index.

[0338] In some embodiments, the highly expressed constructs (e.g., the structural module) can be comprised on one or more gene cassettes, where the use of degradation tags can tune stoichiometry of the translated proteins. Degradation tags target proteins for proteolysis, for example ubiquitin and library of ubiquitin-fusion degradation tags (UbR, UbP, UbW, UbH, Ubl, UbK, UbQ, UbV, UbL, UbD, UbN, UbG, UbY, UbT, UbS, UbF, UbA, UbC, UbE, UbM, 3xUbVR, 3xUbVV, 2xUbVR, 2xUbVV, UbAR, UbW, UbVR, UbAV, 2xUbAR, 2xUbAV), auxin-inducible degron (AID), D-element, the PEST sequence, unstructured initiation sites, or short sequences rich in acceptor lysines. Genes on the highly expressed constructs will not have these degradation tags. In some embodiments, relative degradation tags can be used for the other assembly factor genes that need to be expressed at lower levels. This can be used in combination with promoters and transcript stability examples.

[0339] In some embodiments, the constructs can be comprised on one or more gene cassettes, where the use of micro-ORFs upstream of a cassette (ORF encoding gv genes) can be used to reduce the expression of GV proteins. Micro-ORFs are short open reading frames placed upstream of the ORF encoding the protein(s) of interest and result in the suppression of protein expression. They include a kozak / start codon NNNATG, small peptide and stop codon (TGA, TAG, TAA), for example AAAATGGCCGCGCCCAGAGCGTAG (SEQ ID NO: 481), NNNATG(NNN)[TAG / TGA / TAA] (SEQ ID NO: 474) (

[0028] ). For genes that need to be expressed at lower relative stoichiometries (e.g., the AF3 module), micro-ORFs can be placed upstream of their cassette to reduce the expression level of these GV proteins.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0340] In some embodiments, the constructs can be comprised on one or more gene cassettes, where the use of different inducible promoters (chemically or otherwise) can tune stoichiometry of translated proteins. Different promoters that are inducible by different stimuli can be used to drive expression of the structural modules and / or other cassettes. A higher amount of inducer can be used to increase the expression of the highly expressed constructs. For genes that need to be expressed at lower relative stoichiometries, a relatively lower amount of inducer can be used.

[0341] In some embodiments, the constructs can be comprised on one or more gene cassettes, where the presence of enhancing introns can tune stoichiometry of the translated proteins. Intron-mediated enhancement can be used on the highly expressed constructs. For genes that need to be expressed at lower relative stoichiometries, these introns can be omitted. In some embodiments, specific GV cassettes can include these introns to selectively boost target module expression. (

[0029] ,

[0030] )

[0342] In some embodiments, the constructs can be comprised on one or more gene cassettes, where the stoichiometry of the translated proteins can be tuned by different modes of Ribosome entry. Translation of the highly expressed construct can be initiated via the stronger cap-dependent gene expression mediated by the KOZAK sequence. In some embodiments, genes that need to be expressed at lower relative stoichiometries can be initiated via an Internal Ribosome Entry Site (IRES).

[0343] In some embodiments, the relative stoichiometry of proteins expressed from one or more differentially expressed gene modules is modulated by controlling the copy number of the module or of the genetic construct carrying it within the host cell. The term “copy number” as used herein refers to the number of instances of a gene, cassette, or vector present within a cell, genome, or episomal environment. Variation in copy number directly influences transcript abundance and, consequently, protein concentration. By adjusting the copy number of specific GV expression cassettes or vectors, the ratio of different Gvp proteins can be fine-tuned to yield the desired vesicle morphology, size, and collapse threshold.

[0344] Copy-number control can be achieved at several levels, including: (i) Vector backbone selection, wherein the origin of replication determines the plasmid copy number (for example,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT high-copy ColEl origin versus low-copy pl5A or single-copy BAC systems); (ii) Selectable marker pressure, where differential antibiotic concentrations or auxotrophic complementation maintain defined plasmid ratios; (iii) Targeted genomic integration, wherein constructs are inserted into loci of known transcriptional activity and copy number using recombinase- or CRISPR-mediated approaches; and (iv) Episomal replication elements such as EBV oriP / EBNAl or S / MAR sequences that permit tunable maintenance of extrachromosomal copies in target cells. Through these mechanisms, a single cell population can be engineered to contain higher copy numbers of one GV module (for example, the gvpA / B module) and lower copy numbers of another (for example, the gvpC module), thereby establishing the stoichiometric balance required for efficient GV assembly.

[0345] In some embodiments, the desired DI ratio between differentially expressed gen module can be achieved using multiple constructs or vectors each encoding one or more distinct GV module ehich are co-transfected or co-transduced into a host cell. The relative dosage of each construct determines the transcriptional output of its encoded genes. For instance, introducing a threefold molar excess of a gvpA / B expression construct relative to a gvpC construct can produce a vesicle population enriched in shell-forming subunits with reduced outer-surface reinforcement, resulting in GVs that collapse at lower acoustic pressures and exhibit increased nonlinear response. Conversely, increasing the relative copy number of gvpC or accessory genes can yield mechanically stronger vesicles with higher collapse thresholds, n some embodiments, modulating the relative expression of a structural module (comprising, for example, the structurally reinforced chimeric GV structural protein) relative to accessory genes can physically program the structural lattice. As previously described, integrating the specific engineered chimeric structural proteins yields mechanically stronger vesicles characterized by higher critical collapse thresholds (e.g., right-shifted by at least +150 kPa).

[0346] Such modulation of inter-construct ratios enables programmable control of GV phenotype without altering the underlying promoter or regulatory architecture.

[0347] Copy-number-based stoichiometric tuning can also be dynamically regulated by inducible replication or amplification systems, including those based on Cre-lox-mediated excision or amplification, recombinase directionality factors, or drug-responsive replication origins. ForTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT example, a replication origin that activates in response to doxycycline or temperature change can be used to transiently increase expression of one GV module during an assembly phase and reduce it afterward, producing vesicles with defined temporal assembly kinetics.

[0348] Integration of copy-number control with differential regulatory design allows hierarchical expression programs, wherein transcriptional and translational tuning are reinforced by genomic dosage effects.

[0349] Accordingly, the stoichiometric output of GV expression systems described herein can be determined by the combined effect of (i) internal regulatory elements within a differentially expressed gene module and (ii) the relative copy number of those modules or constructs across the cellular population. Together, these parameters permit precise, quantitative modulation of gas vesicle protein composition, ensuring reproducible generation of GV types with defined acoustic, magnetic, or optical properties in eukaryotic and, in particular, mammalian, prokaryotic, or cell-free systems.

[0350] Accordingly in some embodiments, all components of the GVES can be encoded on a single polynucleotide, vector, or cassette or in two or more polynucleotide, vector, or cassette. For example, a single construct can comprise a monocistronic gene expression cassette encoding gvpA and a polycistronic gene expression cassette encoding all assembly factors, gvpNJKFGWV. The individual genes within the polycistronic AF cassette may be operatively linked by 2A peptides, and the gvpA cassette may be linked to the AF cassette by an IRES, all under the regulatory control of a single promoter.

[0351] In some embodiments, the GVES is provided as a system comprising multiple distinct gene expression cassettes, preferably partitioned across separate polynucleotide constructs. In these embodiments, the structural gvp module, the AF1 module, the AF2 module, and the AF3 module can be comprised in separate gene expression cassettes across at least two or three distinct polynucleotides, such as separate vectors. This multi-construct architecture is particularly advantageous for use with delivery vehicles with a limited packaging capacity, such as viral vectors, including but not limited to lentiviral or adeno-associated viral (AAV) vectors.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0352] For example, a complete engineered gas vesicle gene cluster, along with necessary promoters, terminators, and other regulatory elements, can easily exceed the packaging limit of a single viral vector. Distributing these components across separate, smaller constructs ensures that each individual construct is well within the acceptable packaging limit, leading to higher viral titers, improved genomic stability, and more efficient delivery.

[0353] In this multi-construct embodiment, the stoichiometric control — encompassing both the macrooptimization and the Assembly factor microoptimization — is achieved by controlling the relative copy number or multiplicity of infection (MOI) of each distinct polynucleotide delivered to the host cell. For example, to achieve a target Dosage Index (DI) ratio wherein the structural protein is in excess and the assembly factors follow a descending hierarchy (e.g., Structural-DI > AF1-DI > AF2-DI > AF3-DI), a population of host cells is co-transduced with a calculated excess of the structural vector relative to the respective assembly factor vectors.

[0354] This multi-construct, co-transduction approach has been demonstrated to be surprisingly effective in establishing the foundational macrooptimization principle, enabling efficient transduction of hard-to-transduce primary cells (such as primary human T cells) and resulting in a robust, high-yield system. Among multi-vector configurations, this partitioned architecture provided an optimal baseline signal, for instance, a BURST SBR of approximately 118 (mean ± 8.4 s.e.m.) at a 4:1:1 ratio, demonstrating a substantial improvement over 1:1:1 configurations. In these partitioned embodiments, the structural cassette is preferably monocistronic, and the AF cassettes are preferably polycistronic, each under the control of its own respective promoter.

[0355] In alternative embodiments, in configurations where the total nucleotide sequence of the engineered configurable Gas Vesicle Architecture exceeds the packaging capacity of a single selected delivery vehicle (for example, the approximately 4.7 kb packaging limit of an Adeno-Associated Virus (AAV) vector), the differentially expressed gene modules are comprised across a plurality of separate expression cassettes or vectors.

[0356] In such multi-vector configurations, the assembly factor stoichiometric optimization (e.g., AF1-DI > AF2-DI > AF3-DI) and the structural macrooptimization are not necessarily driven by physical 5'-to-3' drop-off on a single transcript. Rather, the targeted stoichiometric hierarchy isTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT achieved by varying the respective Multiplicity of Infection (MOI) or the relative mass ratios of the administered vectors. For example, a first viral vector comprising the structural module and a second viral vector comprising the AF1 module (gvpF, gvpG) can be administered to the target cell at a substantially higher MOI than a third viral vector comprising the AF3 module (gvpJ), thereby mathematically forcing the targeted intracellular Dosage Index ratios.

[0357] In those embodiments the Dosage Index can be measured or correlated with the Transduction-Expression Index under the same Standard Conditions, such that variations in DI account for differential efficiency of delivery, transcription, translation, or assembly affecting TEI values.

[0358] As used herein, the term “Transduction-Expression Index” or “TEI” refers to a dimensionless, normalized measure of end-to-end gene-to-function efficiency under Standard Conditions. The TEI is defined as the functional ultrasound output from an acoustic reporter gene (ARG) system per effective input, normalized to a designated reference system scored as 1.0 under Standard Conditions. Functional output is quantified using suitable ultrasound readouts comprising collapse-based methods such as BURST (Burst Ultrasound Reconstruction with Signal Template ultrasound readouts or non-collapse based methods utilizing nonlinear buckling signals (e.g., xAM or Amplitude Modulation Pulse Sequence), expressed as signal-to-background ratio (SBR) or an equivalent validated metric obtained with the same imaging parameters. The TEI is thereby calculated as the mean BURST SBR per effective expressing cell for the system of interest divided by that of a designated reference system, measured under the same Standard Conditions.

[0359] The Transduction-Expression Index (TEI) is defined according to the relationship:TEI = (Output / Input)system / (Output / Input)Referencewhere Output corresponds to the measured functional ultrasound signal, typically expressed as the mean BURST signal-to-background ratio (SBR) across a designated region of interest and acquisition time window, and Input corresponds to the effective number of cells at the time of imaging contributing to the signal, which represents the number of cells successfully expressing functional gas vesicles, approximated experimentally by the number or fraction of transduced cells (eg., determined by flow cytometry for reporter markers) within the imaged population,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT particularly when comparing systems where delivery efficiency itself might differ. The ratio of the normalized Output to Input, relative to that of the designated reference system, yields a dimensionless TEI value that quantitatively represents the overall efficiency with which an introduced gene or cassette set produces a functional acoustic output encompassing factors influencing both delivery and post-delivery expression and assembly efficacy. The TEI thereby integrates the contributions of delivery efficiency, transcriptional and translational yield, macromolecular assembly, and acoustic performance into a single normalized measure of system efficacy.

[0360] The DI and TEI together provide a dual-parameter framework linking molecular stoichiometry to macroscopic function. The DI captures the relative balance of expression among gene cassettes, reflecting how dosage and expression ratios contribute to the proper assembly of multi-component complexes, whereas the TEI captures system-level performance encompassing delivery, transcription, translation, assembly, and functional output. Variations in DI can therefore explain variance in TEI, as imbalanced stoichiometry among gene products may impair proper assembly and reduce functional output. Used jointly, the two indices enable quantitative analysis and optimization of gene expression systems for desired functional performance under standardized experimental or operational conditions.

[0361] For purposes of the present disclosure, the Dosage Index (DI) and the Transduction-Expression Index (TEI) are each determined or interpreted with respect to Standard Conditions (SC), which establish a reproducible experimental and analytical framework for comparing gene expression systems. As used herein, Standard Conditions encompass a defined set of physical, biological, and instrumental parameters, including but not limited to the identity and configuration of the imaging apparatus, transducer frequency and pressure, pulse sequence, acquisition frame count, environmental temperature, imaging depth, and data analysis pipeline. The use of Standard Conditions ensures that variations observed in DI or TEI arise from differences in biological or genetic parameters rather than from experimental artifacts or inconsistent measurement settings. Under SC, the reference system — typically a single-construct acoustic reporter gene (ARG) expressing a defined gas vesicle (GV) cluster — is assigned a TEI value of 1.0, providing a baseline for normalization and comparative evaluation.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0362] Under Standard Conditions, variations in DI modulate the expression and assembly components that contribute to TEI. For example, an increase in the DI of a structural gene relative to a scaffold or chaperone gene may disrupt stoichiometric balance, resulting in aberrant complex formation and a corresponding reduction in TEI. Conversely, maintenance of DI values within an empirically determined optimal range, typically between about 0.8 and 1.2, supports balanced expression and efficient assembly, yielding TEI values comparable to or exceeding the reference standard. Accordingly, the combined use of DI and TEI under Standard Conditions provides a quantitative and mechanistic framework for optimizing gene expression systems, enabling predictive adjustment of gene dosage, promoter strength, or cassette ratios to achieve desired functional outcomes.

[0363] In embodiments herein described, the polynucleotide constructs of the GVES, GVRMC, or GVRGC, including the gvpA / B module, the AF1 module, and the AF2 module, whether as separate constructs or combined onto one or more constructs, are provided as part of a vector or "delivery vehicle." The term “vector” indicates a molecule configured to be used as a vehicle to artificially carry foreign genetic material into a cell, where it can be replicated and / or expressed. An expression vector is a vector configured to carry and express the material in a cell under appropriate conditions.

[0364] In some embodiments, a suitable vector can comprise a recombinant plasmid, a recombinant non-viral vector, or a recombinant viral vector. These vectors comprise the polynucleotide constructs and the appropriate regulatory elements such as promoters, enhancers, and terminators compatible with the target cell intended to heterologously express the GV type.

[0365] In preferred embodiments, particularly for delivery into primary cells, immune cells, and for in vivo applications, the vectors are viral vectors. Exemplary viral vectors include, but are not limited to, lentiviral vectors, adeno-associated viral (AAV) vectors, adenoviral vectors, retroviral vectors (including gammaretroviral vectors), baculoviral vectors, and herpes simplex virus (HSV) vectors. As demonstrated in the Examples, lentiviral vectors are particularly suitable for delivering the multi-construct system to hard-to-transduce cells, such as primary human T cells, and achieving robust expression and are thus particularly preferred.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0366] In other embodiments, the polynucleotide constructs can be delivered as non-viral vectors. Examples of non-viral delivery methods include, but are not limited to, plasmid DNA, "naked" DNA or RNA (such as mRNA), lipid nanoparticles (LNPs), liposomes, polyplexes (e.g., using polyethyleneimine or PEI), dendrimers, electroporation, sonoporation, magnetofection, or particle-based delivery (e.g., gene gun). The polynucleotide construct can also be part of a transposon system, such as piggyBac or Sleeping Beauty, which facilitates genomic integration upon delivery.

[0367] In particularly preferred embodiments for in vivo therapeutic applications or transient reporting, the messenger RNA (mRNA) or DNA encoding the engineered configurable Gas Vesicle Architecture is encapsulated within lipid nanoparticles (LNPs) or polymer-based nanocarriers. Delivery via mRNA-LNPs enables rapid, high-efficiency expression of the gas vesicle proteins in the target cell cytoplasm without the risk of permanent genomic integration, which is highly advantageous for transient functional imaging and circumvents viral packaging constraints.

[0368] Accordingly, in certain preferred embodiments, the genetically engineered Gas Vesicle Expression System (GVES) is provided as a combination of two or more distinct polynucleotide constructs, such as a set of separate vectors, wherein the differentially expressed gene modules are partitioned across said constructs. This multi-construct or multi-vector architecture is particularly advantageous for delivering large, multi-gene clusters, such as the engineered configurable Gas Vesicle Architecture integrating the chimeric structural proteins and the U / N assembly complex, using delivery vehicles that have a limited packaging capacity. For example, the total size of a single construct encoding all GVGC modules and necessary regulatory elements may exceed the optimal packaging limit of viral vectors, such as lentiviral vectors (approximately 8-10 kb), which can lead to reduced viral titers and inefficient gene delivery.

[0369] In certain embodiments, particularly where delivery vehicles with strict packaging limits are utilized, the GVES is configured as a set of three or more distinct polynucleotide constructs or vectors. In this configuration, a first polynucleotide construct comprises the structural gvp differentially expressed gene module (e.g., encoding the chimeric GvpA / B), a second polynucleotide construct comprises the AF1 gvp differentially expressed gene module (e.g.,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT encoding highly expressed assembly factors such as GvpF and GvpG), and one or more additional polynucleotide constructs comprise the AF2 and AF3 gvp differentially expressed gene modules (e.g., encoding moderately expressed GvpN and GvpJ, and strictly limited GvpK). This partitioning allows each individual construct to remain well within the physical packaging limits of the selected delivery vector, thereby facilitating high-titer viral production, preserving genomic stability, and ensuring efficient co-delivery. This multi-construct approach enables the precise stoichiometric control required for both macrooptimization and Assembly factor microoptimization to be achieved at the level of delivery by controlling the relative copy number or dosage of each vector introduced into the host cell.

[0370] In some preferred embodiments, a method for expressing Gas Vesicles involves cotransducing the target cell population with a composition comprising the set of vectors at a predetermined relative ratio. For example, to achieve the desired Dosage Index (DI) ratio wherein the gvpA / B-DI is at least 2-fold higher than the AF1-DI and AF2-DI, the cells can be transduced with a relative ratio of transducing units (e.g., Multiplicity of Infection or MOI) of the first (gvpA / B) vector to the second (AF1) and third (AF2) vectors in a preferred range from 2:1:1 to 4:1:1. Delivering the vectors at a preferred ratio of approximately 4:1:1, for instance, has been shown to be highly effective, resulting in robust expression and a superior functional ultrasound signal compared to non-stoichiometric ratios or baseline, unoptimized single-vector systems. This multi-vector delivery strategy has proven surprisingly effective for engineering hard-to-transduce cells, such as primary T cells, and achieves a significantly higher Transduction-Expression Index (TEI) than comparable baseline single-construct systems lacking the engineered configurable Gas Vesicle Architecture under Standard Conditions. The polynucleotide constructs in this set are preferably viral vectors, such as lentiviral vectors, but can also include adeno-associated viral (AAV) vectors, or non-viral vectors.

[0371] In some preferred embodiments, to practice this multi-vector embodiment, a method for expressing Gas Vesicles involves co-transducing the target cell population with a composition comprising the set of vectors at a predetermined relative ratio. For example, to achieve the desired macrooptimization and Assembly factor microoptimization wherein the gvpA / B-Dl is at least 2-fold higher than any AF-DI, and the AF Dis follow a descending hierarchy (AF1-DI > AF2-DI >Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT AF3-DI), the cells are transduced with a relative ratio of transducing units (e.g., Multiplicity of Infection or MOI) of the structural vector to the assembly factor vectors that physically mirrors this hierarchy. Delivering the vectors at a structural-to-assembly ratio of approximately 4:1:1 or 5:1:1, for instance, has been shown to be highly effective, resulting in robust expression and a superior functional ultrasound signal compared to non- stoichiometric ratios or unoptimized, native single-vector systems. This tailored multi-vector delivery strategy has proven surprisingly effective for engineering hard-to-transduce cells, such as primary T cells, and achieves a significantly higher Transduction-Expression Index (TEI) than comparable unoptimized singleconstruct systems lacking the engineered configurable Gas Vesicle Architecture under Standard Conditions. The polynucleotide constructs in this set are preferably viral vectors, such as lentiviral vectors, but can also include adeno-associated viral (AAV) vectors, or non-viral vectors.

[0372] In embodiments herein described, engineered configurable Gas Vesicle Architecture comprising a GVGC in two or more gene cassettes located on one or more polynucleotide construct herein described operatively connected to regulatory sequences can be introduced to a host allowing expression of the GV constructs and producing of gas vesicles in the host.

[0373] As used herein, the term “host” refers to a multicellular biological organism comprising the GVES or related components. A host can be a plant an animal and in particular a vertebrate and non-human mammal (e.g., a mouse, rat, non-human primate, pig, or dog) used in research or preclinical diagnostics, or a human subject in clinical applications. The host provides the target site (e.g., an organ, a tumor, or the central nervous system) where the engineered host cells reside and where nonlinear acoustic detection is performed.

[0374] The term “host cell” refers to an individual cell, or a population of cells, that is genetically engineered to express the GVES. A host cell can be an isolated cell (e.g., in vitro culture), a primary cell (e.g., primary neurons or human T-cells), or a cell comprised within a host (in vivo). The host cell is the functional unit responsible for: (i) receiving the stoichiometric genetic payload; (ii) maintaining the Target Dosage Index (DI) Ratio; and (iii) assembling gas vesicles until the nonlinear visibility threshold is crossed.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0375] It is to be understood that a host can comprise multiple types of host cells, only a subset of which may be engineered to express the GVES. For example, in a mammalian host containing a tumor, the host cells engineered to express the GVES may be therapeutic T-cells that have homed to said tumor (the target site) for the purpose of nonlinear acoustic monitoring of cell-based therapy dynamics.

[0376] A significant advantage of the engineered configurable Gas Vesicle Architecture within a multi-construct system is its ability to provide good transduction efficiency and surprisingly robust gene expression in mammalian cells that are typically considered difficult to engineer. This includes, in particular, clinically relevant primary cells and immune cells, such as primary human T cells. Prior to the present invention, achieving stable, high-yield expression of the complete polycistronic GV gene cluster in these cell types was a significant technical barrier. The combination of overcoming viral packaging limits, which provides for more efficient delivery, and the stoichiometric optimization, which provides for a high functional yield per cell, enables for the first time the robust and detectable expression of acoustic reporters in these therapeutically important cell populations.

[0377] As used herein, the term “immune cell” refers to any cell of hematopoietic origin that participates in innate or adaptive immune responses and that can be genetically engineered to express gas vesicle constructs (GVES, GVPC, or GVRGC) as described herein. Immune cells include, without limitation, lymphoid lineage cells such as T lymphocytes (including CD4+helper T cells, CD8+cytotoxic T cells, yd T cells, and regulatory T cells), B lymphocytes, and natural killer (NK) cells; myeloid lineage cells such as monocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, and mast cells; and precursor or progenitor forms thereof derived from bone marrow or hematopoietic stem cells. Immune cells can be obtained directly from a mammalian host (for example, human peripheral blood mononuclear cells, spleen, thymus, lymph node, or bone marrow) or generated ex vivo through expansion and differentiation of stem or progenitor cells.

[0378] Immune cells can be engineered using viral or non-viral vectors to express GV constructs for use as contrast-generating reporter cells, biosensors, or therapeutic carriers. In particular, immune cells expressing GVs can be used to report localization, migration, activation, or cytotoxicTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT events in vivo under ultrasound or MRI, as described in U.S. Pat. Nos. 11,761,008 B2 and 11,446,523 B2. The intracellular environment of immune cells provides post-translational machinery (such as proteases, kinases, and redox regulators) that can influence the structure or modification state of the GvpC protein, thereby generating immune-cell-specific GV types with unique acoustic or magnetic characteristics. Accordingly, “immune cell” in the sense of the disclosure encompasses both unmodified and engineered immune cells of innate or adaptive lineages, including autologous, allogeneic, or xenogeneic sources, suitable for research, diagnostic, or therapeutic use.

[0379] As used herein, the term “primary cell” refers to a cell that has been directly isolated from a mammalian tissue or organ and retains the differentiated characteristics, genetic profile, and physiological responses of its in vivo origin. Primary cells are distinct from immortalized or transformed cell lines in that they have limited replicative potential and more faithfully recapitulate the biochemical and structural context of native tissue. Primary cells can be harvested from a wide range of mammalian tissues, including but not limited to blood, liver, lung, heart, brain, kidney, pancreas, skin, muscle, and connective tissues, using mechanical, enzymatic, or perfusion-based dissociation methods followed by culture under defined growth conditions.

[0380] Exemplary primary cell types include hepatocytes, cardiomyocytes, neurons, astrocytes, microglia, fibroblasts, endothelial cells, keratinocytes, adipocytes, pancreatic 0-cells, renal epithelial cells, and smooth or skeletal muscle cells. Primary immune cells such as macrophages, dendritic cells, monocytes, T cells, B cells, and NK cells are also encompassed. Primary cells can be genetically engineered in vitro or ex vivo using transfection, transduction, electroporation, or mRNA delivery methods to express GV gene clusters or related constructs (GVES, GVPC, or GVRGC) to form gas vesicle structures or reporters within their cytoplasm.

[0381] As described in U.S. Pat. No. 11,761,008 B2, mammalian primary cells engineered with GV constructs can assemble stable gas vesicles that retain detectability under MRI or ultrasound while preserving cellular physiology. In certain embodiments, GV expression in primary cells can be regulated by inducible or tissue-specific promoters to ensure controlled expression and compatibility with therapeutic applications. Accordingly, “primary cell” in the sense of the present disclosure encompasses any non-immortalized mammalian cell that is directly isolated from aTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT tissue and capable of genetic modification to express, assemble, or host gas vesicle nanostructures in vitro, ex vivo, or following reintroduction into a mammalian subject.

[0382] In particular in some embodiments, the method comprises introducing into the mammalian cell a genetically engineered Gas Vesicle expression system (GVES) herein described (for example, via the single polycistronic lentiviral vector or the multi-vector architectures. The introducing step is performed for a time and under condition sufficient to allow expression of the gvp genes and assembly of the engineered gas vesicles in the mammalian cell and in particular within the mammalian cell cytoplasm.

[0383] In some embodiments, the method comprises introducing into a cell of the mammalian host a genetically engineered Gas Vesicle expression system (GVES) herein described in which the gvp genes encode for proteins of the gas vesicle type. The introducing is performed for a time and under condition to allow expression of the gvp genes in the mammalian cell, thereby achieving a functional intracellular concentration of assembled gas vesicles.

[0384] Expression of GV constructs in a mammalian cell can be performed by cloning one or more polynucleotides encoding naturally occurring GV proteins or homologs thereof that are required for production of GVs (comprising gvpB, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and gvpU and other proteins known to those skilled in the art and described herein) into one or more suitable constructs configured to express the heterologous GV proteins in the mammalian cell. Polynucleotides encoding GV protein genes can be cloned using commercially available reagents from vendors such as Qiagen, Invitrogen, Applied Biosystems, Promega, New England BioLabs and others, following standard molecular biology methods known in the art, such as those described herein. As would be understood by those skilled in the art, polynucleotides encoding GV protein genes can be obtained from several different sources. For example, polynucleotides encoding GV proteins can be obtained by isolating genomic DNA or cDNA encoding GV proteins from microorganisms whose genomes encode GV proteins genes, and / or express GV proteins RNA. RNA can be isolated from a cell that expresses GV proteins genes, and cDNA produced by reverse transcription using standard techniques and commercial kits. Genomic DNA can be purified from the cell, and cDNA or genomic DNA encoding one or more GV proteins isolated, following methods known to those in the art. In addition or in the alternative, polynucleotidesTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT comprising one or more gas vesicle genes can be synthesized using oligonucleotide and polynucleotide synthetic methods known in the art. For example, if rare mammalian codons are identified following purification of genomic DNA from the cell, rare mammalian codons are preferably edited to improve expression in the target cell. PCR-based amplification of one or more GV protein genes can be performed using appropriately designed primer pairs (e.g. using PrimerDesign or other programs known to those skilled in the art). PCR-based amplification can be followed by ligation (e g. using T4 DNA ligase) of a polynucleotide encoding gas vesicle gene amplicon into an appropriate construct in a plasmid suitable for propagation in bacteria or archaea, such as transformation-competent A. coli DH5alpha or other competent E Coli type, followed by growth of transformed cell cultures, purification of the plasmid for confirmation of the cloned gene by DNA sequence analysis, among other methods known to those skilled in the art. Expression vectors can comprise plasmid DNA, viral vectors, or non-viral vectors, among others known to those skilled in the art, comprising appropriate regulatory elements such as promoters, enhancers, and post-transcriptional and post-translational regulatory sequences that are compatible with the mammalian cell intended to heterologously express the GV, as would be understood by a skilled person. In particular, in embodiments described herein, expression vectors suitable for regulating heterologous expression of GVs comprise those having promoters and other regulatory elements known to skilled persons that are compatible with mammalian cells, including cell lines, primary cells cultured in vitro such as petri dishes or introduce the GV gene circuits inside the animal to genetically engineer cells directly inside the animal and described above. Promoters can be constitutively active or inducible (and chosen to be selectively expressed in different cell types).

[0385] In particular, in some embodiments described herein, production of a GV gene sequences can be codon-optimized (for example to remove rare mammalian codons) for expression in the mammalian cell type according to methods identifiable by a skilled person. As would be understood by those skilled in the art, the term “codon optimization” as used herein refers to the introduction of synonymous mutations into codons of a protein-coding gene in order to improve protein expression in expression systems of a particular organism, such as human, in accordance with the codon usage bias of that organism. The term “codon usage bias” refers to differences in the frequency of occurrence of synonymous codons in coding DNA. The genetic codes of different organisms are often biased towards using one of the several codons that encode a given amino acidTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT over others, and use the one codon with a greater frequency than expected by chance. Optimized codons in organisms reflect the composition of their respective genomic tRNA pool. The use of optimized codons can help to achieve faster translation rates and high accuracy (and ultimately higher recombinant protein yield).

[0386] In some embodiments, one or more statistical methods proposed and used to analyze codon usage bias the field of bioinformatics and computational biology can be used for codon optimization in the sense of the disclosure. Methods such as the 'frequency of optimal codons' (Fop), the Relative Codon Adaptation (RCA) or the 'Codon Adaptation Index' (CAI) are used to predict gene expression levels, while methods such as the 'effective number of codons' (Nc) and Shannon entropy from information theory are used to measure codon usage evenness. Multivariate statistical methods, such as correspondence analysis and principal component analysis, are widely used to analyze variations in codon usage among genes. There are many computer programs to implement the statistical analyses enumerated above, including Codon W, GCUA, INCA, and others identifiable by those skilled in the art. Several software packages are available online for codon optimization of gene sequences, including those offered by companies such as GenScript, EnCor Biotechnology, Integrated DNA Technologies, ThermoFisher Scientific, among others known those skilled in the art. Those packages can be used in providing GV proteins with codon usage ensuring optimized expression in various prokaryotic cell systems as will be understood by a skilled person. In particular, codon optimization in embodiments herein described can be used primarily to remove or limit the use of rare codons, or keep codon usage above -10%).

[0387] Mammalian cell used herein to include a GVES of the disclosure refers to a mammalian cell which can be transduced, infected, transfected or transformed with a vector under certain culture conditions. The vector can be plasmid, a viral particle, or others identifiable to a person skilled in the art. The term mammalian cell refers to cells isolated from an animal (mammal) tissue and expanded in culture for use as therapeutic and research tools.

[0388] In some embodiments, the transformed mammalian cells can comprise one or more cells such as T-cells, hematopoietic stem cells, mesenchymal stem cells, neural precursor cells, macrophages, fibroblasts or cardiomyocytes and any cell where one can express reporter genes (e g. Green fluorescent protein (GFP)).Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0389] In some embodiments the Stoichiometric GVES of the disclosure can be used in connection with CAR-T and / or CAR-P cells, in particular in connection with the diagnostic and / or therapeutic applications.

[0390] As used herein, the term “chimeric antigen receptor” (CAR) refers to a synthetic, modular receptor engineered to endow a host cell — typically an immune cell such as a T lymphocyte, natural killer (NK) cell, or macrophage — with the ability to recognize a defined antigen in a manner independent of the endogenous major histocompatibility complex (MHC). A CAR-T cell is a T lymphocyte genetically modified to express such a receptor, while a CAR-P cell refers to a chimeric antigen receptor phagocyte, typically derived from a macrophage, monocyte, or dendritic-cell lineage engineered to perform antigen-specific phagocytosis, secretion, or i mmunomodul ati on .

[0391] A CAR construct generally comprises: (i) an extracellular antigen-binding domain, frequently a single-chain variable fragment (scFv) derived from a monoclonal antibody, nanobody, or ligand; (ii) a hinge or spacer region providing flexibility and optimal receptor geometry; (iii) a transmembrane domain anchoring the receptor in the cell membrane; and (iv) one or more intracellular signaling domains that trigger cellular activation. For CAR-T cells, the signaling domain typically includes a CD3( / chain fused to one or more co-stimulatory motifs such as CD28, 4-1BB (CD137), 0X40, or ICOS, thereby controlling the intensity, persistence, and cytokine profile of the immune response. CAR-P receptors employ analogous architectures but couple the antigen-recognition domain to phagocytic signaling motifs such as those from Megfl 0, FcRy, or CD3t^ to induce engulfment rather than cytolytic activity.

[0392] CAR-T and CAR-P cells can be generated ex vivo by transducing isolated immune cells with viral or non-viral vectors carrying a CAR expression cassette under a constitutive or inducible promoter. The engineered cells are then expanded and re-introduced into the subject, where they perform antigen-specific recognition and effector functions. Expression of a CAR can be combined with gas vesicle expression systems (GVES) or other reporter modules described herein to enable real-time imaging of CAR-cell localization, viability, and activation.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0393] CAR-T cells have demonstrated clinical efficacy in the treatment of hematologic malignancies, including B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma, by targeting cell-surface antigens such as CD 19, CD20, CD22, BCMA, and CD 138. Additional constructs are in development for solid tumors, targeting antigens including HER2, EGFR, GD2, mesothelin, MUC1, and PSMA. CAR-P systems are particularly suited for therapeutic applications requiring phagocytosis or antigen presentation, such as clearance of tumor cells, amyloid plaques, or infected cells, and for re-education of the tumor microenvironment via secretion of cytokines or checkpoint modulators (e.g., IL-12, IL-15, or PD-L1 inhibitors).

[0394] Further therapeutic embodiments include dual-CAR or tandem-CAR architectures, in which a single cell expresses two or more CARs recognizing different antigens to enhance selectivity or prevent tumor escape; switchable CARs activated by small molecules or bispecific adaptors; and safety-switch CARs containing suicide genes or protease-cleavable inactivation domains. CAR-P macrophages may additionally be engineered with synthetic receptors that recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to enable antimicrobial or anti-inflammatory therapy.

[0395] In certain embodiments, CAR-T or CAR-P cells are employed as diagnostic or reporter cells to detect the presence, distribution, or activity of a specific antigen in vivo. In embodiments where the engineered immune cells are coupled with genetically encoded reporters such as the engineered configurable Gas Vesicle Architecture (GVES, GVPC, or GVRMC) described herein, the engineered immune cells can be non-invasively visualized using ultrasound or magnetic resonance imaging (MRI). Such CAR-reporter hybrids allow monitoring of cell trafficking, tumor infiltration, persistence, or activation state within a subject over time.

[0396] Additional diagnostic embodiments include the use of CAR-P cells as antigen-capturing biosensors within implantable or in vitro platforms, where antigen engagement triggers phagocytosis, secretion of detectable metabolites, or expression of imaging reporters. CAR-T or CAR-P cells engineered with inducible GV expression can therefore provide dual-function therapeutic and diagnostic (“theranostic”) capabilities, enabling real-time assessment of therapeutic efficacy and immune dynamics within target tissues.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0397] Accordingly, CAR-T and CAR-P technologies, alone or in combination with gas-vesicle reporter constructs described herein, can be configured for therapeutic, diagnostic, or integrated theranostic use. The combination of targeted immune activation and non-invasive imaging enables precision monitoring of engineered immune-cell behavior and facilitates adaptive therapeutic control. Such embodiments include GV-expressing CAR-T cells that report antigen encounter via ultrasound contrast enhancement or MRI phase-shift, CAR-P macrophages designed to visualize phagocytic activity in inflamed or neoplastic tissue, and multiplexed CAR-reporter cell systems used to stratify patient response or guide combination immunotherapies.

[0398] In some embodiments, the transformed mammalian cells can be part of a tissue in vivo or ex vivo.

[0399] In some embodiments, the transformed mammalian cells can be isolated mammalian cells such as mammalian cell lines. Mammalian cell lines used herein refer to human or non-human mammalian recombinant expression systems capable of producing post-translational modifications which closely resemble those in mammalian cells in vivo. Exemplary non-human mammalian cell lines include CHO-K1, mouse myeloma cell lines such as NS0, SP2 / 0, rat myeloma cell lines such as YB2 / 0, baby hamster kidney (BHK), N2A cells, HeLa, Jurkat, NIH3T3, and others identifiable to a person skilled in the art. Human mammalian cell lines are immortalized cells propagated in vitro from primary explants of human tissue or body fluid. Exemplary human cell lines include HEK293 and its derivatives, HT-1080, PER.C6, Huh-7 as well as others identifiable to a person skilled in the art.

[0400] In some embodiments, the transformation can occur in an individual of a mammalian species such as Homo sapiens or Musmusculus, for example, among others. In some embodiments, mammalian cells in the sense of the disclosure comprise stem cells, progenitor cells, induced pluripotent stem cells, and others identifiable by a skilled person.

[0401] In some embodiments, the Gas Vesicle Expression System (GVES) of the present disclosure can be used as an enhanced nonlinear contrast acoustic detection system and configured for enhanced nonlinear contrast and related detection in a target cell.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0402] The term nonlinear signal also referred to as a nonlinear acoustic scattering signal, a and nonlinear acoustic scattering, is an acoustic output from a gas vesicle that is not linearly proportional to the incident acoustic pressure. This signal encompasses any detectable change in the acoustic field where the output power or frequency content does not scale 1 : 1 with the input. It encompasses both harmonic responses and acoustic transients. This signal is measured by identifying acoustic data that deviates from the fundamental incident frequency or displays sudden intensity shifts (e.g., step-functions) that are statistically distinguishable from the linear scattering of the surrounding tissue background.

[0403] An acoustic output or “acoustic response” refers to the total detectable acoustic field generated by the interaction of an incident ultrasound wave with a gas vesicle. Variations of acoustic output include steady-state outputs, such as continuous periodic oscillation, and transient outputs, which are time-limited. Measurement of the acoustic output is performed using an ultrasound transducer to capture backscattered pressure waves, which are then processed in either the time domain (to identify transients) or the frequency domain (to identify harmonics).

[0404] A harmonic response (or harmonic) is a type of nonlinear signal characterized by frequency components other than the fundamental incident frequency. This is an umbrella term encompassing integer harmonics (e.g., second or third harmonics), sub-harmonics, and ultraharmonics. Variations include those generated by reversible mechanical shell oscillations (e.g., buckling) or irreversible mechanical events (e.g., collapse). Measurement is typically achieved through frequency-domain filtering or pulse-inversion sequences designed to cancel the fundamental linear output while isolating nonlinear frequency distortions.

[0405] An acoustic transient is a time-limited, non-periodic acoustic output generated during a non-equilibrium event. It is characterized by a sudden, high-magnitude change in backscattered intensity over a short temporal window. Variations include "illumination" spikes during initial buckling or the singular "pop" of a collapse event. Transients are measured by monitoring the temporal intensity profile of a voxel and identifying localized deviations (spikes or drops) that occur on a millisecond or microsecond timescale.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0406] The physical driver of the nonlinear signal is a pressure-induced state change, defined as a physical transition of a gas vesicle shell from a first structural configuration to a second structural configuration. Pressure-induced state change can be categorized and comprise Reversible State Change and Irreversible State Change.

[0407] The wording Reversible State Change as used herein with respect to gas vesicles indicates A transition where the shell undergoes deformation — such as buckling or inward bowing — but retains structural integrity and returns to its original state. This typically generates repetitive transients or steady-state harmonics, measured via non-destructive imaging like xAM.

[0408] The wording Irreversible State Change used herein with respect to gas vesicles indicates a permanent alteration of the shell, such as mechanical collapse, rupture, or fragmentation. This generates a singular, high-magnitude acoustic transient (a "collapse signal"), measured by the permanent disappearance of the scattering source from the voxel.

[0409] Specific nonlinear signals comprise BURST signals and xAM signals.

[0410] A BURST (Burst Ultrasound Reconstruction of Spatiotemporal Transients) signal is a specific embodiment of an irreversible acoustic transient resulting from the mechanical collapse of gas vesicles induced by a pressure step-change. In a BURST signal, the acoustic output of a voxel undergoes a sudden step-function change from a high-scattering state to a zero-scattering state. This is measured and isolated by calculating a voxel-wise Pearson correlation coefficient between the observed temporal intensity profile and a predetermined temporal template vector representing the collapse event. By integrating the structurally reinforced chimeric gas vesicle structural proteins, the critical collapse pressure triggering the BURST signal can be right-shifted (e g., by at least +150 kPa), enabling the multiplexed spatial differentiation of distinct gas vesicle populations.

[0411] An xAM (cross-amplitude modulation) signal is a specific embodiment of a nonlinear signal resulting from a reversible state change, such as the buckling or non-linear deformation of a gas vesicle shell. Unlike signals that rely on permanent structural failure, an xAM signal is generated by operating gas vesicles within a pressure regime that induces nonlinear mechanical oscillations without surpassing the collapse threshold. This signal is an acoustic output thatTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT typically manifests as a harmonic response or a repetitive sequence of transients. Variations of xAM signals include those generated by specific pulse-modulation sequences where the non-linear components are isolated by comparing the acoustic response of full-amplitude pulses to a summation of half-amplitude pulses. Measurement of an xAM signal is performed by calculating the residual signal after subtracting the linear fundamental components, thereby isolating the nonlinear distortions produced by the reversible buckling of the gas vesicle shell. As with the BURST signal, integrating the chimeric structural proteins allows the pressure regime required to induce this reversible xAM buckling to be programmed and distinctly right-shifted.

[0412] Th term “enhanced nonlinear contrast” as used herein refers to the physical increase in the magnitude of nonlinear acoustic backscattering resulting from the optimized yield and density of gas vesicle nanostructures within a target cell . This enhancement is fundamentally enabled by the engineered configurable Gas Vesicle Architecture herein described. Specifically the stoichiometric Dosage Index (DI) of the gas vesicle expression system (GVES), wherein the partitioning of gene modules maximizes the intracellular production of gas vesicles beyond what is achievable with non-stoichiometric or native gene clusters within a same target cell.

[0413] The term “target cell” in the sense of the disclosure indicates a cell within a target site under investigation, as will be understood by a skilled person upon reading of the present disclosure. Accordingly as used herein, the term "target cell" refers to any prokaryotic or eukaryotic cell capable of being genetically engineered to express the Gas Vesicle Expression Systems (GVES) described herein. Target cell in the sense of the disclosure comprise populations of a same or different cell type.

[0414] As used herein, the term “cell type” refers to a distinct category of cells characterized by a specific morphological, physiological, and transcriptional profile. While most cells within a host share the same genomic DNA, a specific cell type is defined by its differential gene expression, which dictates its unique functional properties, metabolic flux, and intracellular environment.

[0415] In some embodiments target cells encompass bacterial cells (e.g., engineered probiotics such as Escherichia, Bacillus or Lactobacillus used as living diagnostics), archaeal cells, In someTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT embodiments the target cells comprise eukaryotic cells such as animal cells and plant cells, fungal cells, and mammalian cells.

[0416] In preferred embodiments, target cells comprise eukaryotic cells and in particular mammalian cells including primary cells and immune cells as will be understood by a skilled person upon reading of the present disclosure.

[0417] In some preferred embodiments target cells can comprises primary cells and in particular primary mammalian cells.

[0418] In some embodiments the primary cells can comprise hepatocytes, cardiomyocytes, neurons (including excitatory, inhibitory, and dopaminergic neurons), neuronal cells, glial cells (astrocytes, microglia), fibroblasts, and immune cells of hematopoietic origin (T lymphocytes, B lymphocytes, NK cells).

[0419] In some preferred embodiments the target cells can comprise neurons and associated glial cells of the central and peripheral nervous systems. Neurons, including excitatory glutamatergic neurons, inhibitory GABAergic neurons, dopaminergic neurons, and sensory neurons, represent a high-priority target cell for the high-resolution mapping of neural activity. Because the mammalian brain is a high-noise environment characterized by significant acoustic scattering and static tissue clutter, neurons engineered with the gas vesicle expression system must be configured to cross the threshold density to transition into the nonlinear visibility regime. Other primary cells within this category include astrocytes, microglia, oligodendrocytes, hepatocytes, cardiomyocytes, skeletal muscle cells, and fibroblasts.

[0420] In some preferred embodiments target cells can comprise immune cells of hematopoietic origin, which are integral to the adaptive and innate immune responses. This category includes lymphoid lineage cells such as T lymphocytes, B lymphocytes, and natural killer cells, as well as myeloid lineage cells such as monocytes, macrophages, and dendritic cells. A particularly significant embodiment within this subgenus is the therapeutic engineered cell, exemplified by chimeric antigen receptor T cells or chimeric antigen receptor phagocytes. These cells are engineered to navigate to specific target sites and report their localization or activation state via the attainment of the gas vesicle threshold density. A particularly preferred embodiment is aIllTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT therapeutic engineered cell, such as a chimeric antigen receptor T (CAR) T cell or phagocyte, configured to cross the nonlinear visibility threshold upon activation as will be understood by a skilled person upon reading of the present disclosure.

[0421] In some embodiments, target cells can delivered, and / or comprised with target tissues of an individual. Target tissues include specific physiological compartments such as the mammalian brain, liver, kidney, lungs, heart, or the circulating blood. In preferred embodiments of high resolution of the present disclosure target sites are characterized as high-noise environments where physiological background signals and tissue clutter typically obscure standard acoustic reporters.

[0422] In preferred embodiments of the enhanced nonlinear contrast for acoustic detection systems described herein, the target sites are characterized as high-noise or opaque environments where physiological background signals, tissue clutter, and intrinsic acoustic scattering typically obscure standard linear acoustic reporters. Exemplary environments characterized as high-noise or opaque include deep-seated tissues and biological compartments where the density, heterogeneity, or depth of the medium presents a significant barrier to signal isolation. An “opaque environment”, in the sense of the present disclosure, refers to a tissue site where the overlapping acoustic properties of the surrounding biological matrix — such as the dense neural networks of the intact mammalian brain or the complex vascularization of internal organs — inhibits the clear differentiation of conventional reporter signals from background artifacts.

[0423] The engineered configurable Gas Vesicle Architecture herein described, can be configured as an enhanced nonlinear contrast system for acoustic detection in a target cell, enables the detection of an increased nonlinear signal due to the robust, order-of-magnitude increased yield (e g., at least a 10-fold volumetric increase) obtained following delivery of the engineered system herein described. This system is particularly preferred in opaque environments and / or in environments characterized by high noise.

[0424] In particular, the GVES herein described is most preferably used within opaque biological environments where the density or depth of the medium typically obscures standard linear acoustic reporters.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0425] A skilled person will understand that the enhanced gas vesicle yield and / or enhanced transfection efficiency in a target cell enabled by the engineered configurable Gas Vesicle Architecture allows the target cell to produce GVs in amounts that not only attain, but robustly surpass a threshold density of assembled gas vesicles. This enables the system to rapidly cross a nonlinear visibility threshold, thereby facilitating high-resolution nonlinear acoustic detection across different types of target cells, target tissues, and target sites.

[0426] As used herein, the "threshold density" represents a cell-specific physical constant defining the concentration of gas vesicles per unit volume required to attain and cross a nonlinear visibility threshold and trigger a transition from a linear acoustic scattering regime to a nonlinear visibility regime within a set target site.

[0427] As used herein, the term “nonlinear visibility threshold” refers to a critical physical and statistical limit defined by the intracellular density of assembled gas vesicles required to trigger a transition from an acoustically invisible state (rest state) to an acoustically detectable state (nonlinear visibility) within a target site. This threshold is reached when the concentration of gas vesicles per unit volume within a target cell is sufficient to produce a threshold-triggered increase in nonlinear scattering that is statistically distinguishable from the environmental noise floor (e.g., static tissue clutter and physiological background).

[0428] A skilled person will understand that this threshold is a functional variable that depends on the environmental noise floor and static clutter characteristic of the specific target site (e.g., the differing static tissue clutter in the mammalian brain versus circulating blood), as well as the sensitivity of the nonlinear imaging modality (e g., BURST or xAM).

[0429] Given a selected imaging modality, rapidly reaching and surpassing the nonlinear visibility threshold of target cells within a target site is the primary functional result of the engineered configurable Gas Vesicle Architecture, specifically the integration of the structural macrooptimization, the assembly factor microoptimization, and the engineered U / N assembly complex. This optimization ensures that the protein yield is sufficient to produce a robust nonlinear acoustic signature that is statistically distinguishable from the background artifacts of that specific site.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0430] As used herein, the term “nonlinear acoustic signature” refers to the unique, identifiable "texture" or profile of the acoustic backscatter produced by the gas vesicles. This signature varies in the target cells and is selectively influenced by: (i) the type of target cell (e.g., neuronal vs. immune); (ii) the number of cells within a resolution element of the target site; (iii) the acoustic properties of the target site itself; and (iv) the structural composition of the gas vesicle shell, such as the distinct, right-shifted acoustic collapse pressure profile generated by the integration of a chimeric GvpA / B structural protein.

[0431] This signature serves as the "template" for the adaptive demixing algorithm herein described, which utilizes voxel-wise Pearson correlation to isolate this specific genetic reporter signal from the broader noise floor. Because the signature is unique to the gas vesicles produced under a specific DI ratio and structural configuration (for example, multiplexing driven by the +150 kPa right-shifted acoustic collapse pressure of the chimeric GvpA / B shell), the system can distinguish genetic signals with a statistical confidence that remains robust even in high-noise environments like the mammalian brain. Because the signature is site- and shell-dependent, the adaptive demixing algorithm utilized in some embodiments described herein is configured to perform voxel-wise Pearson correlation to isolate this specific signature from the site-specific noise. This ensures that the reconstruction of the 3D activity map remains accurate regardless of the depth or opacity of the biological environment.

[0432] A skilled person will understand upon reading of the present disclosure that rapidly reaching this threshold density is the primary functional requirement for isolating genetic reporter signals of target cells from the physiological background noise (e.g., tissue clutter or motion artifacts) of high-noise environments such as the mammalian brain.

[0433] In previous implementations, gas vesicle expression systems were configured to achieve an intracellular density sufficient to merely cross a defined nonlinear visibility threshold. However, because the engineered configurable Gas Vesicle Architecture described herein provides an intracellular yield increased by at least an order of magnitude, the system functionally exceeds this baseline threshold by a substantial margin.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0434] This maximized intracellular yield translates directly to enhanced spatiotemporal imaging capabilities. Functionally, this order-of-magnitude increase allows for a significantly lower limit of detection (LOD) within a target tissue, enabling the nonlinear acoustic detection of fewer engineered cells per volumetric imaging voxel compared to reference systems. Furthermore, because the engineered target cells accumulate the required threshold density of gas vesicles at an accelerated rate, the system provides enhanced temporal resolution, allowing for the acoustic detection of rapid biological events or transient genetic circuit activations that would otherwise remain acoustically sub -threshold.

[0435] In embodiments herein described, the attainment of the threshold density is enabled by the precise stoichiometric configuration of the Gas Vesicle Gene Cluster (GVGC). This can be achieved through a highly optimized single-transcript polycistronic architecture, or by partitioning the GVGC into at least three separate Gas Vesicle Polynucleotide Constructs (GVPC): a structural module (e.g., gvpA'B) and at least two assembly factor modules (AF1, AF2 / AF3). Both the optimized single-transcript architecture and the multi-construct partitioned architecture result in enhanced gas vesicle yield (e.g., an intracellular volumetric yield increase of at least 10-fold) and / or enhanced transfection efficiency for delivering the system to a target cell. These stoichiometrically configured architectures result in a Transduction-Expression Index (TEI) that is at least 5-fold higher than the TEI achieved by an unoptimized single-construct system expressing the same gene cluster without the stoichiometric configuration of the gvp genes. This architectural improvement allows a statistically significant portion of the target cell population to reach or exceed the threshold density and nonlinear visibility threshold for certain target cells, thereby providing an increased non-linearity and enhanced contrast that enables high-resolution mapping.

[0436] In preferred embodiments where the polynucleotide constructs of the GVES of the disclosure are comprised within vectors subject to a physical packaging constraint, the highly compressed polycistronic arrangement, or alternatively the three-construct split, further results in a significant increase in visibility due to the increase in yield of GV and transfection efficiency provided by the GVES under Standard Conditions.

[0437] A skilled person will understand that the packaging limit of the vector will impact the related TEI in standard, unoptimized one-construct or two-construct systems, which are known orTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT expected to result in GV production that is statistically below the threshold density for many target cells.

[0438] A skilled person will also understand that regardless of whether the target cell is a prokaryotic bacterium or a eukaryotic neuron, by achieving the threshold density and nonlinear visibility threshold of a target cell, an enhanced nonlinear contrast gas vesicle expression system can allow the target cell to transition from an acoustically invisible state to an acoustically visible state in the nonlinear regime, enabling high-resolution mapping even in challenging biological environments.

[0439] In particular, in the engineered configurable Gas Vesicle Architecture of the disclosure, the configuration is directed to obtain a target DI ratio and / or a target Transduction-Expression Index (TEI), which are selectively calibrated based on the physiological and metabolic characteristics of a target mammalian cell to ensure the nonlinear visibility threshold of the specific target cell type is surpassed. It has been observed that different cell types possess varying assembly capacities, defined as the maximum density of gas vesicles a cell can support before reaching metabolic exhaustion or physical packing limits. Consequently, the disclosure provides a cellspecific correspondence between the GVES architecture and the target cell, wherein the DI ratio is tuned to maximize the nonlinear acoustic yield relative to the cell's specific volume and background noise environment.

[0440] For example, in neuronal cells, an overall DI ratio of approximately 5:1:1 is preferred. This higher structural-to-assembly ratio is configured to drive rapid protein accumulation during transient windows of activity (e.g., neuronal bursting), ensuring that the threshold density is reached quickly enough to capture high-resolution temporal dynamics. In another example, in immune cells (such as T-cells or CAR-T cells), a DI ratio in the range of approximately 2:1:1 to 4:1:1 can be preferred to balance the requirement for acoustic visibility with the metabolic demands of cellular proliferation and migration. In these embodiments, the lower DI ratio ensures that the cell remains viable and functionally active while still providing sufficient nonlinear signal amplification, as will be understood by a skilled person.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT

[0441] As used herein, the term “approximately” or “about,” when used in reference to a Dosage Index (DI) ratio (e.g., a ratio of approximately 5:1:1), refers to a range of values that retains the essential functional property of the specified ratio: namely, the ability of the target cell to cross the nonlinear visibility threshold under Standard Conditions.

[0442] In particular, “approximately” encompasses a variance of ±10%, and preferably ±5%, of the numerical value of each component within the ratio. For example, a DI ratio of approximately 5:1:1 includes ratios where the structural module (e.g., gvpA / B) maintains a relative Dosage Index of 4.0 to 6.0 relative to the assembly factor modules (e.g., AF1, AF2) being maintained at approximately 0.8 to 1.2.

[0443] Furthermore, the term "approximately" indicates that the stoichiometric balance is maintained such that the primary structural protein remains in a significant molar excess (at least 2-fold, and preferably 4-fold to 5-fold) over the assembly factors, sufficient to drive the formation of the gas vesicle nanostru...

Claims

Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT CLAIMS1. A genetically engineered Gas Vesicle Expression System (GVES) configured for expressing in a target cell within a target site a gene cluster of gvp genes encoding gas vesicle (GV) proteins capable of forming a GV type, the GVES comprising:a) at least one structural gvp differentially expressed gene module comprising a gvpA / B gene under control of a promoter and additional regulatory regions allowing expression of a GvpA / B protein in the target cell with a structural Dosage Index (DI);b) at least one first assembly factor (AF1) gvp differentially expressed gene module comprising an AF1 set of gvp genes encoding a first set of GV assembly factors AF1 comprising at least a gvpG gene and a gvpF gene, allowing expression with an AF1 Dosage Index (DI);c) at least one second assembly factor (AF2) gvp differentially expressed gene module comprising an AF2 set of gvp genes encoding a second set of GV assembly factors AF2 comprising at least a gvpN gene, allowing expression with an AF2 Dosage Index (DI); and d) at least one third assembly factor (AF3) gvp differentially expressed gene module comprising an AF3 set of gvp genes encoding a third set of GV assembly factors AF3 comprising at least a gvpK gene, allowing expression with an AF3 Dosage Index (DI);wherein the gvp gene modules are operably linked by regulatory sequences allowing co-expression of the GV proteins with a Target Dosage Index (DI) ratiowherein the structural -DI is greater than the AF1-DI, the AF1-DI is greater than the AF2-DI, and the AF2-DI is greater than the AF3-DI .

2. The genetically engineered GVES of claim 1, wherein the target cell is a eukaryotic cell.

3. The genetically engineered GVES of claim 2, wherein the eukaryotic cell is a mammalian cell.

4. The genetically engineered GVES of claim 3, wherein the mammalian cell is a primary cell selected from hepatocytes, cardiomyocytes, neurons, astrocytes, microglia, fibroblasts, endothelial cells, keratinocytes, adipocytes, pancreatic P-cells, renal epithelial cells, and skeletal muscle cells.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 5. The genetically engineered GVES of claim 3, wherein the mammalian cell is a primary immune cell of hematopoietic origin selected from T lymphocytes, B lymphocytes, natural killer (NK) cells, monocytes, macrophages, and dendritic cells.

6. The genetically engineered GVES of claim 1, wherein the target cell is a prokaryotic cell.

7. The genetically engineered GVES of claim 6, wherein the prokaryotic cell is an engineered probiotic bacterium.

8. The genetically engineered GVES of any one of claims 1 to 7, wherein the target site is an in vitro cell culture or an ex vivo tissue.

9. The genetically engineered GVES of any one of claims 1 to 7, wherein the target site is an in vivo physiological environment within a host.

10. The genetically engineered GVES of claim 9, wherein the host is a mammalian host.

11. The genetically engineered GVES of claim 9 or 10, wherein the target site is a high-noise or opaque biological environment.

12. The genetically engineered GVES of any one of claims 9 to 11, wherein the target site is selected from a mammalian brain, a tumor microenvironment, a liver, a kidney, a heart, a lung, a gastrointestinal tract, and circulating blood.

13. The genetically engineered GVES of any one of claims 1 to 12, wherein the structural-DI is configured to be at least 2-fold higher than the AF1-DI and the AF2-DI.

14. The genetically engineered GVES of claim 13, wherein the structural-DI is configured to be at least 4-fold to 5-fold higher than the AF1-DI and the AF2-DI.

15. The genetically engineered GVES of any one of claims 1 to 14, wherein the Target DI ratio of structural-DI : AF1-DI : AF2-DI : AF3-DI operates within a range of approximately (2-12) : (2-8) : (0.5-2) : (0.125-1).Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 16. The genetically engineered GVES of claim 15, wherein the Target DI ratio is approximately 4:2:l:0.5.

17. The genetically engineered GVES of claim 15, wherein the Target DI ratio is approximately 6:2:1:0.25.

18. The genetically engineered GVES of any one of claims 1 to 17, wherein the gvp differentially expressed gene modules are arranged sequentially along a single polycistronic construct.

19. The genetically engineered GVES of claim 18, wherein the gvp genes are separated by tunable genetic elements comprising self-cleaving 2A peptides and / or Internal Ribosome Entry Sites (IRES) configured to enforce the Target DI ratio.

20. The genetically engineered GVES of claim 19, wherein the self-cleaving 2A peptides are selected from the group consisting of P2A, T2A, E2A, F2A, BmCPV 2A, and BmIFV 2A.

21. The genetically engineered GVES of any one of claims 1 to 17, wherein the gvp differentially expressed gene modules are partitioned across a plurality of at least three distinct delivery vectors.

22. The genetically engineered GVES of claim 21, wherein the delivery vectors are viral vectors selected from lentiviral vectors and adeno-associated viral (AAV) vectors.

23. The genetically engineered GVES of claim 22, wherein the Target DI ratio is configured to be achieved by co-transducing the target cell at a relative Multiplicity of Infection (MOI) mirroring the Target DI ratio.

24. The genetically engineered GVES of claim 21, wherein the delivery vectors are non-viral vectors comprising messenger RNA (mRNA) or plasmid DNA.

25. The genetically engineered GVES of claim 24, wherein the non-viral vectors comprise messenger RNA (mRNA) encapsulated within lipid nanoparticles (LNPs) configured for transient expression in the target cell.

26. The genetically engineered GVES of any one of claims 1 to 25, wherein the Target DI ratio enables the target cells to surpass a threshold density of assembled gas vesicles, transitioning theTitle: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT target cell from an acoustically sub-threshold state to triggering a detectable nonlinear acoustic scattering signature.

27. A chimeric gas vesicle (GV) structural protein comprising:an N-terminal core structural shell domain derived from a first gas vesicle structural protein of a first species or a derivative thereof, comprising an alphal-betal-beta2-alpha2 secondary structure topology, and having a Core C-terminus terminating at the C-terminal end of the alpha2 helix; anda C-terminal stabilizing tail domain derived from a second gas vesicle structural protein of a second species or a derivative thereof, comprising a structurally disordered C-terminal region natively located downstream of an alpha2 helix of said second gas vesicle protein;wherein the stabilizing tail domain is directly covalently attached to the Core C-terminus of the core structural shell domain.

28. The chimeric GV structural protein of claim 27, wherein the first gas vesicle structural protein is a GvpA, and the second gas vesicle structural protein is a GvpB.

29. The chimeric GV structural protein of claim 27, wherein the first gas vesicle structural protein is a GvpB, and the second gas vesicle structural protein is a GvpA.

30. The chimeric GV structural protein of claim 27, wherein the first gas vesicle structural protein is a GvpA, and the second gas vesicle structural protein is a GvpA.

31. The chimeric GV structural protein of claim 27, wherein the first gas vesicle structural protein is a GvpB, and the second gas vesicle structural protein is a GvpB.

32. The chimeric GV structural protein of any one of claims 27 to 31, wherein the first species is selected from Anabaena flos-aquae, Halobacterium salinarum, Microcystis aeruginosa, Planktothrix rubescens, and Trichodesmium erythraeum.

33. The chimeric GV structural protein of any one of claims 27 to 32, wherein the second species is selected from Bacillus megaterium, Serratia sp. ATCC 39006, Desulfovibrio vulgaris, and Streptomyces coelicolor.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 34. The chimeric GV structural protein of any one of claims 27 to 33, wherein the core structural shell domain is derived from Anabaena flos-aquae GvpA, wherein the stabilizing tail domain is derived from Bacillus megaterium GvpB, and wherein the chimeric GV structural protein comprises an amino acid sequence having at least 70% overall sequence identity to SEQ ID NO: 482.

35. The chimeric GV structural protein of claim 34, wherein the core structural shell domain independently comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to amino acids 1 to 63 of SEQ ID NO: 482.

36. The chimeric GV structural protein of claim 34 or 35, wherein the stabilizing tail domain independently comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to amino acids 64 to 88 of SEQ ID NO: 482.

37. The chimeric GV structural protein of any one of claims 27 to 36, comprising sequence variance consisting of functionally equivalent conservative amino acid substitutions, wherein a substituted amino acid possesses a side-chain van der Waals volume within + / - 30%, a net formal charge at pH 7.4 within + / - 1, and a Kyte-Doolittle hydropathy index within + / - 1.5 units relative to the corresponding residue of the reference sequence.

38. The chimeric GV structural protein of any one of claims 27 to 37, wherein the core structural shell domain consists of an absolute length of 50 to 80 amino acids, and wherein the stabilizing tail domain consists of an absolute length of 10 to 60 amino acids.

39. The chimeric GV structural protein of any one of claims 27 to 38, wherein the stabilizing tail domain comprises a truncation of at least 5, at least 10, or at least 15 amino acids from the extreme C-terminus relative to the wild-type second gas vesicle protein.

40. An engineered polynucleotide encoding the chimeric GV structural protein of any one of claims 27 to 39, wherein the polynucleotide comprises a direct fusion junction lacking any intervening synthetic linker sequence between a nucleotide sequence encoding the core structural shell domain and a nucleotide sequence encoding the stabilizing tail domain.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 41. An engineered gas vesicle comprising a protein shell formed from a plurality of the chimeric GV structural proteins of any one of claims 27 to 39, wherein the structural integration of the stabilizing tail domain physically reinforces the engineered gas vesicle, providing an intrinsic median critical collapse pressure right-shifted by at least +50 kPa, or at least +100 kPa relative to a reference gas vesicle consisting of a protein shell lacking the stabilizing tail domain.

42. The engineered gas vesicle of claim 41, wherein the intrinsic median critical collapse pressure is right-shifted by at least +150 kPa, at least +200 kPa, or at least +250 kPa.

43. The engineered gas vesicle of claim 41 or 42, wherein the structurally reinforced protein shell exhibits a theoretical buckling limit extending between 600 kPa and 1.5 MPa, between 700 kPa and 1.2 MPa, or between 800 kPa and 1.0 MPa.

44. The engineered gas vesicle of any one of claims 41 to 43, wherein the engineered gas vesicle exhibits a high-density clustering phenotype characterized by an interwoven bundling architecture intersecting at variable non-parallel angles ranging from 15 degrees to 90 degrees.

45. The engineered gas vesicle of claim 44, wherein the variable non-parallel angles range from 30 degrees to 90 degrees, or from 45 degrees to 75 degrees.

46. A genetically engineered Gas Vesicle Expression System (GVES) comprising: at least one assembly factor gvp differentially expressed gene module comprising a gvpU gene and a gvpN gene under the control of one or more heterologous promoters configured to co-express a GvpU assembly protein and a GvpN assembly protein in a target cell; wherein the GvpN assembly protein comprises a beta-barrel hexamerization domain and conserved Walker A and Walker B nucleotide-binding motifs, and wherein the GvpU assembly protein is configured to physically interact with the beta-barrel hexamerization domain of the GvpN assembly protein to form a stabilized GvpN-GvpU assembly complex.

47. The genetically engineered GVES of claim 46, wherein the target cell is a mammalian cell.

48. The genetically engineered GVES of claim 46 or 47, wherein the gvpU gene and the gvpN gene are stoichiometrically configured to co-express the GvpU assembly protein and the GvpN assembly protein at a functional molar ratio ranging from 0.5:1 to 3.0:1.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 49. The genetically engineered GVES of claim 48, wherein the functional molar ratio ranges from 0.8:1 to 2.0:1.

50. The genetically engineered GVES of claim 48, wherein the functional molar ratio is approximately 1:1.

51. The genetically engineered GVES of any one of claims 46 to 50, wherein the GvpN assembly protein and the GvpU assembly protein are derived from the same target species, selected from the group consisting of Bacillus megaterium, Anabaena flos-aquae, Halobacterium salinarum, Microcystis aeruginosa, and Serratia sp. ATCC 39006.

52. The genetically engineered GVES of any one of claims 46 to 51, wherein the formation of the GvpN-GvpU assembly complex increases an intracellular volumetric yield of assembled gas vesicles by at least 3-fold, at least 5-fold, at least 8-fold, or at least 10-fold relative to a baseline expression system lacking the gvpU gene.

53. The genetically engineered GVES of any one of claims 46 to 52, wherein the formation of the GvpN-GvpU assembly complex maintains an acoustic collapse threshold of the assembled gas vesicles within a variance of + / - 15%, + / - 10%, or + / - 5% (+ / - 75 kPa, + / - 50 kPa, or + / - 25 kPa) relative to said baseline expression system.

54. An engineered configurable Gas Vesicle Architecture comprising at least two of: a) the genetically engineered GVES of any one of claims 1 to 26; b) the engineered polynucleotide of claim 40 encoding the chimeric GV structural protein; and / or c) the genetically engineered GVES of any one of claims 46 to 53 comprising the ipf / gene and the gvpN gene.

55. The engineered configurable Gas Vesicle Architecture of claim 54, comprising the synergistic combination of (a) and (b).

56. The engineered configurable Gas Vesicle Architecture of claim 54, comprising the synergistic combination of (a) and (c).

57. The engineered configurable Gas Vesicle Architecture of claim 54, comprising the synergistic combination of (b) and (c).Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 58. The engineered configurable Gas Vesicle Architecture of claim 54, comprising the synergistic combination of (a), (b), and (c).

59. The engineered configurable Gas Vesicle Architecture of claim 58, wherein an AF3 Dosage Index (DI) of the Target DI ratio is quantitatively modified to a higher relative level than a wildtype reference DI to prevent morphological assembly bottlenecking of a volumetrically enhanced gas vesicle pool.

60. The engineered configurable Gas Vesicle Architecture of any one of claims 54 to 59, wherein the Architecture provides a functional acoustic output characterized by a BURST Signal-to-Background Ratio (SBR) greater than 100 under Standard Conditions.

61. The engineered configurable Gas Vesicle Architecture of claim 60, wherein the target cell is a mammalian cell, and wherein the Architecture maintains target mammalian cell viability at a Toxicity / Viability Index statistically equivalent to an unengineered control cell population under Standard Conditions.

62. The engineered configurable Gas Vesicle Architecture of any one of claims 54 to 61, further comprising a gvpC differentially expressed gene module encoding an outer scaffold protein, wherein the outer scaffold protein physically interfaces with the chimeric GV structural proteins to further right-shift the median critical acoustic collapse pressure in a dose-dependent manner.

63. The engineered configurable Gas Vesicle Architecture of claim 62, wherein the outer scaffold protein comprises a protease-cleavable variant (URoC), wherein protease-mediated cleavage of the outer scaffold protein dynamically alters the mechanical reinforcement of the protein shell.

64. The engineered configurable Gas Vesicle Architecture of any one of claims 54 to 63, wherein the differentially expressed gene modules are arranged sequentially on a single polycistronic transcript, and wherein the single polycistronic transcript is physically sized to fit within a single lentiviral vector having a functional packaging limit of 8.0 to 10.0 kilobases.

65. A genetically engineered Gas Vesicle Reporting Molecular Component (GVRMC) comprising: the engineered configurable Gas Vesicle Architecture of any one of claims 54 to 64; and an activity-dependent target regulatory region operably linked to the Gas Vesicle Architecture,Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT wherein the target regulatory region is configured to be activated by a molecular component of a genetic circuit in response to an intracellular biochemical event.

66. The genetically engineered GVRMC of claim 65, wherein the activity-dependent target regulatory region comprises a mammalian promoter selected from the group consisting of Fos, Arc, Npas4, NF AT, NF-KB, and Synapsin.

67. The genetically engineered GVRMC of claim 65 or 66, further comprising an activitydependent trigger-filter genetic circuit comprising: an activation-dependent promoter operably linked to a sequence encoding a transactivator protein fused to a post-translational degradation element (degron); wherein the degron is selected from a PEST sequence, an ornithine decarboxylase (ODC) domain, an auxin-inducible degron (AID), a destabilizing domain (DD), or a light-oxygen-voltage (LOV) domain.

68. The genetically engineered GVRMC of claim 67, wherein the transactivator protein initiates expression of the Gas Vesicle Architecture upon a biological trigger, and wherein the degron facilitates the rapid clearance of the transactivator protein in the absence of said trigger to maintain an acoustic zero-baseline characterized by a threshold-triggered increase in nonlinear signal-to-noise ratio (SNR) of at least 10-fold, 20-fold, 50-fold, or 100-fold upon activation.

69. A genetically engineered target cell comprising the engineered configurable Gas Vesicle Architecture of any one of claims 54 to 64, or the genetically engineered GVRMC of any one of claims 65 to 68.

70. The genetically engineered target cell of claim 69, wherein the target cell is a mammalian cell.

71. The genetically engineered target cell of claim 70, wherein the mammalian cell is a primary cell selected from hepatocytes, cardiomyocytes, neurons, astrocytes, microglia, fibroblasts, endothelial cells, keratinocytes, adipocytes, pancreatic P-cells, renal epithelial cells, and skeletal muscle cells.

72. The genetically engineered target cell of claim 70, wherein the mammalian cell is a primary immune cell expressing a Chimeric Antigen Receptor (CAR) selected from a CAR-T cell and a CAR-P cell.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 73. A method for multiplexed volumetric nonlinear acoustic imaging of a target site, the method comprising:a) providing a target site comprising a first cell population and a second cell population, wherein the first cell population expresses a first gas vesicle type having a first critical acoustic collapse pressure (Pci), and the second cell population expresses the engineered configurable Gas Vesicle Architecture of any one of claims 54 to 64 to produce a second gas vesicle type comprising the chimeric GV structural protein and having a second critical acoustic collapse pressure (Pc2), wherein Pc2 is right-shifted and greater than Pci;b) applying a first ultrasound pulse sequence to the target site at a first acoustic pressure (Pfirst) wherein Pci < Pfirst < Pc2, thereby selectively collapsing the first gas vesicle type and generating a first acoustic image specific to the first cell population; andc) subsequently applying a second ultrasound pulse sequence to the target site at a second acoustic pressure (Psecond) wherein Psecond > Pc2, thereby selectively collapsing the second gas vesicle type and generating a second acoustic image specific to the second cell population.

74. The method of claim 73, wherein the target site is an in vivo target site within a host.

75. The method of claim 74, wherein the host is a mammalian host.

76. The method of claim 75, wherein the in vivo target site is a high-noise biological environment selected from a mammalian brain, a tumor microenvironment, a liver, a kidney, a heart, a lung, a gastrointestinal tract, and circulating blood.

77. The method of any one of claims 73 to 76, wherein the first gas vesicle type comprises a protein shell genetically lacking an outer scaffold protein GvpC (AC), thereby lowering the first critical acoustic collapse pressure (Pci) relative to a wild-type reference.

78. The method of any one of claims 73 to 76, wherein the first gas vesicle type comprises a protein shell wherein an outer scaffold protein GvpC has been chemically stripped, partially removed, or truncated.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 79. The method of claim 78, wherein said stripping or removal increases the mechanical compliance of the protein shell and enhances the nonlinear acoustic scattering profile of the first gas vesicle type at low acoustic pressures relative to an intact wild-type reference.

80. The method of any one of claims 73 to 79, wherein the engineered configurable Gas Vesicle Architecture of the second cell population comprises the gvpC module of claim 62, and wherein the outer scaffold protein physically interfaces with the chimeric GV structural protein to further right-shift the second critical acoustic collapse pressure (Pc2) in a dose-dependent manner.

81. The method of claim 80, wherein the outer scaffold protein comprises the protease-cleavable variant (URoC) of claim 63, and wherein an intracellular biochemical event comprising protease-mediated cleavage of the outer scaffold protein dynamically alters the mechanical reinforcement of the protein shell, shifting the second critical acoustic collapse pressure (Pc2) to a lower detectable acoustic collapse pressure to provide a dynamic ratiometric readout of intracellular protease activity.

82. The method of any one of claims 73 to 81, wherein the target site further comprises a third cell population expressing a third gas vesicle type having an intermediate collapse pressure (Pc_int) wherein Pci < Pc int < Pc2, and wherein the method further comprises applying an intermediate ultrasound pulse sequence to selectively collapse the third gas vesicle type, enabling high-order spatial tri-plexing.

83. The method of any one of claims 73 to 82, wherein generating the first acoustic image and the second acoustic image comprises isolating nonlinear gas vesicle transients from background tissue scattering by calculating a voxel-wise Pearson correlation coefficient by comparing a temporal intensity profile of a voxel across an N-frame 4D spatiotemporal data stack to a temporal template vector representing a mechanical gas vesicle collapse event.

84. The method of claim 83, wherein the temporal template vector is defined as Y = [1, 0, 0, ..., 0], representing a sharp signal peak in a first collapse frame followed by a lack of signal in subsequent frames.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 85. The method of claim 83 or 84, further comprising generating a 3D signal map by applying a statistical indicator function derived from a t-distribution with N-2 degrees of freedom to the calculated correlation coefficients.

86. The method of claim 85, wherein applying the statistical indicator function retains only voxels exceeding a numeric threshold to achieve a background rejection rate exceeding 99.9%.

87. The method of claim 86, wherein the background rejection rate exceeds 99.99% or 99.999%.

88. The method of any one of claims 83 to 87, further comprising performing a temporal window integration for collapse events spanning multiple frames by summing voxel sequences across a defined collapse window.

89. The method of any one of claims 73 to 88, further comprising administering hyperpolarized 129Xe gas to the target site, and performing chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) to detect a signal reduction specific to the presence of the gas vesicle types, validating the acoustic imaging.

90. A composition for the stoichiometric expression and nonlinear detection of gas vesicles, comprising the engineered configurable Gas Vesicle Architecture of any one of claims 54 to 64, and a pharmaceutically acceptable vehicle configured for delivery to a target site in a host.

91. The composition of claim 90, wherein the host is a mammalian host.

92. The composition of claim 90 or 91, wherein the composition is formulated for systemic administration to the host.

93. The composition of claim 92, wherein the systemic administration route is selected from intravenous, intra-arterial, and intraperitoneal routes.

94. The composition of claim 90 or 91, wherein the composition is formulated for local or topical administration to the host.

95. The composition of claim 94, wherein the local administration route is selected from intradermal, subcutaneous, intranasal, and intratumoral injection.Title: “Chimeric Structural Proteins...”Inventors: Mengtong Duan et al. Attorney Docket: P3343-PCT 96. The composition of any one of claims 90 to 95, wherein the engineered configurable Gas Vesicle Architecture is comprised within a viral vector selected from a lentiviral vector and an adeno-associated viral (AAV) vector.

97. The composition of any one of claims 90 to 95, wherein the engineered configurable Gas Vesicle Architecture is comprised within a non-viral vector selected from plasmid DNA and messenger RNA (mRNA).

98. The composition of claim 97, wherein the messenger RNA (mRNA) is encapsulated within a lipid nanoparticle (LNP) configured for transient expression in a target cell.

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