Methods for identifying and selecting self-replicating RNA molecules for biomedical applications

Abstract

This disclosure relates, in general, to methods for identifying and / or selecting self-replicating RNA (srRNA) delivery systems suitable for biomedical applications. More specifically, this disclosure relates to methods for identifying and / or selecting srRNA delivery systems using combinations of key quality characteristics for srRNA vaccines and biological products. It also provides srRNA compositions, formulations, and methods for inducing pharmacodynamic effects in subjects requiring them, as well as methods for preventing and / or treating various health conditions.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 502,219, filed May 15, 2023. The disclosure of the application referenced above, including all figures, is hereby expressly incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to the fields of molecular virology and immunology, and more particularly to methods for identifying and / or selecting self - replicating RNA (srRNA) delivery systems suitable for biomedical applications.

Background Art

[0003] RNA technology has come to the forefront of innovative medicine in recent years and is being explored for a wide range of treatments, including preventive and therapeutic vaccines, expression of biological - agent proteins, and gene therapy. In addition to the conventional mRNA platforms currently approved for preventive SARS - CoV2 vaccines, srRNA is increasingly being used as a vaccine and therapeutic modality for in - situ production of proteins.

[0004] Nucleic acid vaccines, such as srRNA vaccines, are a different approach in vaccine development. Unlike conventional vaccines that deliver the target antigen either as a protein structure in the form of a purified or recombinant protein or as part of an attenuated or inactivated pathogen, nucleic - based vaccines deliver DNA or RNA encoding the target antigen (e.g., immunogen). The generation of a protective immune response depends on the cellular uptake and expression of the delivered nucleic acid that precedes the immunological presentation of the target antigen. Synthetic srRNA vaccines are currently being evaluated clinically for infectious diseases and oncology. Prototypic srRNA vectors in clinical development are derived from alphaviruses, particularly Venezuelan equine encephalitis virus (VEEV).

[0005] Synthetic srRNAs offer several advantages, namely an improved safety profile based on the absence of genomic integration or cell transformation, and simplified manufacturing. Another safety advantage of srRNAs is the reduced effective human dose. Because srRNAs amplify within host cells, even low doses result in higher and more persistent protein expression, making them more beneficial as biologics compared to mRNA. Furthermore, srRNA-based vaccines are beneficial compared to mRNAs due to their ability to induce strong cell-mediated immunity, exemplified by the strong CD8+ and CD4+ T-cell response crucial for effective tumor treatment. Finally, the absence of a viral shell allows for repeated administration without inducing / reducing anti-vector immunity, and enables encoding multiple larger target genes that are typically limited by the packaging ability of viral particles. Despite these inherent advantages, recent clinical candidates have highlighted challenges in the development of fully synthetic srRNA products.

[0006] Because structural proteins in the alphaviral genome are removed from synthetic srRNA vectors, formulation is necessary for optimal in vivo delivery. However, the relatively large size of srRNA molecules compared to known mRNA vectors presents unique challenges in formulating srRNA, whether as a vaccine or a biologic. These challenges necessitate costly and time-consuming screening of numerous delivery systems to identify the appropriate and optimal one. Identifying a suitable delivery system for srRNA products is crucial for their successful development as biomedical compositions. The delivery strategy must be tailored to the specific application of the srRNA molecule and the product being delivered.

[0007] Furthermore, a set of quality control measures (for chemistry, control, and manufacturing) are required to identify desirable product characteristics during the process, release, and stability evaluation of srRNA vaccine candidates or biological products, ensuring a reproducible and safe product for preclinical and clinical use. In addition to safety and efficacy, these quality control measures are typical requirements for all successful treatments.

[0008] There is a need for efficient methods to identify or screen effective srRNA delivery systems for prophylactic and therapeutic biomedical compositions. [Overview of the Initiative]

[0009] This disclosure provides a reliable analytical evaluation method based on a newly identified combination of quality characteristics that can be rapidly performed to predict a suitable delivery system for biomedical applications. A suitable delivery system is important, for example, to ensure the optimal in vivo activity of formulated srRNA and thereby ensure the effective delivery of srRNA vaccines or biological products (e.g., drug products).

[0010] Accordingly, the present disclosure relates, in general terms, to methods for selecting or identifying srRNA delivery systems suitable for biomedical applications, and to compositions comprising such srRNA delivery systems. In particular, as described in more detail below, some embodiments of the present disclosure provide a method for identifying or selecting an srRNA delivery system for biomedical applications, comprising: a) formulating an input srRNA composition together with a nonviral delivery vehicle to produce an srRNA delivery system comprising a formulated srRNA composition; b) measuring the potency of the formulated srRNA composition relative to an unformulated input srRNA composition; c) measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition relative to the amount of full-length srRNA molecules in the input srRNA composition; and d) selecting an srRNA delivery system as suitable for biomedical applications if the formulated srRNA composition in (b) retains at least about 25% of the potency of the unformulated input srRNA composition.

[0011] In some embodiments of this method, the srRNA delivery system in (d) may be selected as suitable for biomedical applications if the formulated srRNA composition in (b) retains at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of the potency of the unformulated srRNA composition.

[0012] In some embodiments for identifying or selecting an srRNA delivery system, the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition in (c) is a reduction of less than 40% relative to the amount of full-length srRNA molecules in the input srRNA composition. In some embodiments of the method, the reduction percentage in (c) is less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%, relative to the amount of full-length srRNA molecules in the input srRNA composition.

[0013] In some embodiments of a method for selecting or identifying a delivery system for biomedical applications, the input srRNA composition comprises double-stranded RNA (dsRNA) molecules and single-stranded RNA (ssRNA) molecules. In some embodiments, the method further includes a step of measuring the percentage of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) in the input srRNA composition. In some embodiments, the step of measuring the percentage of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) in the input srRNA composition is performed by an immunoblot assay.

[0014] In some embodiments of the methods provided herein, the percentage of dsRNA to ssRNA is less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.5%, or less than about 0.25%.

[0015] In some embodiments of the methods of the present disclosure, the step of measuring the potency of the formulated srRNA composition in (b) is performed in vivo or in vitro. In some embodiments, the step of measuring the potency of the formulated srRNA composition includes detection of RNA replication, detection of viral protein expression, and / or detection of heterologous gene expression. In some embodiments, the step of measuring the potency of the formulated srRNA composition includes immunoblotting analysis, fluorescence flow cytometry analysis, enzyme-linked immunoassay analysis, immunogenicity analysis, bioactivity analysis, and / or efficacy in a disease model. In some embodiments, the step of measuring potency includes evaluating RNA replication efficiency. In some embodiments, evaluating RNA replication efficiency includes a monoclonal antibody. In some embodiments, the monoclonal antibody is J2. In some embodiments, replication efficiency is determined by measuring the frequency of cells having dsRNA per ng of transfected RNA in an in vitro potency assay.

[0016] In some embodiments, the step of measuring the potency of the formulated srRNA composition in (b) includes an in vivo potency assay. In some embodiments, the in vivo potency assay is performed in animal cells. In some embodiments, the in vivo potency assay is performed in mammalian cells.

[0017] In some embodiments of the methods of the present disclosure, the step of measuring the percentage of full-length srRNA molecules in step (c) includes assaying the degradation of full-length srRNA molecules during the formulation process of step (a). In some embodiments, the step of measuring the percentage of full-length srRNA molecules in (c) includes gel electrophoresis and / or capillary electrophoresis.

[0018] In one aspect of this disclosure, the Specified provides a method for identifying a self-replicating RNA (srRNA) delivery system for biomedical applications, comprising: (a) measuring the percentage of double-stranded RNA (dsRNA) molecules in an input srRNA composition relative to single-stranded RNA (ssRNA) molecules in an input srRNA composition; (b) formulating the input srRNA composition together with a non-viral delivery vehicle to produce an srRNA delivery system containing the formulated srRNA composition; (c) measuring the potency of the formulated srRNA composition relative to an unformulated input srRNA composition; and (d) measuring the full-length srRNA molecules in the formulated srRNA composition. The present invention provides a method comprising the steps of: measuring the percentage change in the amount of a given input srRNA composition relative to the amount of full-length srRNA molecules; and selecting an srRNA delivery system as suitable for biomedical applications if the percentage of dsRNA relative to ssRNA in (e)(i)(a) is less than approximately 2.5%, the formulated srRNA composition in (ii)(c) retains at least approximately 25% of the potency of the unformulated input srRNA composition, and the percentage change in (iii)(d) is a percentage decrease of less than approximately 40% relative to the amount of full-length srRNA molecules in the unformulated input srRNA composition.

[0019] In some embodiments of the methods of the present disclosure, the step of measuring the percentage of dsRNA to ssRNA in (b) comprises an immunoblotting assay, the step of measuring potency comprises an in vitro potency assay, and the step of measuring the percentage change in the amount of full-length srRNA between the formulated srRNA and the input srRNA composition in (d) comprises capillary electrophoresis.

[0020] In some embodiments of the method of this disclosure, the percentage of dsRNA to ssRNA is about 0.0% to about 2.5%, about 0.5% to about 2.0%, and about 1.0% to about 1.5%.

[0021] In some embodiments, the percentage change in (d) is less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some embodiments, the percentage change is between about 5% and about 40%, or between about 10% and about 30%, or between about 20% and about 25%.

[0022] In some embodiments of the methods provided herein, the step of measuring the potency of the formulated srRNA composition in (b) is performed in vivo or in vitro. In some embodiments, the step of measuring the potency of the formulated srRNA composition includes detection of RNA replication, detection of viral protein expression, and / or detection of heterologous gene expression. In some embodiments, the step of measuring the potency of the formulated srRNA composition includes immunoblotting analysis, fluorescence flow cytometry analysis, enzyme-linked immunoassay analysis, immunogenicity analysis, bioactivity analysis, and / or efficacy in a disease model. In some embodiments, the step of measuring potency includes evaluating RNA replication efficiency. In some embodiments, evaluating RNA replication efficiency includes a monoclonal antibody. In some embodiments, the monoclonal antibody is J2.

[0023] In some embodiments of the methods provided herein, replication efficiency is determined by measuring the frequency of cells with dsRNA per ng of transfected RNA in an in vitro potency assay. In some embodiments, the step of measuring the potency of the formulated srRNA composition in (b) includes an in vivo potency assay. In some embodiments, the ex vivo potency assay is performed in animal cells. In some embodiments, the ex vivo potency assay is performed in mammalian cells.

[0024] In some embodiments of the methods of the present disclosure, the step of measuring the percentage of full-length srRNA molecules in (c) is by gel electrophoresis or capillary electrophoresis.

[0025] Non-limiting examples of non-viral delivery vehicles include polymeric nanoparticles, or lipid-based nanoparticles (LNPs), liposomes, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, physiological buffers, or any combination thereof. In some embodiments, the LNP comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is from about 100:1 to about 3:1, from about 70:1 to about 10:1, or from about 16:1 to about 4:1.

[0026] In some embodiments of the methods provided herein, the non-viral delivery vehicle includes a physical delivery system, and the srRNA is formulated as "naked" srRNA.

[0027] In some embodiments of the methods of the present disclosure, the selected srRNA delivery system is formulated as an immunogenic formulation. In some embodiments, the selected srRNA delivery system is formulated as a non-immunogenic formulation.

[0028] In some embodiments, the selected srRNA delivery system is formulated as a biological product. In some embodiments, the selected srRNA delivery system is formulated as a vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is a prophylactic vaccine.

[0029] In another embodiment, the Disclosure provides a composition comprising an srRNA delivery system obtained by any one of the methods of the Disclosure.

[0030] In a further embodiment, the Disclosure provides a pharmaceutical composition comprising an srRNA delivery system obtained by any one of the methods of the Disclosure and a pharmaceutically acceptable excipient.

[0031] In yet another embodiment of this disclosure, the Specified provides a method for preventing or treating a health condition in a subject, comprising administering to the subject prophylactically or therapeutically a composition of the Disclosure. In some embodiments, the composition induces a pro-inflammatory or anti-inflammatory response in the subject. In some embodiments, the composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the composition does not induce an inflammatory response and / or anti-inflammatory response in the subject.

[0032] In some embodiments, the composition induces an immune response in the subject. In some embodiments, the composition does not induce an immune response in the subject.

[0033] In yet another embodiment, the Disclosure provides a method for inducing a pharmacodynamic effect in a subject, comprising administering the composition of the Disclosure to the subject prophylactically or therapeutically. In some embodiments, the composition induces an immune response in the subject. In some embodiments, the composition does not induce an immune or pro-inflammatory and / or anti-inflammatory response in the subject.

[0034] The above summary is illustrative and not intended to limit the scope of this invention. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, purposes and features of this disclosure will be fully apparent from the drawings and detailed description and the claims. [Brief explanation of the drawing]

[0035] [Figure 1] This is a graphical representation of an example workflow for screening / identifying / selecting srRNA delivery systems, comprising the steps of: formulating an input srRNA composition with a nonviral delivery vehicle to produce an srRNA delivery system having the formulated srRNA composition; measuring the potency of the formulated srRNA; and selecting the srRNA delivery system as suitable for biomedical applications if the formulated srRNA composition retains at least about 25% of the potency of the unformulated input srRNA composition.

[0036] [Figure 2] This is a graphical representation of an example workflow for screening / identifying / selecting srRNA delivery systems, comprising the steps of: formulating an input srRNA composition with a nonviral delivery vehicle to produce an srRNA delivery system having the formulated srRNA composition; measuring the potency of the formulated srRNA; measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA relative to the amount of full-length srRNA in the input srRNA composition; and selecting the srRNA delivery system as suitable for biomedical applications if the formulated srRNA composition retains at least about 25% of the potency of the unformulated input srRNA composition. The workflow also includes a step of confirming the suitability of the srRNA delivery system if the percentage change in the formulated srRNA composition is less than a 40% decrease relative to the amount of full-length srRNA molecules in the input srRNA composition.

[0037] [Figure 3] A graphic diagram of a non-limiting example of a screening workflow of some embodiments of the method disclosed herein, comprising: (i) measuring the percentage of double-stranded RNA (dsRNA) to single-stranded RNA (ssRNA) in an input srRNA composition (Assay 1); (ii) measuring the potency of formulated srRNA molecules in an srRNA delivery system relative to the potency of srRNA molecules in the input srRNA composition (Assay 2); and (iii) measuring the change in the amount of full-length srRNA molecules relative to full-length srRNA between the formulated srRNA in the input RNA composition and full-length srRNA (Assay 3).

[0038] [Figure 4] This is a graphical representation of a sigmoid curve of the dose-dependent frequency of dsRNA-positive cells after transfection with srRNA, as measured by an in vitro potency assay of an embodiment of the method of the present disclosure. [Modes for carrying out the invention]

[0039] This disclosure relates, in general, to methods for screening, selecting, or identifying self-replicating RNA (srRNA) delivery systems suitable for biomedical applications such as the delivery of vaccines or biological products. These methods enable the efficient identification of suitable delivery systems during the formulation process, addressing the challenge of formulating large, unstable srRNA molecules into effective and potent therapeutic or prophylactic compositions. In particular, the methods for selecting or identifying srRNA delivery systems for biomedical applications provided herein include measuring combinations of quality characteristics (or criteria) of srRNA input compositions before and after formulation with a delivery vehicle to generate suitable delivery systems.

[0040] This disclosure identifies three key quality characteristics (QAs) and the use of various combinations thereof for identifying a (potent and effective) srRNA delivery system as suitable for biomedical applications. These QAs include the integrity of the formulated srRNA, assessed by measuring the change in (1) the amount (e.g., percentage) of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) in the input srRNA composition after in vitro transcription (IVT) of srRNA, and / or (2) the potency of the formulated srRNA composition relative to the unformulated input srRNA composition (e.g., srRNA vector replication efficiency), and / or (3) the change in the amount (e.g., percentage) of full-length srRNA molecules in the formulated srRNA composition relative to the unformulated input srRNA composition. Focusing on these three QAs may enable rapid and reliable screening of many vaccine or drug candidates.

[0041] The first quality characteristic is the amount of dsRNA present relative to ssRNA after in vitro transcription of srRNA, which can be an indicator of the quality and immunogenicity of the input srRNA composition. Enzymatic in vitro transcription (IVT) is commonly used to produce srRNA and can be used to produce the srRNA of this disclosure from linearized DNA templates. In vitro synthesis of transcription RNA, primarily using phage RNA polymerase (RNAP), is robust and well-established in the large-scale production of synthetic RNA. However, the introduction of such synthetic in vitro transcribed mRNA into cell or animal models can produce an immune response to the synthetic molecule, thereby potentially activating cytoplasmic sensors such as RIG-I and MDA5, which can activate the innate immune system in response to viral dsRNA, for example. Such results can be undesirable. Recent studies have identified two major types of byproducts in the IVT reaction that form dsRNA molecules. The first type of dsRNA molecule may be formed by the 3' extension of the run-off product annealing to a complementary sequence within the run-off transcript body either cis (folding back onto the same RNA molecule) or trans (annealing to a second RNA molecule) to form an extended double helix. The second type of dsRNA molecule may be formed by the hybridization of an antisense RNA molecule to the run-off transcript. Antisense RNA molecules have been reported to be formed in a promoter and run-off transcript-independent manner. Accordingly, in accordance with this disclosure, we quantify the percentage of dsRNA in an input srRNA composition, which is a quality characteristic that may be considered when screening srRNA delivery systems for suitability for biomedical applications, whether formulated as a vaccine or a biologic.

[0042] The second quality characteristic is the potency of the formulated srRNA composition relative to the unformulated input srRNA composition (e.g., the replication efficiency of the srRNA vector), which can be measured by an in vitro potency assay. An example of an in vitro potency assay is a transfection assay that evaluates the self-replication capacity of srRNA. In the assay, replication efficiency is measured by capturing the expression of intermediate dsRNA and equivalent proteins in individual cells with an antigen-specific monoclonal antibody, as will be discussed in more detail below. By evaluating the amplification / replication capacity of srRNA in such an in vitro potency assay, it is possible to verify that the srRNA delivery system has the appropriate full-length sequence, RNA capping, and strand integrity.

[0043] The third quality characteristic includes evaluating the integrity of the formulated srRNA by measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition compared to the unformulated input srRNA composition.

[0044] Accordingly, the methods of this disclosure for selecting / identifying srRNA delivery systems may include one or more of the following steps: a) measuring the percentage of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) after in vitro transcription (IVT) of srRNA; b) measuring the potency of a formulated srRNA composition relative to an unformulated input srRNA composition; and c) measuring the percentage change in the amount of full-length srRNA in the formulated srRNA composition relative to the amount of full-length srRNA in the unformulated input srRNA composition. In some embodiments, if the measurement of the percentage of dsRNA in step a) indicates an undesirable amount of dsRNA (as described in more detail below), the synthesis of new srRNA may be repeated, or the srRNA may not be formulated. However, if the amount of dsRNA is within an appropriate range (as also described in detail below), the input srRNA composition is formulated together with a nonviral delivery system. After formulation with a non-viral delivery system and necessary downstream processing, the potency of the formulated srRNA composition and the percentage change in the amount of full-length srRNA molecules in the srRNA composition relative to the amount of full-length srRNA in the unformulated srRNA input composition are measured. The inventors found that the percentage change in potency and the amount of full-length srRNA is a sufficient indicator and a good predictor of the in vivo activity of the formulated srRNA composition.

[0045] As shown in Figure 2 and as described below in detail, an example workflow of the method of the present disclosure includes (a) obtaining an input srRNA composition containing srRNA produced, for example, by an in vitro transcription (IVT) reaction, and (b) measuring the percentage of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) in the input srRNA composition. If the percentage of dsRNA is less than about 2.5%, the remaining steps are carried out: (c) formulating the srRNA with a nonviral delivery vehicle to produce an srRNA delivery system; measuring the replication efficiency of the formulated srRNA relative to the srRNA before formulation (i.e., in the input srRNA composition); and measuring the percentage change in full-length RNA after formulation. In some embodiments, the workflow may include selecting the srRNA delivery system as suitable for biomedical applications if the formulated srRNA composition in (b) retains at least about 25% of the potency of the unformulated srRNA composition.

[0046] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0047] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have meanings that are generally understood by those skilled in the art to which this disclosure pertains. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be construed as being substantially different from those generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly employed using conventional methodologies.

[0048] The singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “a cell” includes one or more cells, including a mixture thereof. “A and / or B” is used herein to include all options of “A,” “B,” “A or B,” and “A and B.”

[0049] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other stated or existing values ​​within that stated range are understood to be included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are included in this disclosure, subject to any limits specifically excluded in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in this disclosure. In this specification, certain ranges are indicated by a number preceded by the term “about,” which, when used herein, has its usual meaning of “approximately.” The term “about” is used to literally support the exact number it precedes, and any number that is close to or approximates the number preceded by the term. When determining whether a number is close to or approximates a specifically enumerated number, an unenumerated number that is close or approximates may be a number that, in the context in which it is presented, provides a substantially equivalent to the specifically enumerated number. Where the degree of approximation is not clear from the context, "approximately" means either within plus or minus 10% of the given value, or rounded to the nearest significant figure, and in all cases includes the given value.

[0050] As used herein, the term “naked” refers to nucleic acids that are substantially free of other macromolecules such as lipids, polymers, and proteins. “Naked” nucleic acids, such as self-replicating RNA, are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated, absorbed, or bound to liposomes, microparticles, nanoparticles, cationic emulsions, etc.

[0051] When the term “recombinant” is used in relation to cells, nucleic acids, proteins, or vectors, it indicates that the cells, nucleic acids, proteins, or vectors have been modified or produced by human intervention, such as by laboratory methods, or are the result of such modifications. Therefore, recombinant proteins and nucleic acids, for example, include proteins and nucleic acids produced by laboratory methods. Recombinant proteins may contain amino acid residues not found in the natural (non-recombinant or wild-type) form of the protein, or may contain modified, for example, labeled amino acid residues. The term can include any modification of a peptide, protein, or nucleic acid sequence. Such modifications include any chemical modification of a peptide, protein, or nucleic acid sequence containing one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the creation of fusion proteins, such as fusion proteins containing antibody fragments; and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. When used in relation to cells, the term "recombinant" is not intended to include naturally occurring cells, but rather to include cells that have been manipulated / modified to contain or express polypeptides or nucleic acids that would not be present in the cell if not manipulated / modified.

[0052] As used herein, the term “self-replicating RNA” refers to RNA containing all the genetic information necessary to induce its own amplification or self-replication within a permissible cell. To induce its own replication, the RNA molecule contains (1) a polymerase, replicase, or other protein that can interact with a viral or host cell-derived protein, nucleic acid, or ribonucleoprotein to catalyze the RNA amplification process, and (2) a cis-acting RNA sequence necessary for the replication and transcription of the RNA encoded in the subgenome replicon. These sequences can, during the replication process, bind to its self-coding protein, or to a non-self-coding cell-derived protein, nucleic acid, or ribonucleoprotein, or to a complex between any of these components. For the purposes of this disclosure, an alphavirus srRNA molecule generally contains elements in the following order: a 5' viral or defective interfering RNA sequence required for cis replication; a sequence encoding a biologically active alphavirus non-structural protein (e.g., nsP1, nsP2, nsP3, and nsP4); a promoter for subgenomic RNA (sgRNA); a 3' viral sequence required for cis replication; and a polyadenylate tract (Poly(A)). Furthermore, the term srRNA generally refers to a positive polarity molecule, or "message" sense, and srRNA may be of a length different from that of all known naturally occurring alphaviruses. In some embodiments of this disclosure, the srRNA does not contain a sequence of at least one structural viral protein, and / or the sequence encoding a structural gene may be replaced with a heterologous sequence. In these examples, if the srRNA is packaged into a recombinant alphavirus particle, it may contain one or more sequences, so-called packaging signals, that work to initiate interaction with the alphavirus structural proteins that cause the particle to form.

[0053] The aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments of “comprising,” “consisting,” and “consisting essentially of.” As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is comprehensive or open-ended and does not exclude additional unlisted elements or steps of a method. As used herein, “consisting of” excludes any elements, steps, or components not specified in the claimed composition or method. As used herein, “consisting essentially of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claimed composition or method. Any enumeration of the term “comprising” herein, particularly in descriptions of the components of a composition or the steps of a method, is understood to include compositions and methods that are essentially composed of, and comprise, the enumerated components or steps.

[0054] The terms “effective amount,” “therapeutic effective amount,” or “pharmaceutical effective amount” of the compositions of this disclosure, such as nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, generally refer to an amount sufficient to achieve a specified purpose (e.g., to achieve an effect to be administered, to stimulate an immune response, to prevent or treat a disease, or to alleviate one or more symptoms of a disease, disorder, infection, or health condition) compared to the absence of the composition. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be called a “therapeutic effective amount.” “Alleviation” of symptoms means a reduction in the severity or frequency of symptoms, or the disappearance of symptoms. The exact amount of a composition containing the "therapeutic dose" varies depending on the therapeutic purpose and can be determined by techniques known to those skilled in the art (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0055] As used herein, the term “pharmaceutically acceptable excipient” refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering the compound of interest to a subject. Thus, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, salines, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are suitable for pharmaceutically effective administration. Auxiliary active compounds (e.g., antibiotics and additional therapeutic agents) may also be incorporated into the composition.

[0056] For clarity, it should be understood that certain features of the Disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if any combination were explicitly disclosed individually. In addition, all subcombinations of various embodiments and their components are also specifically encompassed by the Disclosure and are disclosed herein as if any such subcombination were explicitly disclosed individually.

[0057] The terms “cell,” “cell culture,” and “cell line” refer not only to specific target cells, cell cultures, or cell lines, but also to the offspring or potential offspring of such cells, cell cultures, or cell lines, regardless of the number of transfers or passages during culture. It should be understood that not all offspring are strictly identical to the parent cells. This is because certain modifications may occur in subsequent generations due to either mutation (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that offspring may not actually be identical to the parent cells, but as long as the offspring retain the same function as the original cells, cell cultures, or cell lines, they are still included in the scope of the terms used herein.

[0058] While various features of this disclosure can be described in the context of a single embodiment, these features may be provided separately or in any suitable combination. Conversely, while this disclosure may be described herein in the context of separate embodiments for clarity, it can also be implemented in a single embodiment.

[0059] II. Method of Disclosure A. Methods for screening or identifying srRNA delivery systems As discussed above, some embodiments of this disclosure provide methods for screening, selecting, or identifying srRNA delivery systems for biomedical applications.

[0060] In one embodiment, this specification provides a method for selecting or identifying a self-replicating RNA (srRNA) delivery system for biomedical applications, comprising: a) formulating an input srRNA composition together with a non-viral delivery vehicle to produce an srRNA delivery system having a formulated srRNA composition; b) measuring the potency of the formulated srRNA composition relative to an unformulated input srRNA composition; c) measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition relative to the amount of full-length srRNA molecules in the input srRNA composition; and d) selecting an srRNA delivery system as suitable for biomedical applications if the formulated srRNA composition in (b) retains at least about 25% of the potency of the unformulated srRNA composition.

[0061] In another embodiment, the present disclosure is a method for identifying a self-replicating RNA (srRNA) delivery system for biomedical applications, comprising: (a) measuring the percentage of double-stranded RNA (dsRNA) molecules in an input srRNA composition relative to single-stranded RNA (ssRNA) molecules in an input srRNA composition; (b) formulating the input srRNA composition with a non-viral delivery vehicle to produce an srRNA delivery system containing the formulated srRNA composition; (c) measuring the potency of the formulated srRNA composition relative to an unformulated input srRNA composition; and (d) measuring the amount of full-length srRNA molecules in the formulated srRNA composition. The present invention provides a method comprising: measuring the percentage change in the amount of full-length srRNA molecules in an input srRNA composition; and selecting an srRNA delivery system as suitable for biomedical use if the percentage of dsRNA to ssRNA in (e)(i)(a) is less than approximately 2.5%, the formulated srRNA composition in (ii)(c) retains at least approximately 25% of the potency of the unformulated input srRNA composition, and the percentage change in (iii)(d) is a decrease of less than approximately 40% relative to the amount of full-length srRNA molecules in the unformulated input srRNA composition.

[0062] Non-limiting exemplary embodiments of the disclosed method may include one or more of the following features:

[0063] 1. Input srRNA composition The input srRNA compositions of this disclosure are reference compositions comprising a starting material or a reference material or an unformulated srRNA molecule of interest. In some embodiments, the input srRNA composition to be formulated is the same as the input srRNA composition obtained by an in vitro transcription assay used to produce the srRNA molecule.

[0064] In some embodiments, the srRNA molecule is intended to be used as a vaccine. In some embodiments, the srRNA molecule is intended as a biological agent for delivering a selected payload.

[0065] In some embodiments, srRNA may be transcribed using an in vitro transcription assay, as will be discussed in more detail below. In some embodiments, srRNA may be one or more single-stranded positive-sense RNA (+ssRNA) viral genomes. In some embodiments, srRNA may be derived from an alphavirus. In some embodiments, srRNA may be manipulated or assembled from multiple constructs or multiple genomes. In some embodiments, srRNA may be synthesized. Those skilled in the art will understand that any technique for assembling nucleic acid fragments may be used, for example, by Gibson assembly technique or by ligation or PCR-based procedures such as fusion PCR.

[0066] The input srRNA may be present in any suitable buffer. Non-limiting examples include citrate buffer or other aqueous buffers. In some embodiments, the input srRNA is in 1 mM citrate buffer. In some embodiments, the input srRNA is in 1 mM citrate buffer at pH 6.5.

[0067] 2. Transcription or synthesis of srRNA The methods of this disclosure provide srRNA. srRNA can be transcribed using assays known to those skilled in the art. An example of such assay is, for instance, an enzymatic in vitro transcription assay (IVT) from a linearized DNA template using phage RNA polymerase (RNAP). Such assays typically produce byproducts in the IVT reaction, thereby forming undesirable dsRNA molecules. A first type of dsRNA molecule may be formed by the 3' extension of a run-off product annealing to a complementary sequence within the body of the run-off transcript either cis (folding back onto the same RNA molecule) or trans (annealing to a second RNA molecule) to form an extended double helix. A second type of dsRNA molecule may be formed by the hybridization of an antisense RNA molecule to a run-off transcript. As will be discussed in more detail below, quantifying the percentage of dsRNA in an input srRNA composition is useful in identifying or selecting srRNA delivery systems suitable for biomedical applications. In some embodiments, if the amount of dsRNA is greater than approximately 2.5% of the total RNA (dsRNA and ssRNA) in the input srRNA composition, it is desirable to repeat the srRNA IVT.

[0068] 3. Nonviral delivery vehicle One aspect of the present disclosure relates to a method for selecting / identifying srRNA delivery systems for biomedical applications, comprising the step of formulating an input srRNA composition together with a nonviral delivery vehicle to produce an srRNA delivery system having the formulated srRNA input composition.

[0069] Non-limiting examples of non-viral delivery vehicles that can be used in the methods of this disclosure include, for example, the following publications: Yan Y. et al. (2022). Non-viral vectors for RNA delivery. J Control Release. 2022 Feb;342:241-279; Miron-Barroso S, et al. (2021). Nanotechnology-Based Strategies to Overcome Current Barriers in Gene Delivery. Int J Mol Sci. 2021 Aug 9;22(16):8537; Chaudhary N, Weissman D, Whitehead KA. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021 Nov;20(11):817-838. Erratum in: Nat Rev Drug Discov.; Shuai Q. et al. (2021). mRNA delivery via non-viral carriers for biomedical applications. Int J Pharm.2021 Sep 25;607:121020;Ibba ML.et al.(2021).Advances in mRNA non-viral delivery approaches.Adv Drug Deliv Rev.2021 Oct;177:113930;Tarach P et al.(2021)Recent Advances in Preclinical Research Using PAMAM Dendrimers for Cancer Gene Therapy.Int J Mol Sci.2021 Mar 13;22(6):2912;and Ulkoski D.et al.(2019).Recent advances in polymeric materials for the delivery of RNA therapeutics.Expert Opin Drug Deliv.These are described in 2019 Nov;16(11):1149–1167, and are all incorporated herein by reference in their entirety.

[0070] Suitable delivery vehicles for the methods and compositions of the present disclosure may be any non-viral delivery system that can protect the srRNA payload from degradation and effectively diffuse across the cell membrane of a host cell, for example, by endocytosis. Exemplary suitable delivery vehicles for the methods and compositions of the present disclosure include, but are not limited to, physiological buffers, liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, microspheres, immunostimulatory complexes (ISCOMs), and conjugates of bioactive ligands that can facilitate delivery and / or enhance the immune response. These compounds are readily available to those skilled in the art; see, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes are also used and are known in the art. Adjuvants may protect antigens (e.g., srRNA molecules) from rapid dispersion by sequestering them in local deposits, or adjuvants may include substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. A person skilled in the art can make an appropriate selection from, for example, those listed below.

[0071] In some embodiments, the delivery vehicle may comprise one or more of the following: physiological buffers, liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, microspheres, immunostimulatory complexes (ISCOMs), bioactive ligand conjugates, or any combination thereof.

[0072] Exemplary types of lipids suitable for the delivery vehicles described herein include cationic lipids, ionizable cationic lipids, anionic lipids, neutral lipids, and combinations thereof. Neutral lipids such as the fusion phospholipid DOPE or membrane component cholesterol may be included in LNPs as “helper lipids” to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to low stability and rapid clearance, as well as the occurrence of inflammatory or anti-inflammatory responses. LNPs may also contain hydrophobic lipids, hydrophilic lipids, or lipids that are both hydrophobic and hydrophilic.

[0073] In some embodiments, the LNPs of this disclosure may comprise one or more ionizable lipids. Examples of ionizable lipids suitable for the compositions and methods of this disclosure are those described in PCT International Publication No. 2020252589(A1) and International Publication No. 2021000041(A1), and Love KT et al, Proc Natl Acad Sci USA, Feb. 2, 2010 107(5)1864-1869, which are incorporated herein by reference in their entirety.

[0074] In some embodiments, the LNP of this disclosure comprises one or more lipid compounds described above in Love KT et al, 2010, such as C16-96, C14-110, and C12-200. In some embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of this disclosure comprises C12-200.

[0075] In some embodiments, the LNPs of this disclosure comprise one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In particular, LN16 and MD1. In another example, a certain type of LNP comprises a GalNAc moiety, which is bound to the outside of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of the srRNA of this disclosure to the liver.

[0076] In some embodiments, the LNPs of this disclosure comprise one or more neutral lipids. As described above, neutral lipids, also known as “structural lipids” or “helper lipids,” may also be incorporated into lipid formulations and lipid particles in some embodiments. Lipid formulations and lipid particles may contain one or more structural lipids in about 10 to 40 mol% of the composition. Appropriate structural lipids support the formation of particles during manufacturing. Structural lipids refer to any one of a number of lipid species that exist in anionic, uncharged, or neutral amphoteric forms at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0077] Examples of structural lipids include amphoteric lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane. Examples include -1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0078] In another embodiment, the structural lipid may be any lipid that is negatively charged at physiological pH. Examples of such lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0079] Non-limiting neutral lipids suitable for the compositions and methods of this disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of this disclosure comprise one or more ionizable lipid compounds described in PCT International Publication No. 2020252589(A1) and International Publication No. 2021000041(A1), which are incorporated herein by reference in whole.

[0080] In some embodiments, the LNPs of this disclosure include C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMOLIE-DPyPE, or GL67A-DOPE-DMP-polyethylene glycol (PEG). In some embodiments, LNPs can be produced by combining lipids in any number of molar ratios. Furthermore, LNPs can be produced by combining polynucleotides with lipids in a wide range of molar ratios.

[0081] In some embodiments of the delivery vehicle described herein, which includes LNPs, the mass ratio of lipids to nucleic acids in the LNP delivery vehicle is about 100:1 to about 3:1, about 70:1 to 10:1, or 16:1 to 4:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 8:1. In some embodiments, the lipid-based nanoparticles (LNPs) have an average diameter of about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or less than about 25 nm. In some embodiments, the LNPs have an average diameter in the range of about 70 nm to 100 nm. In some embodiments, the LNP has an average diameter in the range of approximately 88 nm to approximately 92 nm, 82 nm to approximately 86 nm, or approximately 80 nm to approximately 95 nm.

[0082] In some embodiments, LNP refers to any particle having a diameter of 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or less than 25 nm. Alternatively, nanoparticles may be in the size range of 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm.

[0083] Stabilizers may be included in embodiments of lipid formulations to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or facilitate the formation of intermolecular hydrophobic-hydrophilic interactions. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadeca-9-enoate), Myrj52 (polyoxyethylene(40) stearate), and Brij® S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-bound lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0084] In some embodiments, the stabilizer constitutes about 0.1 to 3 mol% of the total lipid mixture. In some embodiments, the stabilizer constitutes about 0.5 to 2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at more than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6 to 10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10 mol% of the lipid mixture.

[0085] Steroids can also be included in lipid compositions for specific uses, and the lipid particles produced therefrom may contain sterols such as cholesterol and plant sterols.

[0086] Polymer delivery vehicle In one embodiment of this disclosure, the delivery vehicle may be a polymer (polymer vector). The polymer or polymer vector may include homopolymers and copolymer vectors. Non-limiting examples of polymer vectors include polyethyleneimine (PEI; linear PEI, branched PEI, pluronic linked PEI, crosslinked PPEI, PEI-lipid hybrids), polyethyleneimine hybrids, poly(acrylate), polyester, poly(β-aminoether), poly(amideamine), poly(aspartamide), polypeptide (poly(amino acid)(PAA)), polyL-lysine (PLL), PLL-stabilized polyriboinosinate (polyICLC), chitosan-based systems, poly(glycomidoamine)( Examples include PGAA, glycopolymers, poly(d-glucaramidoamines), methacrylamide-based glycopolymers, tartaric acid and glucarate-based poly(glycomidoamines), stimulus-responsive polymers, bioreducible derivatives of poly(amidoamines), bioreducible derivatives of PEI, poly(2-dimethylaminoethyl methacrylate), and bioreducible polymers containing poly(L-lysine), pH-responsive polymers containing polyesters and polyacrylates, charge-altering release transporters, pH-responsive gene vectors containing combinations of dimethylaminoethyl methacrylate (DMAEMA), propyl acrylic acid (PPAA), and butyl methacrylate (BMA), charge-altering release transporters (CART), ser-CART, ATP-responsive polymers, high molecular weight amphoteric electrolytes, and protein-based polymers.

[0087] In some embodiments, the input srRNA composition is formulated with poly(amideamine)(PAMAM) dendrimers (Tarach and Janaszewka, 2021). PAMAM dendrimers are repeatedly branched three-dimensional molecules made from amide and amine subunits, possessing unique physicochemical properties.

[0088] In some embodiments, the selected srRNA delivery system is formulated as a biological agent. Non-limiting examples of biological agents include cytokines, chemokines, and other soluble immunomodulators, enzymes, peptides and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors, and gene silencing / editing molecules.

[0089] In some embodiments, the selected srRNA delivery system is formulated as an immunogenic product. In some embodiments, the selected srRNA delivery system is formulated as a non-immunogenic product.

[0090] In some embodiments, the selected srRNA delivery system is formulated as a vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is a prophylactic vaccine.

[0091] In some embodiments, the srRNA input composition of this disclosure can be formulated with a nonviral delivery vehicle (mixing and complex formation, Figure 3) to produce an srRNA delivery system, after which the formulated srRNA composition can be subjected to downstream processing. Non-limiting examples of downstream processing include purification by tangential flow filtration, dialysis, filtration, size exclusion chromatography, and affinity chromatography.

[0092] 4. Measurement of effectiveness During the formulation process of an input srRNA composition, the srRNA may lose its potency. Therefore, this disclosure also provides a method for selecting / identifying an srRNA delivery system for biomedical applications, which includes a step of measuring the potency of a formulated srRNA composition compared to an unformulated input srRNA composition.

[0093] The potency of srRNA can be measured using various methods and / or assays known to those skilled in the art. In some embodiments, the step of measuring the potency of a formulated srRNA composition includes immunoblotting analysis, fluorescence flow cytometry analysis, enzyme-linked immunoassay analysis, immunogenicity analysis, bioactivity analysis, and / or efficacy in a disease model.

[0094] In some embodiments of the methods of this disclosure, the potency of the formulated sRNA composition is measured using an in vitro potency assay that measures replication efficiency by capturing intermediate double-stranded RNA produced during srRNA replication. In some embodiments, measuring replication efficiency includes assaying protein expression in individual cells. The protein expression assay may include using an antibody that detects srRNA replication intermediates. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is J2. J2 is a commercially available anti-dsRNA IgG2a mouse monoclonal antibody from English & Scientific Consulting (Scicons).

[0095] In some embodiments, the in vitro efficacy assay may be as follows: The test srRNA can be diluted and directly electroporated into cells. In some embodiments, if the test srRNA (input srRNA composition) is encapsulated or adsorbed to a non-viral delivery system, the srRNA can be extracted from the delivery system using a surfactant (or other extraction method) before electroporating the srRNA into cells. After sufficient incubation, the cells can be fixed and immunoassayed using a fluorophore-conjugated antibody (J2) that specifically detects the dsRNA replication intermediate of the vector. Signal-positive cells indicate the presence of fully functional srRNA, which can be quantified by fluorescence flow cytometry. The assay readout is the frequency of positive cells per ng of transfected RNA. The dose-response frequency of cells transfected with dsRNA+ can be observed, producing a sigmoid curve (as shown in Figure 3) similar to the standard curve produced by enzyme-linked immunosorbent assays (ELISA), which are widely used for the quantification of other biological molecules. Using srRNA reference standards can reduce variability in cell-based assays and enable comparisons of potency between assays. The srRNA standard may be an aliquot of a large preparation stored at -80°C, and the potency of each test RNA is defined in comparison to the standard.

[0096] In some embodiments, a similar strategy can be used to quantify viral replicon particle (VRP) titers by infecting cells with a series of dilutions of the particles, followed by an immunoassay with a J2 antibody. The potency of the srRNA delivery system of this disclosure can be measured by an in vivo assay. As will be understood by those skilled in the art, an in vivo assay that can be used to measure the potency of the srRNA delivery system may be any assay that evaluates protein expression or immune response in cells after administration of the srRNA delivery system (a formulated synthetic srRNA construct with or without a control containing an unformulated vector), as discussed in Example 4 below. In some embodiments, the potency of the srRNA delivery system of this disclosure is evaluated by measuring the expression of a reporter gene at the injection site in a subject. In some embodiments, the reporter gene is luciferase. In some embodiments, the subject is a mouse. In some embodiments, in vivo imaging of luciferase activity is performed using IVIS instruments at various time points. In some embodiments, secreted proteins such as agonists, antagonists, or monoclonal antibodies can be assayed at systemic protein levels by sequential blood collection, subsequent serum preparation, and ELISA analysis of the expressed proteins.

[0097] In some embodiments, the efficacy of the srRNA delivery system of this disclosure can be evaluated by measuring the immune response (e.g., B cells and T cells) to the encoded antigen in a subject. In some embodiments, the subject is a mouse.

[0098] In some embodiments, the efficacy of the srRNA delivery system of the Disclosure can be evaluated by measuring the encoded agonist / antagonist or monoclonal antibody in the systemic circulation of a subject such as a mouse. In some embodiments, the efficacy of the srRNA delivery system of the Disclosure can be evaluated as detailed in Example 4. In some embodiments, total antigen-specific IgG in response to injection of the srRNA delivery system is measured. In some embodiments, antigen-specific IgG is measured using an ELISA protocol known in the art.

[0099] In some embodiments of the methods disclosed herein, the srRNA delivery system is considered suitable for biomedical applications if the formulated srRNA composition retains at least about 25% of the potency of the unformulated srRNA composition (starting material or srRNA input composition). In some embodiments, the delivery system is considered suitable if the composition retains at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or any value in between, the potency of the unformulated input srRNA composition.

[0100] In some embodiments, the delivery system is considered appropriate if the srRNA composition retains an efficacy of approximately 25% to 100%, approximately 30% to 95%, approximately 35% to 85%, approximately 40% to 80%, approximately 45% to 75%, approximately 50% to 70%, approximately 55% to 65%, or any range in between, relative to the efficacy of the unformulated input srRNA composition.

[0101] In some embodiments, the srRNA delivery system in (d) is selected as suitable for biomedical applications if the formulated srRNA composition in (b) retains at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the potency of the unformulated input srRNA composition.

[0102] 5. Measurement of changes in full-length srRNA This disclosure provides a method for selecting / identifying srRNA delivery systems for biomedical applications, comprising the step of measuring the percentage change in the amount of full-length molecules in a formulated srRNA composition relative to the amount of full-length srRNA molecules in an input srRNA composition (Assay 3 in Figure 2).

[0103] The amount of full-length srRNA can be measured using various methods and / or assays known to those skilled in the art. The step of measuring the percentage change in the amount of full-length srRNA molecules may include assaying the degradation of full-length srRNA molecules during srRNA formulation.

[0104] In some embodiments, the step of measuring the percentage change in the amount of full-length srRNA molecules can be performed by capillary electrophoresis, which enables the quantification of full-length srRNA. Capillary electrophoresis is well known to those skilled in the art as an alternative to conventional slab electrophoresis for the separation of nucleic acid fragments.

[0105] Capillary electrophoresis (CE) separates and identifies labeled nucleic acid fragments by passing them through polymer-filled capillaries. The higher electric field applied in CE enables faster and higher-throughput separation. This process allows for the analysis of single bases with very small sample volumes.

[0106] In some embodiments, the delivery system is selected as suitable for biomedical applications if the percentage reduction in the amount of full-length srRNA molecules in the formulated srRNA composition is less than about 40% relative to the amount of full-length srRNA molecules in the input srRNA composition. In some embodiments, the percentage reduction relative to the amount of full-length srRNA molecules in the input srRNA composition is less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than about 10%, less than about 5%, or any value in between.

[0107] 6. Measurement of the percentage of double-stranded RNA molecules relative to single-stranded RNA molecules. This disclosure provides a method for selecting / identifying srRNA delivery systems for biomedical applications, comprising the step of measuring the amount (e.g., as a percentage) of double-stranded RNA (dsRNA) molecules relative to single-stranded RNA (ssRNA) molecules (Assay 1 in Figure 2).

[0108] The amount of dsRNA relative to ssRNA can be measured by methods known to those skilled in the art. In some embodiments, the step of measuring the amount of dsRNA relative to ssRNA may be performed by an immunoblotting assay (Assay 1 in Figure 2). Immunoblotting assays are used to detect the presence of unwanted dsRNA molecules in synthesized mRNA preparations. Based on the use of dsRNA-specific monoclonal antibodies, such assays enable highly sensitive and selective detection of dsRNA molecules, regardless of their nucleotide composition and sequence. Some commercially available kits are highly specific, as they can detect dsRNA in nucleic acid extracts even in the presence of other nucleic acids in excess 1,000 to 10,000 times. Immunoblotting assays function on the principle of sandwich ELISA, as well as a monoclonal antibody against dsRNA as a catcher antibody and another antibody as a detection antibody, enabling detection and / or characterization of dsRNA by MAB even in the presence of large excesses of other nucleic acids (e.g., Schonborn et al. 1991, which is incorporated herein by reference in its entirety).

[0109] In some embodiments, the percentage of dsRNA to ssRNA in the input srRNA composition may be less than about 2.5%, measured using a dsRNA immunoblot assay. In some embodiments, the percentage of dsRNA to ssRNA may be less than about 2.4%, less than about 2.3%, less than about 2.2%, less than about 2.1%, less than about 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or any value in between.

[0110] In some embodiments, the percentage of dsRNA to ssRNA may be in the range of approximately 0.0% to approximately 2.5%, approximately 0.1% to approximately 2.4%, approximately 0.2% to approximately 2.3%, approximately 0.3% to approximately 2.2%, approximately 0.4% to approximately 2.1%, approximately 0.5% to approximately 2.0%, approximately 0.6% to approximately 1.9%, approximately 0.7% to approximately 1.8%, approximately 0.8% to approximately 1.7%, approximately 0.9% to approximately 1.6%, approximately 1.0% to approximately 1.5%, approximately 1.1% to approximately 1.4%, approximately 1.2% to approximately 1.3%, or any range in between, as determined using a dsRNA immunoblot assay.

[0111] In some embodiments, the percentage of dsRNA to ssRNA in the input srRNA composition is measured after in vitro transcription / generation of the srRNA and before its formulation. In some embodiments, if the amount of dsRNA is outside the range described above, a new srRNA composition may be generated.

[0112] B. Methods for preventing or treating health conditions in the subject. This disclosure provides a method for preventing or treating a health condition in a subject, comprising administering to the subject, prophylactically or therapeutically, an effective amount of a composition comprising an srRNA delivery system obtained by the method of this disclosure.

[0113] The Disclosure also provides a method for inducing a pharmacodynamic effect in a subject, which includes administering the composition of the Disclosure to the subject prophylactically or therapeutically.

[0114] The methods described herein include, for example, glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile diabetes, Reiter's disease, Behçet's disease, immune complex type nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, lupus erythema It may be useful for the treatment and / or prevention of immune, autoimmune, or inflammatory diseases such as todes, rheumatoid arthritis (RA), ankylosing spondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, small vessel vasculitis, Omen syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpesvirus-related diseases, human viral infections, coronaviruses, other enteroviruses, herpesviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV) or adenovirus infections, bacterial pneumonia, wounds, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C. Non-limited examples of inflammatory diseases include asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.

[0115] In some embodiments of methods for preventing or treating a health condition in a subject, the health condition is a proliferative disorder or microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection) or cancer. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection).

[0116] In some embodiments of methods for preventing or treating a health condition in a subject, the health condition is, for example, a disease or condition affecting fewer than 200,000 people in the United States and a rare disease as defined by the Orphan Drug Act (www.fda.gov / patients / rare-diseases-fda), and / or inflammation and / or autoimmune disorders. In some embodiments, the subject has or is suspected of having a condition associated with inflammation and / or autoimmune disorders and / or rare diseases (including, but not limited to, familial Mediterranean fever, adult Still's disease, rheumatoid arthritis, and osteoarthritis). In some embodiments, the health condition is a proliferative disorder (e.g., cancer) and a chronic infection (e.g., a viral infection).

[0117] As discussed above, a therapeutically effective dose includes an amount of the therapeutic composition sufficient to promote a specific effect when administered to a subject such as an individual who has, is suspected of having, or is at risk of having, a health condition such as a disease or infection. In some embodiments, the effective dose includes an amount sufficient to prevent or delay the onset of symptoms of a disease or infection, alter the course of symptoms of a disease or infection (for example, but not limited to slowing the progression of symptoms of a disease or infection), or reverse the symptoms of a disease or infection. In any case, it is understood that an appropriate effective dose can be determined by a person skilled in the art using routine experiments.

[0118] The effectiveness of treatment can be determined by a skilled clinician. However, treatment is considered effective if at least one or all of the signs or symptoms of the disease or infection improve or go into remission. Effectiveness can also be measured by whether the symptoms of the individual, as assessed by the need for hospitalization or medical intervention, do not worsen (e.g., the progression of the disease stops or at least slows down). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes all treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals), and includes (1) suppressing the disease or infection, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease or infection, e.g., causing regression of symptoms; and (3) preventing the onset of symptoms or reducing the likelihood thereof.

[0119] The general methods described herein are for illustrative purposes only. Other alternative methods and substitutes will be apparent to those skilled in the art upon consideration of this disclosure and are included in the spirit and scope of this application.

[0120] III. Composition delivery system This disclosure provides, in particular, compositions comprising an srRNA delivery system, wherein the srRNA delivery system is obtained by a method of this disclosure suitable for biomedical applications.

[0121] The term “delivery system” is used herein to describe a system that is produced after an input srRNA composition has been formulated with any of the delivery vehicles described herein, thereby producing a delivery system containing the formulated srRNA composition.

[0122] Therefore, the srRNA delivery system of this disclosure comprises the srRNA molecule of interest and a delivery vehicle. Examples of delivery vehicles are described in detail above.

[0123] In some embodiments, the delivery system is considered suitable for biomedical applications if it can ultimately deliver the target payload to the individual that needs it. The delivery system may be suitable for biomedical applications if it can deliver a potent and effective amount of the desired payload to the target cell.

[0124] A delivery system can be suitable for a vaccine if it can enable, for example, an encoded antigen-specific immune response, correction of inflammation, expression of the encoded protein within target cells, systemic circulation in an organism, or expression of the encoded protein in a host cell or target.

[0125] The delivery system may be suitable for the biological agent being delivered, depending on the biological agent being delivered. For example, a suitable delivery system may be one that is well tolerated by the host cell or organism (e.g., the adverse events it produces are at an acceptable level), or one that induces inflammation or does not induce inflammation.

[0126] The srRNA delivery systems of this disclosure can be formulated for the delivery of target payloads. Non-limiting examples of payloads include small molecules, peptides, proteins, and nucleic acids, including small activated RNA (saRNA), long non-coding RNA (lncRNA), antisense oligonucleotides (ASOs), small interfering molecules (siRNA), microRNA, and DNA.

[0127] srRNA delivery systems can be formulated as biological agents. Examples of biological agents include, but are not limited to, recombinant proteins, hormones, monoclonal antibodies, cytokines, growth factors, gene therapy products, vaccines (e.g., therapeutic or prophylactic), cell-based products, stem cell therapies, gene silencing / editing therapies, and tissue engineering products.

[0128] In some embodiments, the srRNAs of this disclosure can be formulated as prophylactic or therapeutic compositions. For example, the compositions of this disclosure can be formulated to improve, prevent, treat, or manage health conditions such as immune disorders or microbial infections.

[0129] Pharmaceutical composition This specification provides pharmaceutical compositions comprising an srRNA delivery system obtained by the method of this disclosure and a pharmaceutically acceptable excipient (e.g., a carrier).

[0130] In some embodiments, the compositions of the Disclosure are formulated together with a delivery vehicle. The delivery vehicle may be a nonviral delivery vehicle. In some embodiments, the delivery vehicle is in lipid-based nanoparticles (LNPs). Examples of LNPs are discussed in more detail above. In some embodiments, the srRNA of the Disclosure is formulated in polymer nanoparticles. The srRNA of the Disclosure may also be used in a naked form. In some embodiments, the srRNA is formulated in liposomes. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0131] In some embodiments, srRNA is formulated as a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine.

[0132] In some embodiments, the pharmaceutical composition of this disclosure is an immunogenic composition, for example, a composition that can stimulate an immune response in a subject. In some embodiments, the pharmaceutical composition does not induce an immune response in a subject.

[0133] In some embodiments, the composition is substantially non-immunogenic to the subject, for example, a composition that minimally stimulates the immune response in the subject. In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biological agent.

[0134] In some embodiments, the composition induces a pro-inflammatory or anti-inflammatory response in the subject. In some embodiments, the composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the composition does not induce an inflammatory response and / or anti-inflammatory response in the subject.

[0135] In some embodiments, the disclosed pharmaceutical composition is formulated to suit its intended route of administration. In some embodiments, the pharmaceutical composition is formulated for one or more of the following: intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, oral administration, and parenteral administration.

[0136] Examples of parenteral administration routes include, for example, intravenous, intranodal, intradermal, intratumoral, intraarticular, subcutaneous, transdermal (topical), transmucosal, vaginal, and rectal administration. Solutions or suspensions used for parenteral administration may contain the following components: sterile diluents such as sterile water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (for example, to about 7.2 to 7.8, e.g., pH 7.5). Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0137] Suitable pharmaceutical compositions for injection include sterile aqueous solutions or dispersions, and sterile powders for the preparation of sterile injection solutions or dispersions immediately before use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, New Jersey), or phosphate-buffered saline (PBS). In these cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, and / or sodium chloride are generally contained in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0138] Sterile injection solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components from those listed above.

[0139] In some embodiments, the pharmaceutical compositions of the Disclosure can be administered to a subject in an effective amount to stimulate an immune response in a composition having a pharmaceutically acceptable carrier. Generally, the subject can be immunized by an initial series of injections (or administration via one of the other routes described below), and then a booster may be administered to enhance the defense obtained by the original series of administrations. The initial series of injections and the subsequent booster are administered in doses and for a duration necessary to stimulate an immune response in the subject. In some embodiments, the administered composition increases interferon production in the subject. In some embodiments of the methods disclosed, the subject is a mammal. In some embodiments, the mammal is a human.

[0140] Suitable pharmaceutically acceptable carriers for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the preparation of sterile injection solutions or dispersions on demand. In these cases, the composition must be sterile and fluid enough to be easily injected. The composition must also be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Microbial activity can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.

[0141] When pharmaceutical compositions are adequately protected as described above, they can be administered orally, for example, with an inert diluent or an absorbable food carrier. Pharmaceutical compositions and other components may also be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into an individual's diet. For oral therapeutic administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers.

[0142] In some embodiments, the srRNAs of this disclosure can be delivered to cells or targets by lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many humans have pre-existing immunity to viral particles, but not to LNPs. Furthermore, adaptive immune responses to LNPs are unlikely to occur, which allows for repeated administration of LNPs. LNPs are discussed above.

[0143] The dosage, toxicity, and therapeutic efficacy of the pharmaceutical compositions disclosed herein are, for example, determined by the LD50 (lethal dose for 50% of the population) and ED50. 50 The therapeutic index (LD) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the dose that is therapeutically effective in 50% of the population. The dose ratio between toxicity and therapeutic effect is the therapeutic index. 50 / ED 50 This can be expressed as a ratio. Compounds exhibiting a high therapeutic index are generally appropriate. Compounds exhibiting toxic side effects may be used, but care must be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0144] For example, data obtained from cell culture assays and animal studies can be used when formulating dosage ranges for human use. Dosages of such compounds are generally given to EDs that have little to no toxicity.50 The concentration is within the range of circulating concentrations in the body. The dose can be varied within this range depending on the dosage form and route of administration used. For all compounds used in the methods disclosed herein, the therapeutically effective dose can first be estimated from a cell culture assay. The dose is measured in the IC50 in cell culture. 50 Animal models can be formulated to obtain a range of circulating plasma concentrations, including, for example, the concentration of the test compound that achieves maximum half-dose inhibition of symptoms. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0145] The pharmaceutical compositions described herein may be administered at least once per week, including at least once per day and at least once every other day. Those skilled in the art will understand that the dosage and timing required to effectively treat a subject may be influenced by certain factors, including but not limited to the severity of the disease, previous treatments, the subject's overall health and / or age, and other pre-existing diseases. Furthermore, treatment of a subject with a therapeutically effective amount of the polyvalent polypeptides and polyvalent antibodies of this disclosure may be a single treatment or a series of treatments. In some embodiments, the compositions are administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, for example, 9 days, followed by another 5 days of administration every 8 hours. With respect to nucleic acid constructs, the therapeutically effective amount (e.g., effective dose) of the nucleic acid construct depends on the nucleic acid construct selected. For example, a single dose in the range of about 0.001 to 0.1 mg / kg patient body weight may be administered. In some embodiments, doses of about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, a single dose ranging from approximately 0.03 μg to 300 μg / kg patient body weight may be administered. In some embodiments, a single dose ranging from approximately 0.3 mg to 3 mg / kg patient body weight may be administered.

[0146] In some embodiments, the pharmaceutical composition is incorporated into a therapeutic composition for use in a method of preventing or treating a subject who has, is suspected of having, or is at high risk of developing a microbial infection. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the microbial infection is a fungal infection. In some embodiments, the microbial infection is a viral infection.

[0147] In some embodiments, the compositions of the Disclosure are administered individually to a target as a single therapy (monotherapy) or as a primary therapy in combination with at least one additional therapy (e.g., a second-line therapy). In some embodiments, the second-line therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second-line therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the primary and second-line therapies are administered synchronously. In some embodiments, the primary therapy is administered simultaneously with the second-line therapy. In some embodiments, the primary and second-line therapies are administered sequentially. In some embodiments, the primary therapy is administered before the second-line therapy. In some embodiments, the primary therapy is administered after the second-line therapy. In some embodiments, the primary therapy is administered before and / or after the second-line therapy. In some embodiments, the primary and second-line therapies are administered alternately. In some embodiments, the primary and second-line therapies are administered together in a single formulation.

[0148] For clarity, it is understood that certain features of the Disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if any combination were explicitly disclosed individually. In addition, all subcombinations of various embodiments and their components are also specifically encompassed by the Disclosure and are disclosed herein as if any such subcombination were explicitly disclosed individually.

[0149] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. All patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0150] In carrying out the present invention, unless otherwise indicated, the prior art of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which is known to those skilled in the art, will be used. Such techniques are described in Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as "Sambrook" in this specification); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements up to 2014); Bollag, D. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. Get et al. (1995). Viral Vectors: Gene Therapy. and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: the Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferre, F. & Gibbs, R. (1994).This topic is fully explained in literature such as PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. Let al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley (including supplements up to 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, and their disclosures are incorporated herein by reference. Further embodiments are disclosed in more detail in the following embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of this disclosure or the claims.

[0151] Example 1: In vitro transfer (IVT) RNA samples are prepared by in vitro transcription (IVT) using a plasmid DNA template linearized by enzymatic digestion. In these examples, the DNA is linearized either by NotI, which cleaves downstream of the T7 terminator, or by SapI, which cleaves at the poly(A) end. In vitro transcription is performed using bacteriophage T7 polymerase, either with a 5' ARCA cap (HiScribe® T7 ARCA mRNA kit, NEB) or by adding a 5' cap 1 after uncapped transcription (HiScribe® T7 High Yield RNA Synthesis Kit, NEB) (Vaccinia Capping System, mRNA Cap 2'-0-Methyltransferase, NEB). The RNA product is then purified using phenol / chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB). The RNA concentration in the RNA sample is measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific). These RNA samples are used as input RNA compositions (e.g., srRNA compositions) in the assays described below.

[0152] Example 2: Immunoblot assay The amount of dsRNA in an input srRNA composition can be measured by immunoblotting assay. Immunoblotting assays are used to detect the presence of unwanted dsRNA molecules in synthesized mRNA preparations, based on the use of dsRNA-specific monoclonal antibodies. Some commercially available kits offer highly specific detection, as they can detect dsRNA in nucleic acid extracts even when other nucleic acids are present in 1,000 to 10,000-fold excess. Generally, immunoblotting assays are based on the sandwich ELISA principle, where a monoclonal antibody against dsRNA acts as a catcher antibody, and another antibody acts as the detection antibody.

[0153] Example 3: In vitro efficacy assay The replicating vector is detected using an in vitro efficacy assay that measures replication efficiency by capturing the expression of intermediate dsRNA and equivalent proteins in individual cells with the antigen-specific monoclonal antibody J2. The test srRNA is diluted and electroporated directly into cells. If the test srRNA (input srRNA composition) is encapsulated or adsorbed to a non-viral delivery system, the srRNA is extracted from the delivery system using a surfactant (or other extraction method) before electroporating the srRNA into cells. After sufficient incubation, the cells are fixed and immunoassayed using a fluorophore-conjugated antibody (J2) that specifically detects the dsRNA replication intermediate of the vector. Signal-positive cells indicate the presence of fully functional srRNA, which can be quantified by fluorescence flow cytometry. The assay readout is the frequency of positive cells per ng of transfected RNA. The dose-response frequency of cells transfected with dsRNA+ can be observed, generating a sigmoid curve similar to the standard curve produced by enzyme-linked immunosorbent assays (ELISA), which are widely used for the quantification of other biological molecules as shown below. Using an srRNA reference standard can reduce the variability of cell-based assays and allow for comparisons of potency between assays. The srRNA standard may be aliquots of large preparations stored at -80°C, and the potency of each test RNA is defined in comparison to the standard.

[0154] In some embodiments, a similar strategy can be used to quantify viral replicon particle (VRP) titers, by serially diluting the particles to infect cells and then performing an immunoassay using a J2 antibody.

[0155] Example 4: In vitro efficacy assay This embodiment describes in vivo experiments that can be performed to evaluate protein expression or immune response after administration using synthetic srRNA constructs (e.g., both unformulated and formulated vectors).

[0156] These experiments involve designing and then evaluating synthetic srRNA constructs.

[0157] Mice and injection. Female C57BL / 6 or BALB / c mice were purchased from Charles River Laboratories or Jackson Laboratory. On the day of administration, 0.01–40 μg of material was intramuscularly injected into one or both quadriceps femoris muscles. The vector was administered either unformulated in saline, or formulated with LNP or polymer. Animals were monitored for body weight and other general observations throughout the course of the study.

[0158] Immunogenicity testing. For immunogenicity testing, the drug was administered to animals on day 0 and on days 14, 21, 35, 42, or 56. For ELISPOT or ICS analysis, the spleen was collected 14 days after the second dose, and serum was isolated for antibody testing on day 14 and 14 days after the second dose.

[0159] ELISpot. To measure the magnitude of the antigen-specific T cell response, ELISpot analysis of IFNγ was performed using the Mouse IFNγELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions for use. Briefly, splenocytes were isolated and placed in a medium containing 1–5 × 10⁶ peptides representing one of the T cell epitopes for the encoded protein, PMA / ionomycin as a positive control, or DMSO as a pseudo-stimulant. 6 The cells were resuspended at a concentration of cells / mL.

[0160] Intracellular cytokine staining (ICS). To measure the magnitude and quality of the antigen-specific T cell response, intracellular cytokines produced as a result of T cell stimulation by an antigen are measured by immunostaining. Briefly, splenocytes are isolated and 1-5 × 10¹⁶ cells are placed in a medium containing a peptide representing one of the T cell epitopes for the encoded protein, PMA / ionomycin as a positive control, or DMSO as a pseudo-stimulant. 6The cells were resuspended at a concentration of cells / mL. After activation ex vivo for 1 hour, a Golgi inhibitor was added to the culture to capture cytokines within the cells. Subsequently, standard immunohistochemistry protocols were used to identify CD4+ and CD8+ T cells and measure cytokine expression and co-expression in the samples. The representative major cytokines of activated T cells are IFNγ and / or IL-2 and / or TNF. The immunohistochemically stained samples were analyzed using a standard flow cytometer.

[0161] Antibodies. Antibody responses to measure all antigen-specific IgG are measured using a previously published ELISA protocol.

[0162] Protein expression. In protein expression studies, the protein is administered to animals on day 0, and protein expression or bioluminescence is evaluated on days 1, 3, 7, and 10, etc., until the signal disappears. If a reporter protein such as luciferase is encoded, in vivo imaging of luciferase activity is performed using IVIS instruments at the indicated time. For secreted proteins such as agonists, antagonists, or monoclonal antibodies, systemic protein levels can be assayed by serial blood collection, followed by serum preparation and ELISA analysis of the expressed protein.

[0163] Example 5: Capillary electrophoresis This assay is used to measure the percentage of full-length RNA in a formulated srRNA composition, i.e., the integrity of the srRNA after formulation. This is done by measuring the amount or percentage of full-length RNA molecules in the srRNA formulation using capillary electrophoresis (SCIEX-PA 800 Plus Pharmaceutical Analysis System). Capillary electrophoresis allows for the quantification of full-length srRNA after formulation.

[0164] Example 6: Protein Expression This assay measures the expression of proteins, such as proteins expressed from srRNA. BHK-21 or Vero cells (e.g., 4D-Nucleofector®, Lonza) are transformed with RNA by electroporation. After 18-20 hours post-transformation, the cells are fixed and permeabilized (eBioscience® Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), and stained with APC conjugate anti-HA mouse monoclonal antibody (2B7, Abcam). The frequency of HA protein+ cells and the mean fluorescence intensity (MFI) of HA protein in individual cells are quantified by fluorescence flow cytometry.

[0165] Example 7: Replication This assay evaluates viral replication efficiency by measuring the frequency of cells containing dsRNA per ng of transfected RNA. BHK-21 or Vero cells (e.g., 4D-Nucleofector®, Lonza) are transformed with srRNA by electroporation. 17–20 hours after transformation, the cells are fixed and permeabilized (eBioscience® Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), stained with PE conjugate anti-dsRNA mouse monoclonal antibody (J2, Scicons), and the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells are quantified by fluorescence flow cytometry.

[0166] While certain alternatives to those described herein are disclosed, various modifications and combinations are possible and intended to be construed within the precise intent and scope of the attached claims. Therefore, there is no intention to limit ourselves to the exact abstract and disclosures presented herein.

Claims

1. A method for selecting / identifying self-replicating RNA (srRNA) delivery systems for biomedical applications, a) A step of compounding an input srRNA composition together with a non-viral delivery vehicle to produce an srRNA delivery system containing the compounded srRNA composition, b) A step of measuring the efficacy of the formulated srRNA composition against an unformulated input srRNA composition, c) A step of measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition relative to the amount of full-length srRNA molecules in the input srRNA composition, d) A method comprising the step of selecting the srRNA delivery system as suitable for biomedical use if the formulated srRNA composition in (b) retains at least about 25% of the potency of the unformulated input srRNA composition.

2. The method according to claim 1, wherein the formulated srRNA composition in (b) retains at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of the potency of the unformulated input srRNA composition, the srRNA delivery system in (d) is selected as suitable for the biomedical application.

3. The method according to claim 1 or 2, wherein the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition in (c) is a decrease of less than about 40% relative to the amount of full-length srRNA molecules in the input srRNA composition.

4. The method according to claim 3, wherein the percentage reduction in (c) is less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than 5% of the amount of full-length srRNA molecules in the input srRNA composition.

5. The method according to any one of claims 1 to 4, wherein the input srRNA composition comprises a double-stranded RNA (dsRNA) molecule and a single-stranded RNA (ssRNA) molecule.

6. The method according to claim 5, further comprising the step of measuring the percentage of double-stranded RNA (dsRNA) relative to single-stranded RNA (ssRNA) in the input srRNA composition.

7. The method according to claim 6, wherein the step of measuring the percentage of double-stranded RNA (dsRNA) to single-stranded RNA (ssRNA) in the input srRNA composition is performed by an immunoblot assay.

8. The method according to claim 6 or claim 7, wherein the percentage of dsRNA relative to ssRNA is less than approximately 2.5%, less than approximately 2.0%, less than approximately 1.5%, less than approximately 1.0%, less than approximately 0.5%, or less than approximately 0.25%.

9. The method according to any one of claims 1 to 8, wherein the step of measuring the efficacy of the formulated srRNA composition in (b) is performed in vivo or in vitro.

10. The method according to claim 9, wherein the step of measuring the efficacy of the formulated srRNA composition includes detection of RNA replication, detection of viral protein expression, and / or detection of heterologous gene expression.

11. The method according to claim 9 or 10, wherein the step of measuring the efficacy of the formulated srRNA composition includes immunoblotting analysis, fluorescence flow cytometry analysis, enzyme-linked immunoassay analysis, immunogenicity analysis, bioactivity analysis, and / or efficacy in a disease model.

12. The method according to claim 10 or 11, wherein the step of measuring the efficacy includes evaluating the RNA replication efficiency.

13. The method according to claim 12, wherein the RNA replication efficiency is evaluated by a monoclonal antibody.

14. The method according to claim 13, wherein the monoclonal antibody is J2.

15. The method according to claim 12, wherein the replication efficiency is determined by measuring the frequency of cells having dsRNA per ng of transfected RNA in an in vitro efficacy assay.

16. The method according to claims 1 to 12, wherein the step of measuring the efficacy of the formulated srRNA composition in (b) comprises an in vivo efficacy assay.

17. The method according to claim 16, wherein the in vivo efficacy assay is performed in animal cells.

18. The method according to claim 16, wherein the in vivo efficacy assay is performed in mammalian cells.

19. The method according to any one of claims 1 to 18, wherein the step of measuring the percentage of full-length srRNA molecules in (c) includes assaying the degradation of full-length srRNA molecules during the formulation process in (a).

20. The method according to any one of claims 1 to 19, wherein the step of measuring the percentage of full-length srRNA molecules in (c) comprises gel electrophoresis and / or capillary electrophoresis.

21. The method according to any one of claims 1 to 20, wherein the nonviral delivery vehicle comprises polymer nanoparticles, or lipid-based nanoparticles (LNPs), liposomes, microspheres, immunostimulatory complexes (ISCOMs), bioactive ligand conjugates, physiological buffers, or any combination thereof.

22. The method according to claim 21, wherein the LNP includes a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

23. The method according to claim 22, wherein the mass ratio of lipids to nucleic acids in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to about 10:1, or about 16:1 to about 4:

1.

24. The method according to any one of claims 1 to 20, wherein the nonviral delivery vehicle comprises a physical delivery system, and the srRNA is formulated as "naked" srRNA.

25. The method according to any one of claims 1 to 24, wherein the selected srRNA delivery system is formulated as an immunogenic preparation.

26. The method according to any one of claims 1 to 24, wherein the selected srRNA delivery system is formulated as a non-immunogenic preparation.

27. The method according to any one of claims 1 to 26, wherein the selected srRNA delivery system is formulated as a biological product.

28. The method according to any one of claims 1 to 26, wherein the selected srRNA delivery system is formulated as a vaccine.

29. The method according to claim 28, wherein the vaccine is a therapeutic vaccine.

30. The method according to claim 28, wherein the vaccine is a preventive vaccine.

31. A method for identifying self-replicating RNA (srRNA) delivery systems for biomedical applications, (a) A step of measuring the percentage of double-stranded RNA (dsRNA) molecules in the input srRNA composition relative to single-stranded RNA (ssRNA) molecules in the input srRNA composition, (b) A step of compounding an input srRNA composition together with a nonviral delivery vehicle to produce an srRNA delivery system containing the compounded srRNA composition, (c) A step of measuring the efficacy of the formulated srRNA composition against an unformulated input srRNA composition, (d) A step of measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA composition relative to the amount of full-length srRNA molecules in the input srRNA composition, A method comprising the step of selecting an srRNA delivery system as suitable for biomedical use if the percentage of dsRNA relative to ssRNA in (e)(i)(a) is less than about 2.5%, the formulated srRNA composition in (ii)(c) retains at least about 25% of the potency of the unformulated input srRNA composition, and the percentage change in (iii)(d) is a decrease of less than about 40% relative to the amount of full-length srRNA molecules in the unformulated input srRNA composition.

32. The method according to claim 31, wherein the step of measuring the percentage of dsRNA to ssRNA in (b) comprises an immunoblotting assay, the step of measuring the efficacy comprises an in vitro efficacy assay, and the step of measuring the percentage change in the amount of full-length srRNA molecules in the formulated srRNA relative to the amount of full-length srRNA molecules in the input srRNA composition in (d) comprises capillary electrophoresis.

33. The method according to claim 31 or 32, wherein the percentage of dsRNA relative to ssRNA is approximately 0.0% to approximately 2.5%, approximately 0.5% to approximately 2.0%, and approximately 1.0% to approximately 1.5%.

34. The method according to any one of claims 31 to 33, wherein the percentage change in (d) is less than approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, or less than approximately 5%.

35. The method according to any one of claims 31 to 33, wherein the percentage change is approximately 5% to approximately 40%, or approximately 10% to approximately 30%, or approximately 20% to approximately 25%.

36. The method according to any one of claims 31 to 35, wherein the step of measuring the efficacy of the formulated srRNA composition in (b) is performed in vivo or in vitro.

37. The method according to claim 36, wherein the step of measuring the efficacy of the formulated srRNA composition includes detection of RNA replication, detection of viral protein expression, and / or detection of heterologous gene expression.

38. The method according to claim 36 or 37, wherein the step of measuring the efficacy of the formulated srRNA composition includes immunoblotting analysis, fluorescence flow cytometry analysis, enzyme-linked immunoassay analysis, immunogenicity analysis, bioactivity analysis, and / or efficacy in a disease model.

39. The method according to any one of claims 36 to 38, wherein the step of measuring efficacy includes evaluating RNA replication efficiency.

40. The method according to claim 39, wherein the RNA replication efficiency is evaluated by a monoclonal antibody.

41. The method according to claim 40, wherein the monoclonal antibody is J2.

42. The method according to claim 41, wherein the replication efficiency is determined by measuring the frequency of cells having dsRNA per ng of transfected RNA in an in vitro efficacy assay.

43. The method according to any one of claims 32 to 42, wherein the step of measuring the potency of the formulated srRNA composition in (b) includes an in vivo potency assay.

44. The method according to claim 37, wherein the ex vivo efficacy assay is performed in animal cells.

45. The method according to claim 44, wherein the ex vivo efficacy assay is performed in mammalian cells.

46. The method according to any one of claims 32 to 36, wherein the step of measuring the percentage of full-length srRNA molecules in (c) is performed by gel electrophoresis or capillary electrophoresis.

47. The method according to any one of claims 32 to 36, wherein the non-viral delivery vehicle comprises polymer nanoparticles or lipid-based nanoparticles (LNPs).

48. The method according to claim 47, wherein the LNP includes a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

49. The method according to claim 48, wherein the mass ratio of lipids to nucleic acids in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to about 10:1, or about 16:1 to about 4:

1.

50. The method according to any one of claims 31 to 46, wherein the nonviral delivery vehicle comprises a physical delivery system and the srRNA is formulated as "naked" srRNA.

51. The method according to any one of claims 31 to 50, wherein the selected srRNA delivery system is formulated as an immunogenic preparation.

52. The method according to any one of claims 31 to 50, wherein the selected srRNA delivery system is formulated as a non-immunogenic preparation.

53. The method according to any one of claims 31 to 52, wherein the selected srRNA delivery system is formulated as a biological product.

54. The method according to any one of claims 31 to 52, wherein the selected srRNA delivery system is formulated as a vaccine.

55. The method according to claim 54, wherein the vaccine is a therapeutic vaccine.

56. The method according to claim 54, wherein the vaccine is a preventive vaccine.

57. A composition comprising an srRNA delivery system obtained by the method described in any one of claims 1 to 56.

58. A pharmaceutical composition comprising an srRNA delivery system obtained by the method of any one of claims 1 to 56, and a pharmaceutically acceptable excipient.

59. A method for preventing and / or treating a health condition in a subject, comprising administering to the subject prophylactically or therapeutically the pharmaceutical composition described in claim 58.

60. The method according to claim 59, wherein the composition induces a pro-inflammatory or anti-inflammatory response in the subject.

61. The method according to claim 60, wherein the composition induces the production of one or more pro-inflammatory molecules in the subject.

62. The method according to claim 59, wherein the composition does not induce an inflammatory response and / or an anti-inflammatory response in the subject.

63. The method according to claim 59, wherein the composition induces an immune response in the subject.

64. The method according to claim 59, wherein the composition does not induce an immune response in the subject.

65. A method for inducing a pharmacodynamic effect in a subject, comprising administering the pharmaceutical composition described in claim 58 to the subject prophylactically or therapeutically.

66. The method according to claim 65, wherein the composition induces an immune response in the subject.

67. The method according to claim 64, wherein the composition does not induce an immune response or a pro-inflammatory response and / or an anti-inflammatory response in the subject.

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