Self-amplifying RNA constructs

Abstract

The present invention relates in general to a platform to selectively eliminate unwanted cells from a population of cells. The invention relates to self-amplifying RNA constructs for cell purification preferably during differentiation and cell engineering.

Description

[0001] SELF-AMPLIFYING RNA CONSTRUCTS

[0002] The present invention relates in general to a platform to selectively eliminate unwanted cells from a population of cells. The invention relates to self-amplifying RNA constructs for cell purification preferably during differentiation and cell engineering.

[0003] Background

[0004] Self-amplifying mRNAs (saRNAs) are a class of RNA molecules that can replicate semi-autonomously within a host cell, enabling prolonged expression of a target gene without the need for repeated administration. Derived from RNA viruses, such as alphaviruses (e.g., Semliki Forest virus, Sindbis virus, Venezuelan Equine Encephalitis virus, Eastern Equine Encephalitis virus, Chikungunya Virus), flaviviruses (Kunjin Virus, West Nile Virus), or other RNA virus families, saRNAs contain viral replication machinery that allows them to replicate their RNA sequence. The elements of a self-amplifying RNA include viral non-structural proteins (often abbreviated as nsP1-4 or NS1-5) and untranslated regions (UTRs) that regulate replication and translation. Alphavirus based saRNAs also contain a sub-genomic promoter (SGP).

[0005] In comparison to conventional messenger RNAs (mRNAs), which undergo rapid degradation and have a limited expression window, self-amplifying RNAs significantly boost gene expression by producing multiple copies of the RNA within the cell. This feature makes saRNAs highly effective in applications requiring sustained protein production, such as gene therapy, vaccine development, and cellular reprogramming. Furthermore, saRNAs are non-integrative and can degrade naturally, reducing the risk of genomic integration or long-term side effects. Their ability to induce high levels of protein expression in a transient manner provides a versatile tool for genetic manipulation in both research and therapeutic settings.

[0006] MicroRNAs (miRNAs) are small, non-coding RNA molecules (18-25 base pairs) that play a crucial role in regulating gene expression by binding to complementary sequences on target messenger RNAs (mRNAs), typically resulting in their degradation or translational repression. Each miRNA can target multiple mRNAs, and their expression patterns are highly cell type-specific, reflecting the physiological state of the cell. miRNAs are pivotal in controlling processes such as differentiation, proliferation, and apoptosis, and they are often uniquely expressed in specific cell types or during certain stages of development.

[0007] Due to their specificity, miRNAs have been extensively studied as biomarkers for identifying cell types and cellular states. The specify of miRNAs has led to the development of "RNA switches" that use miRNA binding sites to control the expression of therapeutic or reporter genes in a cell type-dependent manner. In such systems, miRNA target sequences are incorporated into the 3' untranslated region (UTR), 5’ UTR or other non-coding elements (e.g. introns) of an mRNA, enabling cell-specific regulation. If a particular miRNA is abundant in a cell type, it will bind to the introduced RNA switch, leading to suppression of gene expression in those cells. Conversely, in cells where the miRNA is absent or at low levels, the mRNA is translated, allowing selective gene expression. However most miRNA based RNA switches developed so far are limited by the short half-life of mRNA and so have limited applicability.

[0008] Stem cell-based regenerative therapies hold immense potential for treating a wide range of diseases by enabling the generation of specific cell types that can replace damaged or diseased tissues. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are among the most promising cell sources for these therapies due to their capacity to differentiate into various cell types. However, one of the major challenges associated with stem cell therapies is the production of unwanted or undifferentiated cells during the manufacturing process. These cells can compromise the safety and efficacy of the therapy, as they may reduce therapeutic impact or, in the worst case, form tumours due to uncontrolled growth.

[0009] Current methods for purifying desired cell populations from unwanted or undifferentiated cells face several limitations. Techniques such as cell sorting rely on surface markers, which may not always be expressed in the target cells, making the process inefficient and potentially incomplete. Additionally, the scalability of these techniques is often limited, creating bottlenecks in the production of therapeutic cells. The need for more robust and scalable solutions to remove these unwanted cells is crucial for the continued advancement and safety of stem cell therapies.

[0010] Summary of the invention

[0011] The following discussion is not intended to be limiting but merely to help illustrate general principles of the invention and certain embodiments. The present invention is, at least in part, based on a purification platform comprising self-amplifying RNAs (saRNAs) combined with microRNA responsive elements (MREs) for selective elimination of unwanted cells from a population of cells.

[0012] In a first aspect of the invention, there is provided a self-amplifying RNA (saRNA) molecule comprising:

[0013] (a) a region coding for a viral non-structural protein (nsP);

[0014] (b) a conserved sequence element (CSE);

[0015] (c) an untranslated region (UTR); and

[0016] (d) a polyadenyl sequence wherein the saRNA molecule further comprises a synthetic microRNA (miRNA) responsive element (sMRE); and wherein the saRNA molecule further comprises a transgene. The saRNA molecule may additionally comprise a 5’-cap located upstream of the region coding for one or more viral non-structural protein (nsPs). The 5’-cap may be m7G, optionally m7G(5')ppp(5')(2'OMeA)pG.

[0017] The saRNA molecule may comprise a 5’-UTR or a 3 -UTR, optionally it may comprise both a 5’ UTR and a 3’ UTR. The 5’ UTR may be located upstream of the region coding for one or more viral non- structural protein (nsPs), and downstream of the 5’-cap if present. The 3’ UTR may be located upstream of the conserved sequence element (CSE), and polyadenyl sequence.

[0018] The sMRE may comprise a plurality of miRNA binding sites, for example from 1 to 20 miRNA binding sites, of from 1 to 10 binding sites, suitably, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 miRNA binding sites. The sMRE may comprise tandem miRNA binding sites. Example constructs with multiple miRNA binding sites, include but are not limited to, PLU-R-147 with 2 miRNA binding sites and PLU091 with 4 miRNA binding sites (see Table 1). An example construct with one miRNA binding site, includes but is not limited to, PLU-R-128 (see Table 1).

[0019] Each sMRE may independently be the same or different. Each miRNA binding site may independently be the same or different. Although a single miRNA-binding site can be sufficient to mediate degradation of saRNA-sMRE constructs, the present invention also provides for a combination of unique sMREs within a single construct. For example, two or more distinct sMREs may work synergistically to induce stronger repression / degradation than an individual sMRE alone.

[0020] The sMRE may be positioned on the negative (antisense) strand, effectively targeting the negative saRNA genome strand. An example construct with an sMRE positioned on the negative strand, includes but is not limited to, PLU-R-110 (see Table 1).

[0021] The saRNA molecule may comprise a plurality of sMREs, for example of from 1 to 100 sMREs, suitably, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 sMREs. Example constructs with two or more distinct sMREs, include but are not limited to, PLU-R-1 15 with 2 different sMRE, PLU-R-321 and PLU-R-335 with 3 different sMREs, and PLU-R-315 with 4 different sMREs (see Table 1).

[0022] The saRNA molecule may comprise therefore a plurality of locations for the sMREs in the saRNA molecule, for example 1 , 2, 3, 4, or 5 locations. In one example, the saRNA may comprise 3 locations for sMREs with each location comprising from 1 to 20 miRNA binding sites. The miRNA binding sites can also be present in groups of miRNA binding sites which may therefore comprise different miRNA binding sites, for example at one sMRE there may be a plurality of different groups of miRNA binding sites, e.g., 1 , 2, 3, 4, or 5 different types of miRNA binding sites. Example constructs with multiple locations for sMREs, include but are not limited to, PLU-R-211 with sMRE locations upstream and downstream of the subgenomic promoter (SGP), PLU-R-130 with sMRE locations in the 5’UTR, downstream of the subgenomic promoter, and in the 3’ UTR (see Table 1).

[0023] The saRNA molecule may comprise a plurality of sMREs and / or the sMREs may comprise a plurality of miRNA binding sites. The miRNA binding sites may target distinct miRNA, wherein a single miRNA binding site is specific to a miRNA present in a specific cell type and / or wherein a combination of miRNA binding sites are specific to miRNA present in a specific cell type.

[0024] The microRNA (miRNA) may suitably comprise from 15 to about 30 nucleotides, optionally from 18 to about 25 nucleotides.

[0025] Secondary structural motifs may be incorporated into saRNAs such that sMREs are stem-embedded or loop-embedded, thereby modulating miRNA-binding accessibility and regulatory activity. Example constructs, include but are not limited to, PLU-R-091 , PLU-R-092 and PLU-R-093 (see Table 1).

[0026] The conserved sequence element (CSE) comprises a 5’ conserved sequence element (CSE) and / or a 3’ conserved sequence element (CSE).

[0027] The self-amplifying RNA (saRNA) molecule of the invention may comprise a subgenomic promoter (SGP). The SGP may be located in the saRNA molecule between the nsPs and the CSE. The SGP may be located in the saRNA molecule away from the nsP coding sequence (i.e. decoupled or repositioned). This architectural change separates the functions of the nsP coding region and the SGP, creating flexibility for modular insertion of regulatory elements. Example constructs in which the SGP is located away from the nsP coding sequence, include but are not limited to, PLU-R-162, PLU-R-211 , PLU-R-315 and PLU-R-223 in which the SGP is decoupled from the nsP (see Table 1). The SGP may be the 26S subgenomic promoter (SGP).

[0028] The non-structural protein may suitably be a viral non-structural protein. The non-structural protein (nsP) may be an alphavirus non-structural protein, suitably an alphavirus non-structural protein selected from the group consisting of non-structural protein 1 (nsp1), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and non-structural protein 4 (nsp4), or a combination thereof. The non-structural protein (nsP) may comprise alphavirus nsp1 , nsp2, nsp3 and nsp4. The non-structural protein (nsP) may alternatively be a flavivirus non-structural protein, suitably a flavivirus non-structural protein selected from the group consistding of non-structural protein 1 (NS1), non-structural protein 2A (NS2A), non- structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), non- structural protein 4B (NS4B), and non-structural protein 5 (NS5), or a combination thereof.

[0029] The self-amplifying RNA (saRNA) molecule of the invention may further comprise one or more RNA regulatory elements selected from the group consisting of an internal ribosome entry sequence (IRES), a ribozyme (e.g. Hammerhead, Hepatitis Delta Virus (HDV) ribozymes), a Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE), an AU-rich element (ARE), and an RNAse (e.g. RNAse P) recognition sequence and splicing (donor and acceptor) site. The self-amplifying RNA (saRNA) molecule of the invention may further comprise one or more IRES. Example constructs in which one or more RNA regulatory elements are present, include but are not limited to, PLU-R-247 in which five additional ARE are present, PLU-R-248 in which ten additional ARE are present, and PLU-R-249 in which fifteen additional ARE are present (see Table 1).

[0030] The sMRE may be fully complementary to the miRNA sequence. A reduced level of complementarity (i.e. partial complementarity) is also possible within the scope of the invention, provided that the required level of function of the saRNA molecule is preserved. An example construct in which the sMRE is partially complementary to the miRNA sequence includes but is not limited to PLU-R-152 (see Table 1).

[0031] The self-amplifying RNA (saRNA) molecule may further comprises at least one of element selected from the group consisting of a subgenomic promoter (SGP), a 5’-cap, a 5’-UTR, and a 3’-UTR or a combination thereof. The saRNA molecule may further comprise a SGP, a 5’-cap, a 5’-UTR, and a 3’- UTR. The sMRE may be located at the 5’ UTR, SGP, 3’ UTR or coding sequences (nsP, transgenes) of the saRNA molecule. The sMRE may be located at various different positions within the one or more 5’UTR, one or more SGP, one or more 3’UTR and / or one or more coding sequences (nsP, transgenes) of the saRNA molecule. Example constructs with multiple locations for sMREs, include but are not limited to, PLU-R-21 1 with sMRE locations upstream of the subgenomic promoter (SGP) and in the 3’ UTR, PLU-R-130 with sMRE locations in the 5’UTR, downstream of the subgenomic promoter, and in the 3’ UTR (see Table 1).

[0032] The polyadenyl (polyA) sequence may comprises a sequence of up to 400 nucleotides, up to 250 nucleotides, up to 200 nucleotides, suitably up to 120 nucleotides. Generally suitably polyadenyl sequences may be of around 15 to 60 nucleotides in length, optionally 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42 nucleotides. The polyadenyl sequence may be composed of repeating adenine nucleobases or it may be based on repeating units of the sequence AAUAAA.

[0033] The transgene may be a selection marker selected from the group consisting of a drug resistance gene, a suicide gene, a transcription factor, inhibitor, an immunomodulator, a cell surface marker and / or a fluorescent protein.

[0034] In the self-amplifying RNA (saRNA) molecule of the invention, the drug resistance gene may be an antibiotic resistance gene selected from the group consisting of puromycin N-acetyl-transferase for puromycin resistance (PuroR), an aminoglycoside 3'-phosphotransferase for geneticin resistance (NeoR), Sh ble (from Streptoalloteichus hindustanus) for zeocin resistance (BleoR), hygromycin B phosphotransferase for hygromycin resistance (HygR, for example HygBR), dihydrofolate reductase for methotrexate resistance, and blasticidin S deaminase for blasticidin resistance (BSR), or a combination thereof. The drug resistance gene may be an antibiotic resistance gene, such as puromycin N-acetyl- transferase for puromycin resistance (PuroR) and Sh ble (from Streptoalloteichus hindustanus) for bleomycin, phleomycin and zeocin resistance (BleoR).

[0035] The suicide gene may be selected from the group consisting of Herpes simplex virus-thymidine kinase (HSV-TK), nitroreductase (NTR), cytosine deaminase (CD), carboxypeptidase G2 (CPG2), purine nucleoside phosphorylase (PNP), Granzyme, Caspase-9 (Casp9), Diphtheria Toxin A (DT-A), Fas Ligand (FasL), Ribonuclease (e.g. Barnase), Bcl-2-associated X protein (Bax) and TNF-Related Apoptosis-Inducing Ligand (TRAIL), or a combination thereof.

[0036] The immunomodulator may be selected from the group consisting of E3L, B18R, and Adenovirus VA RNA, or a combination thereof.

[0037] The fluorescent protein may be any generally convenient protein that can be caused to fluoresce such as for example a protein selected from the group consisting of enhanced green fluorescent protein (EGFP), GFP, mCherry, iRFP670, and LSSmApple. Other fluorescent proteins may be used equally.

[0038] The transcription factor may be selected from the group consisting of ASCL1 , POU3F2 (BRN2), MYT1 L, NEUROD1 , GATA4, MEF2C, TBX5, HAND2, FOXA2, HNF4A, PDX1 , NEUROG3 (NGN3), MAFA, MY0D1 , ETV2, FOXC2, FLI1 , CEBPA, PPARG, SOX10, OLIG2, NFIA, LIN28A, SALL4, LMX1A, PITX3, NR4A2 (NURR1), NKX2-5, ISL1 , SOX17, GFI1 , POU4F2 (BRN3B), ATOH1 , GATA6, NKX6-1 , FOXP3, H0XA9, MEIS1 , TAL1 , RUNX1 , CDX2, EOMES, TCF7L2, ESRRB, NR5A2, LHX3, LHX6, PAX6, PAX7, SOX9, PROX1 , TCF21 , ARNTL (BMAL1), NEUROG2 (NGN2), HNF1 B, H0XA5, PAX3, EMX2, LHX2, PITX2, PAX4, SOX11 , IRX3, ISL2, FOXD3, MSX1 , PHOX2B, SP8, GATA3, TCF7, BCL1 1 B, NOTCH1 , IKZF1 , LEF1 , RXR, STAT5, FOXP1 , ID2, MAFB, ZBTB16 (PLZF), GATA1 , BAF60C, JMJD3, NKX2-2, RFX6, HNF1A, ARX, HES1 , GLIS3, ONECUT1 , SPI1 (PU.1), C / EBPp, KLF1 , BCL2, MYOG, HHEX, GATA2, GFI1 B, SNAI2 (Slug), NANOG, SOX7, HNF6, CITED2, HAND1 , HOXB4, ZEB1 , TCF4, HES5, SIX2, IRF8, MNX1 (HB9), HOXC8, HOXC9, PHOX2A, LMX1 B, EN1 , OTX2, TH, FOXA1 , TP63, PAX2, MITF, NRL, SIX3, CEBPG, NKX2-1 (TTF1), RUNX2, SPDEF, TBX20, and NKX3-1.

[0039] The transcription factor may be a lineage-driving transcription factor. The transcription factor may be a transcription factor that is used to direct cells toward a particular fate (e.g. differentiation). The transcription factor may drive cells towards the mesoderm lineage. The transcription factor may drive cells towards the endoderm lineage. The transcription factor may drive cells towards the ectoderm lineage. For example, the transcription factor may be human NGN2 for neuronal induction, see for example construct PLU-R-195 of Table 1 or the transcription factor may be MyoD1 for myogenic induction, see for example constructs PLU-R-321 and PLU-R-335 of Table 1. saRNA comprising lineage-driving transcription factors, in combination with a selection marker transgene, such as a drug resistance gene or a suicide gene, and sMRE complementary to miRNAs present in a specific lineage / cell type enables simultaneous induction and purification of desired cells / lineages.

[0040] There may be multiple (e.g. two or more, optionally 3, 4, or 5) transgenes present in a particular saRNA molecule. For example, an saRNA molecule may include multiple antibiotic resistance genes such as puromycin N-acetyl-transferase for puromycin resistance (PuroR) and Sh ble (from Streptoalloteichus hindustanus) for bleomycin, phleomycin, and zeocin resistance (BleoR). Example constructs in which multiple antibiotic resistance genes are present include but are not limited to, PLU-R-188 and PLU-R- 253 in which PuroR and BleoR are present (see Table 1). For example, an saRNA molecule may include a transcription factor used to direct cells towards a particular fate (e.g. differentiation) and an antibiotic resistance gene, such as puromycin N-acetyl-transferase for puromycin resistance (PuroR). Example constructs, include but are not limited to, PLU-R-195 comprising PuroR and Neurogenin-2 (NGN2) for neuronal induction (see Table 1). For example, an saRNA molecule may include an immunomodulator, a fluorescent protein and a drug resistance gene. Example constructs, include but are not limited to, PLU-R-185 comprising immunomodulator E3L, fluorescent protein enhanced green fluorescent protein (EGFP) and drug resistance gene PuroR (see Table 1). For example, an saRNA molecule may include a transcription factor, a suicide gene, an immunomodulator and a drug resistance gene. Example constructs, include but are not limited to, PLU-R-335 comprising transcription factor hMYOD for myogenic induction, suicide gene herpes simplex virus thymidine kinase (HSV-TK), immunomodulator E3L and drug resistance gene PuroR (see Table 1). Any combination of transgenes is permitted.

[0041] The saRNA may further comprise a sensitising modification. For example, a sensitising modification may be present in the coding region for the non-structural protein (nsP), the subgenomic promoter (SGP), the untranslated regions (UTR) and / or the conserved sequence element (CSE).

[0042] An example of a sensitising modification includes modification of secondary structures. Such secondary structure modification may be achieved, for instance, by replacing the wild-type stem-loop 1 (SL1) with an alternative loop that is smaller and weaker (PLU-R-160), or by introducing a structure that is larger and more complex than the wild-type SL1 (PLU-R-155). The chosen modification may also alter the folding or stability of other stem loops (e.g. stem-loops 2, 3, or 4), thereby further modulating replication efficiency and RNA structural dynamics (PLU-D-157). For example, the secondary structure of the UTR (e.g. 5’UTR) or CSE can effect saRNA replication, stability and transcript persistence. Additionally, these modifications may include sMREs present in single-stranded loop regions and / or double-stranded stem regions. The RNA secondary structure can affect the accessibility of sMREs within saRNA constructs and therefore influences miRNA-dependent regulation.

[0043] Another example of a sensitising modification includes incorporation of one or more RNA regulatory / stability elements, selected from the group consisting of a ribozyme (e.g. Hammerhead, Hepatitis Delta Virus (HDV) ribozymes), an AU-rich element (AREs), and an RNAse (e.g. RNAse P) recognition sequence and splicing (donor and acceptor) site.

[0044] Another example of a sensitising modification may be incorporation of a combination of sMRE specific to a particular cell type. In other words, the combination of distinct sMREs. For example, sMRE-A may be present in a first and second cell type, whereas the combination of sMRE-A and sMRE-B is only present in the first cell type. Such a combination of distinct sMREs may produce synergistic / additive degradation across specific cell types and states.

[0045] The RNA regulatory / stability elements, such as AU-rich elements (AREs) can modulate transcript persistence and expression balance. One or more AREs may be present, for example, 5, 10 or 15 AREs. The AREs may be present in tandem. The AREs may be positioned downstream of the transgene. Example constructs in which one or more AREs are present, include but are not limited to, PLU-R-247 in which five additional ARE are present, PLU-R-248 in which ten additional ARE are present, and PLU- R-249 in which fifteen additional ARE are present (see Table 1).

[0046] The saRNA further may comprise one or more post-translational regulatory elements - such as a degron (e.g. AID system), an ubiquitination signal (e.g. PEST), a phosphorylation site, a SUMOylation site, a protease cleavage site (e.g. TEV), a destabilization domain (e.g. DHFR and FKBP12 F36V), a ribozyme skipping 2A peptide, or a combination thereof. Other coding sequences may be present also that modulate protein stability, localization, or activity after translation. Suitably such sequences may be incorporated into the non-structural protein (nsP) and / or transgene.

[0047] Reference to a 2A peptide includes reference to a 2A-like peptide. The 2A peptide may have the consensus sequence GDVEXNPGP and be of from 18 to 22 amino acids in length (Liu et al Sci. Reports, 7, 2193 (2017)). Exemplary sequences are as follows:

[0048] EGRGSLLTCGDVEENPGP (SEQ ID NO: 1) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 2) QCTNYALLKLAGDVESNPGP (SEQ ID NO: 3) VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 4)

[0049] A linker sequence may be included also at the N-terminal of the 2A peptide also, for example, the linker may have the sequence GSG.

[0050] The self-amplifying RNA (saRNA) molecule may be encapsulated in a lipid nanoparticle (LNP), suitable LNP formulations may be generally composed of four components (i) cholesterol; (ii) a polyethylene glycol (PEG) lipid (e.g., DMG-PEG(2000)); (iii) a phospholipid (e.g., 1 ,2-distearoyl-sn-glycero-3-PC); and (iv) a cationic or ionisable lipid (e.g., SM-102) mixture). Alternatively, saRNA can be formulated in Cationic Lipid Formulation (which may include Lipofectamine 2000 and Lipofectamine MessengerMax®), or as an aqueous solution (e.g., in nuclease-free water, Tris-EDTA (TE) Buffer, EB Buffer).

[0051] The self-amplifying RNA (saRNA) molecule may be delivered into cells using a lipoplex-based transfection or nucleofection. The saRNA may also be delivered into cells by transduction. Any generally suitable means of contacting cells with a saRNA such that it can deliver its effects in cells may be used.

[0052] As will be apparent the saRNA molecule of the invention may comprise further sequences. In one embodiment, the saRNA may therefore comprise the following elements:

[0053] (a) a region coding for a viral non-structural protein (nsP);

[0054] (b) a subgenomic promoter (SGP);

[0055] (c) a conserved sequence element (CSE);

[0056] (d) an untranslated region (UTR); and

[0057] (e) a polyadenyl sequence wherein the saRNA molecule further comprises one or more synthetic microRNA (miRNA) responsive elements (sMREs); and wherein the saRNA molecule further comprises a transgene downstream of the SGP.

[0058] In one embodiment, the saRNA may comprise the following elements:

[0059] (a) a 5’ cap;

[0060] (b) a 5 -UTR;

[0061] (c) a region coding for a viral non-structural protein (nsP);

[0062] (d) a subgenomic promoter (SGP);

[0063] (e) a conserved sequence element (CSE);

[0064] (f) a 3’UTR; and

[0065] (g) a polyadenyl sequence; wherein the saRNA further comprises a synthetic microRNA (miRNA) responsive element (sMREs) and wherein the saRNA further comprises a transgene downstream of the SGP.

[0066] In these embodiments, the components of the saRNA molecule may be as described above for the first aspect.

[0067] To address the limitation in the prior art, the inventors have developed an innovative platform leveraging self-amplifying RNA technology combined with microRNA (miRNA) targeting as defined herein. The platform is designed to selectively eliminate unwanted cells from mixed populations by exploiting the unique miRNA profiles of different cell types. By constructing a "smart molecule" using self-replicating RNA, which incorporates custom miRNA binding sites specific to the unwanted cells, the method provides that only those cells expressing the corresponding miRNAs will lose the protective effect conferred by resistance to a selection stimulus (for example a drug resistance gene) delivered by the molecule. In such an example, exposure to the selection stimulus (for example the drug) can be employed to efficiently remove the unwanted cells, leaving behind a purified population of desired cells (i.e. a negative selection). The invention therefore relates to microRNA-directed selective saRNA suppression and its use in enriching cell populations.

[0068] The saRNA molecules of the present invention have considerable design flexibility, such as:

[0069] • Incorporation of sensitising modifications which can be employed to lowerthe effective threshold of miRNA responsiveness, enabling functional selectivity even at considerably low endogenous miRNA levels.

[0070] • Incorporation of antisense oriented sMREs (effectively targeting the negative saRNA genome strand) which confer functional miRNA-dependent regulation.

[0071] • Decoupling of the SGP from the nSP coding regions to enable independent tuning of replication and transgene expression, permitting upstream placement of sMREs that confine miRNA targeting to genomic RNA, thereby enhancing modularity and control within the saRNA framework

[0072] • Engineering 5’UTR secondary structures to modulate replication output and stability.

[0073] • Incorporation of secondary structural motifs within sMREs to modulate miRNA-binding accessibility and regulatory activity, providing an additional means to adjust system responsiveness.

[0074] Such design flexibility was not known or appreciated prior to the present invention.

[0075] According to a second aspect of the invention there is provided a method of altering the proportion of a first cell type within a population of cells relative to the proportion of a second cell type in the population of cells, where the population of cells is differentiated from a population of undifferentiated cells, the method comprising:

[0076] (a) delivering a self-amplifying RNA (saRNA) molecule into the population of undifferentiated cells, wherein the saRNA comprises a transgene encoding a selection marker selected from the group consisting of a drug resistance gene, a suicide gene, a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein, wherein each saRNA construct comprises a synthetic microRNA (miRNA) responsive element (sMRE) complementary to a miRNA present in the cells of the second cell type in the population;

[0077] (b) causing the undifferentiated cells to differentiate into the population of cells; (c) allowing the sMRE to hybridize to the miRNA and thereby for the saRNA to be degraded in the cells of the second cell type in the population; and

[0078] (d) applying a selection stimulus, whereby after step (d) the proportion of the first cell type in the population of cells is altered relative to the proportion of the second cell type in the population of cells.

[0079] The invention provides for simultaneous induction and selective purification using saRNA-sMRE constructs encoding cell-specific and / or lineage-defining transcription factors and selection mechanisms.

[0080] In this aspect, the undifferentiated population of cells may be a population of induced pluripotent stem (iPS) cells (iPSCs), pluripotent stem (PS) cells or embryonic stem cells (ESC). The undifferentiated cells may include progenitor cells. The population of cells after step (b) may be a heterogenous population of cells comprising undifferentiated and differentiated cells, in which the differentiated cells may be of different cell types. Alternatively, the population of cells after step (b) may be a heterogenous population of differentiated cells of different cell types.

[0081] The Examples of the present invention demonstrate lineage-specific elimination of differentiating iPSCs, across all three germ layers, ectoderm, endoderm and mesoderm.

[0082] Ectodermal cells form two major sub-types of cells, the epidermal ectoderm and the neural ectoderm. Ectodermal cells will produce, included but not limited to, the central nervous system (brain and spinal cord), the neural crest (thereby giving rise to, for example, melanocytes, Schwann cells, enteric nervous system, dorsal root / autonomic ganglia, adrenal medulla, craniofacial cartilage and bone, dentin, cementum, odontoblasts, parts of the dermis in the head and neck, the outflow tract of the heart), the peripheral nervous system, the sensory epithelia of the eye, ear and nose; the epidermis and its appendages (nail and hair), mammary glands, subcutaneous glands and enamel of the teeth.

[0083] Endodermal cells will produce, included but not limited to, lungs, the epithelial lining of the gastrointestinal and respiratory tracts, bladder, urethra, gallbladder, bile ducts, thymus, thyroid, parathyroid, the digestive system and its associated organs such as the pancreas and liver.

[0084] Mesodermal cells will produce, included but not limited to, connective tissue, cartilage, bone, striated and smooth muscle, the heart walls, blood and lymph vessels and associated cells, such as hematopoietic stem and progenitor cells and their derivatives such as myeloid (e.g. monocytes, dendritic cells, megakaryocytes, erythroid cells) and lymphoid cells (T-, B-, NK-Cells), kidney, gonads (ovaries and testes), spleen , microglia and suprarenal (adrenal) cortices. By demonstrating applicability across all three germ layers, the methods of the present invention can be applied to all cell types. The undifferentiated cells may be differentiated into the population of cells by inclusion of a transgene encoding a transcription factor which drives differentiate into a specific cell type / lineage. The transcription factor may drive cells towards the mesoderm lineage. The transcription factor may drive cells towards the endoderm lineage. The transcription factor may drive cells towards the ectoderm lineage. For example, the transcription factor may be human NGN2 for neuronal induction, see for example construct PLU-R-195 of Table 1 or the transcription factor may be MyoD1 for myogenic induction, see for example constructs PLU-R-321 and PLU-R-335 of Table 1. The undifferentiated cells may be differentiated into the population of cells by transfecting the cells with one or more transcription factors and / or culturing the cells under conditions such that the cells are differentiated into a differentiated population of cells.

[0085] The undifferentiated cells may be differentiated into the population of cells by culturing the cells under specific culture conditions comprising one or more growth factors, cytokines, or small molecules which promote differentiation into a specific cell type or lineage. Essentially any method of differentiation is compatible.

[0086] According to a third aspect of the invention, there is provided a method of altering the proportion of a first cell type within a population of cells relative to the proportion of a second cell type in the population of cells, the method comprising:

[0087] (a) delivering a self-amplifying RNA (saRNA) molecule into the population of cells, wherein the saRNA molecule comprises a transgene encoding a selection marker selected from the group consisting of a drug resistance gene, a suicide gene, a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein, wherein each saRNA construct comprises a synthetic microRNA (miRNA) responsive element (sMRE) complementary to a miRNA present in the second cell type;

[0088] (b) allowing the sMRE to hybridize to the miRNA and thereby for the saRNA to be degraded in the cells of the second cell type in the population; and

[0089] (c) applying a selection stimulus whereby after step (c) the proportion of the first cell type in the population of cells is altered relative to the proportion of the second cell type in the population of cells.

[0090] The population of cells may be a heterogenous population of cells comprising undifferentiated and differentiated cells, in which the differentiated cells may be of different cell types. Alternatively, the population of cells may be a heterogenous population of differentiated cells of different cell types.

[0091] In the methods of the second and third aspects of the present invention, the alteration in the level of the first cell type relative to the second cell type may therefore be an increase or a decrease depending on the selection marker used. The first cell type and second cell type may each independently represent a subpopulation of cells (which may be the same or different in terms of the cell lineage and / or phenotype of the cells in the subpopulation) within the population of cells.

[0092] In the methods of the invention, the cell type which is being preferentially enriched for is a desired cell type (the other cell type being an undesired cell type). As will be apparent, depending on the selection stimulus either the first cell type or the second cell type will be enriched for in the performance of the methods of the invention.

[0093] The classification of a cell as a desired cell or as an unwanted cell may be carried out using generally available techniques to identify cells in a population, e.g., using cell surface marker proteins, gene expression profiles etc and / or with reference to cell phenotypes. For example, it may be desired to remove or reduce the proportion of undifferentiated cells in a population of cells after a differentiation protocol has been followed. Alternatively, it may be desired to enrich a population of differentiated cells for cells of a particular lineage, for example if the desired cell type is a cardiomyocyte in a mixed population of differentiated cells including the cardiomyocyte, the invention can be used to enrich the population of cells to increase the relative amount of the cardiomyocyte cells present compared to the other cell types present.

[0094] The alteration in the relative level of the first cell type to the second cell type may result in the elimination of the second cell type from the population, i.e. the percentage of cells of the second cell type remaining is 0%. However, the method of the invention may provide for an enriched population of cells of the first cell type with respect to the second cell type, i.e. the percentage of cells of the first type in the population is greater than 50%, or 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% with respect the cells of the second type in the population of cells. The above definitions therefore apply equally where the relative level of the second cell type is increased compared to the first cell type mutatis mutandis.

[0095] Expressed as a ratio, the elimination of cells of the second type means that the ratio of cells of the first type of the cells of the second type is 100:0. However, in the case where the relative proportion of cells of the first cell type is enriched in the population of cells compared to the second type the ratio may be expressed as greater than 50:50, or 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1 , or 99.5:0.5. The above definitions therefore apply equally where the relative level of the second cell type is increased compared to the first cell type mutatis mutandis.

[0096] In the methods of the second and third aspects of the invention delivering the saRNA molecule to the population of cells may comprising transfection, transduction or any suitable means to introduce the saRNA molecule into the cells. The cells may be from any generally suitable mammalian source, such as for example human cells, but also other from other non-human mammalian sources, e.g., murine.

[0097] The selection stimulus may be the addition of a drug, e.g., an antibiotic, where the selection marker is a drug resistance gene specific for the drug which enables the cell to survive the administration of the drug. The drug resistance gene therefore provides a cell which has received the saRNA, e.g., a transfected cell, with resistance to the selection stimulus provided by the drug thus increasing the relative proportion of a cell which has received the saRNA, e.g., a transfected cell, which does not comprise the miRNA present in the second cell type complementary to the sMRE in the saRNA molecule (i.e., the relative proportion of the desired first cell type is increased) in the population of cells. Alternatively, the miRNA is present at a low level in the second cell type such that the saRNA is not as effective in the second cell type as in the first cell type. As described herein, the drug resistance gene may be an antibiotic resistance gene.

[0098] Alternatively, the selection stimulus may be the use of culture conditions to cause gene expression where the selection marker is a suicide gene. The suicide gene converts a non-toxic prodrug into a toxic drug (either present in the culture medium or can be administered subsequently to saRNA delivery), causing a cell which has received the saRNA, e.g., a transfected cell, to die if it does not possess the miRNA complementary to the sMRE in the saRNA molecule thus increasing the relative proportion of the cell which has received the saRNA, e.g., a transfected cell, which does possess the miRNA present in the second cell type (i.e., the relative proportion of the desired second type of cell is increased) in the population of cells.

[0099] Where the selection marker is a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein other biochemical and / or physical means may be used to select cells using specific binding molecules, e.g., antibodies or similar to remove selected cells, FACS, and / or other means for cell selection using fluorescence detection.

[0100] When selecting for cells according to a method of the invention it may be preferable for the cells of the type being selected for to have an at least 1-fold difference (e.g. 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold) in the amount of miRNA present compared to the non-selected cell type, optionally up to a 10-fold difference or greater, or up to a 15-fold difference or greater, or up to a 100-fold or greater.

[0101] The population of cells may be a population of differentiated cells. The population of cells may be a heterogenous population of cells wherein the cells express different genes, different cell surface markers or different phenotypes.

[0102] The population of cells may comprise cells of a desired cell type and of an undesired type. The cell type may be with respect to the genotype or phenotype of the cells in the population. The population of cells may comprise undifferentiated cells of a desired cell type and of an undesired type. The population of cells may comprise undifferentiated cells and differentiated cells - either of which may be the preferred cell type, i.e. a desired undifferentiated cell type and an undesired differentiated cell type, or an undesired undifferentiated cell type and a desired differentiated cell type.

[0103] The degradation of the saRNA may be caused by translation repression or cleavage wherein the degradation is sufficient to cause inactivation of the saRNA. The degradation of the saRNA may result from miRNA-mediated translational repression or endonucleolytic cleavage, depending on the degree of complementarity between the miRNA and the sMRE sequence. In cases of high, or full complementarity, the RNA-induced silencing complex (RISC) promotes direct cleavage of the saRNA, leading to its rapid degradation. In cases of partial complementarity, the miRNA-RISC complex primarily causes translational repression and accelerated decay of the saRNA without complete cleavage. In both cases, the resulting reduction in saRNA stability and translation directly decreases transgene expression, thereby providing a means to modulate saRNA activity in a miRNA-dependent manner. This mechanism underlies the cell-type-selective and condition-dependent control described herein, enabling the implementation of the previously described methods.

[0104] The methods of the invention may also be used as part of a method to expand and differentiate a population of undifferentiated cells, for example to differentiate a population of iPSCs into a differentiated cell population. Accordingly, a method of the invention may further comprise the steps of further transfecting the cells with one or more transcription factors and / or culturing the cells under conditions such that the cells are differentiated into a differentiated population of cells. Alternatively, the transcription factor may be present in the saRNA molecule such that differentiation of the cells in the cell population occurs simultaneously with the method of the invention described above. The transcription factor may drive cells towards the mesoderm lineage. The transcription factor may drive cells towards the endoderm lineage. The transcription factor may drive cells towards the ectoderm lineage. For example, the transcription factor may be human NGN2 for neuronal induction, see for example construct PLU-R-195 of Table 1 or the transcription factor may be MyoD1 for myogenic induction, see for example constructs PLU-R-321 and PLU-R-335 of Table 1.

[0105] The invention as defined above is in relation to a forward (positive) strand of the saRNA molecule but equally can be understood with reference to the corresponding reverse (negative) strand of the saRNA molecule.

[0106] The saRNA molecules of the present invention can be used in the methods of the present invention.

[0107] Preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. Example constructs of the present invention include those set out in Table 1. This table demonstrates the wide range of design flexibility afforded by the saRNA molecules of the present invention, highlighting the importance of this platform technology. The architecture of the illustrated constructs is provided purely for illustrative purposes. It is within the scope of the invention that the individual components, their arrangement, or their interconnections may be modified, substituted, or repositioned without departing from the spirit or scope of the invention.

[0108] Table 1 : Example saRNA constructs

[0109] Detailed description of the invention

[0110] Definitions

[0111] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0112] The term “a” includes one or more of the elements defined herein, unless the context specifies otherwise.

[0113] The saRNA constructs of the present invention may include one or more conserved sequence elements (CSE), one or more untranslated regions (UTR), one or more synthetic microRNA (miRNA) responsive elements (sMREs), one or more RNA regulatory elements, and / or one or more transgenes.

[0114] As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). As used herein the term “coding region” refers to the region of nucleotides that encode any protein that the saRNA is designed to express. The coding region may encode for one or more viral non-structural proteins (nsPs).

[0115] As used herein, "complementary" means that a nucleobase or region thereof (e.g. of the microRNA) is capable of pairing with a nucleobase of a complementary nucleic acid target sequence (e.g. the sMRE) through base-pair interactions, facilitating targeted binding. The complementary binding between the sMRE and the miRNA may be complete, wherein each nucleotide within the miRNA binding site pairs exactly with its corresponding base in the miRNA sequence, or it may be partial, allowing for a defined degree of mismatch. Such mismatches are permissible provided that they do not hinder the essential recognition and binding by the miRNA. In specific embodiments, the sMRE may have a seed region — typically spanning 6 to 8 nucleotides — characterised by a high degree of complementary binding to the miRNA. Within the seed region mismatches of 1 nucleotide, 2 nucleotides, or 3 nucleotides are acceptable. Outside the seed region, the sMRE may incorporate mismatches with the miRNA sequence; the allowable degree of mismatch is not strictly limited and may range from 0% to 75% of the total length of the miRNA sequence, with mismatches occurring in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions, as long as miRNA recognition and binding are maintained. The complementary region within the sMRE may vary in total length, typically comprising between 8 to 24 nucleotides to facilitate stable and specific miRNA binding. More preferably, the sMRE may be designed with a length of approximately 20 to 22 nucleotides, though shorter or longer sequences may be employed depending on the desired binding strength and specificity. Mismatches in the sMRE outside the seed region may include, but are not limited to, bulges, insertions, or other nucleotide substitutions, allowing for adjustable binding affinity and specificity without loss of the sMREs functional integrity in miRNA recognition. The sMRE may thus be engineered to exhibit a range of complementary binding, containing sequences with at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% complementarity.

[0116] The term "comprising" is used herein to mean including the method steps or elements identified, but that such steps or elements do not comprise an exclusive list and as such there may be present additional steps or elements.

[0117] As used herein, “conserved sequence element” or “CSE” refers to a region of nucleotides that is highly conserved within self-amplifying RNA replicon systems due to its essential role in replication. CSEs serve as replication-associated motifs recognized by the replicase complex and are required for the initiation and regulation of RNA synthesis. In such systems, 5' CSEs participate in positive-strand synthesis, whereas 3' CSEs direct negative-strand synthesis, together ensuring accurate and efficient RNA replication. Modifications to the sequence or structure of a CSE can alter replicase recognition and consequently modulate replication efficiency or fidelity. The CSE may be present within untranslated regions (UTRs). The CSE may be present in the untranslated regions. For all compositions described herein, and all methods using a composition described herein, the compositions can either comprise the listed components or steps, or can "consist essentially of the listed components or steps. When a composition is described as "consisting essentially of the listed components, the composition contains the components listed, and can further contain other components which do not substantially affect the methods disclosed, but do not contain any other components which substantially affect the methods disclosed other than those components expressly listed; or, if the composition does contain extra components other than those listed which substantially affect the methods disclosed, the composition does not contain a sufficient concentration or amount of the extra components to substantially affect the methods disclosed. When a method is described as "consisting essentially of the listed steps, the method contains the steps listed, and can further contain other steps that do not substantially affect the methods disclosed, but the method does not contain any other steps which substantially affect the methods disclosed other than those steps expressly listed. As a nonlimiting specific example, when a composition is described as "consisting essentially of a component, the composition can additionally contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents and other such components which do not substantially affect the methods disclosed.

[0118] As used herein, “degradation” refers to the breakdown or destabilization of molecules, including but not limited to, nucleic acids, proteins and lipids. Degradation may be a result of translation repression or cleavage. In the context of RNA degradation, RNA molecules may be degraded through a variety of mechanisms, including endonucleolytic or exonucleolytic cleavage by nucleases, or via regulated decay pathways such as RNA interference (RNAi) mediated by the RNA-induced silencing complex (RISC). Degradation may also occur through sequence- or structure-dependent elements, such as AU-rich elements (AREs) located in untranslated regions, which recruit RNA-binding proteins and decay complexes to promote deadenylation, decapping, and exonucleolytic degradation. Collectively, these processes reduce RNA stability, abundance, or translational capacity, leading to functional inactivation of the affected RNA molecule.

[0119] As used herein, “degron” refers to a specific sequence or motif within a protein that signals the protein for degradation by the cell's proteolytic machinery, typically the proteasome or lysosome. The degron acts as a "tag" that marks the protein for destruction. Examples of degrons include PEST sequences and ubiquitin tags.

[0120] As used herein “desired cell type” refers to a cell type or a stage-specific cell that is to be targeted, selected or enriched in accordance with the invention. As an example, if the method of the present invention is used for enrichment of cardiomyocytes, the desired cell type is cardiomyocytes. In contrast “undesired cell type” refers to a cell type or a state-specific cell that is not to be targeted, selected or enriched in accordance with the invention. Using the same example, wherein the method of the present invention is used for enrichment of cardiomyocytes, the undesired cell type would be any other cell type, such as cardiac progenitor cells or fibroblasts. In some instances, a desired cell type may be referred to as a first cell type, and an undesired cell type referred to as a second or subsequent (e.g. third or fourth) cell type.

[0121] Examples of cell types, which can be either desired or undesired, depending on the cell type to be enriched, include but are not limited to: T Cells, Hematopoietic Stem Cells (HSCs), CD34+ cells, Hematopoietic progenitor cells, Common Lymphoid Progenitors (CLPs), Lymphoid progenitor cells, Pro- T Cells (Thymocytes), Early T-cell precursors, Double-Negative (DN) Thymocytes, CD4-CD8- T cells, Double-Positive (DP) Thymocytes, CD4+CD8+ T cells, Single-Positive (SP) Thymocytes, CD4+ T cells (Helper T cells), CD8+ T cells (Cytotoxic T cells), B Cells (B Lymphocytes), CD19+ cells, CD20+ cells, Natural Killer (NK) Cells, CD16+CD56+ cells, Dendritic Cells, Antigen-presenting cells (APCs), CD11 c+ cells, Macrophages, Mononuclear phagocytes, CD14+ cells, Granulocytes, Neutrophils, Eosinophils, Basophils, Undifferentiated Stem Cells, Pluripotent stem cells (PSCs), Embryonic stem cells (ESCs), Induced pluripotent stem cells (iPSCs), Other Hematopoietic Lineage Cells, NK Cell Precursors, Mature NK Cells, Common Myeloid Progenitors (CMPs), Granulocyte-Macrophage Progenitors (GMPs), Monocyte Precursors, Monocytes, Neutrophils (CD66b+ cells), Erythroid Cells, Red blood cells, Erythrocytes, Lymphoid Lineage Cells, Neural Progenitor Cells (NPCs), Neural stem cells (NSCs), Floor Plate Progenitors, Midbrain Dopaminergic Neuron Precursors, Dopaminergic Neurons, GABAergic Neurons, Glutamatergic Neurons, Serotonergic Neurons, Glial Cells, Astrocytes, Oligodendrocytes, Microglia, Neural Crest Derivatives, Schwann Cells, Peripheral Neurons, Mesodermal Cells, Endodermal Cells, Optic Vesicle-like Structures, Retinal Progenitor Cells (RPCs), Photoreceptor Precursors, Rods, Cones, Retinal Ganglion Cells, Retinal Pigment Epithelium (RPE) Cells, Non-Retinal Neurons, Cortical Neurons, Midbrain Neurons, Bipolar Cells, Amacrine Cells, Horizontal Cells, Muller Glia, Motor Neurons, Interneurons, Oligodendrocyte Progenitor Cells (OPCs), Spinal Cord Progenitors, Sensory Neurons, Mesodermal Progenitor Cells, Cardiac Progenitor Cells, Cardiogenic mesoderm cells, Cardiomyocytes, Ventricular Cardiomyocytes, Atrial Cardiomyocytes, Pacemaker Cells, Sinoatrial node cells, Nodal cells, Smooth Muscle Cells (SMCs), Endothelial Cells (ECs), Definitive Endoderm Cells, Pancreatic Progenitor Cells, PDX1 + cells, Endocrine Progenitor Cells, Beta Cells, Alpha Cells, Delta Cells, PP Cells, Epsilon Cells, Exocrine Pancreatic Cells, Acinar Cells, Ductal Cells, Hepatocytes, Intestinal Cells, Fibroblasts, Intestinal Epithelial Cells, Keratinocytes, Non-Target Neurons, Hematopoietic Progenitors, Neural Progenitors, Mesenchymal Stem Cells (MSCs), Multipotent mesenchymal stromal cells, Epithelial Cells, Chondrocytes, Adipocytes, Immune Cells, Ectodermal Progenitor Cells, Neuroectodermal Cells, Hepatic Progenitor Cells (Hepatoblasts), Renal Progenitor Cells, Lung Progenitor Cells, Intestinal Stem Cells, Germ Cell-like Cells (PGCLCs), Thymic Epithelial Progenitor Cells, Inner Ear Hair Cells, Platelets, Alveolar Epithelial Cells (Type I and II), Airway Epithelial Cells, Podocytes, Other Nephron Cells, Osteoblasts (Bone Cells), Myocytes (Muscle Cells), Melanocytes, Enteroendocrine Cells, Corneal Epithelial Cells, Corneal Endothelial Cells, Thymic Epithelial Cells, Thyroid Follicular Cells, Sperm Cells, Oocytes, Renal Interstitial Fibroblasts, Mesangial Cells, Pericytes, Ureteric Bud (UB) Progenitor Cells. The cells may be human or non-human. As used herein, “enriching” refers to increasing the proportion of a specific cell type or type of cell within a population of cells.

[0122] As used herein, "expression" means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5 '-cap), translation and post-translational modifications. For example, expression of a protein, i.e. “cargo”, from a transgene

[0123] As used herein, "fully complementary" means that each nucleobase or region thereof (e.g., of the microRNA) is capable of pairing with a nucleobase of a complementary nucleic acid target sequence (e.g. the sMRE). Thus, fully complementary refers to no mismatches or unhybridized nucleobases with respect to its target sequence, i.e. there is complementarity. The term “partially complementary” means that some but not all of the nucleobases are capable of pairing with nucleobases of complementary nucleic acid target sequences. The miRNA sequence may be at least 45%, at least 80%, at least 90%, at least 95%, at least 99% or 100% complementary to the nucleic acid sequence of the sMRE.

[0124] As used herein, "hybridization" means the pairing of complementary sequences. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0125] As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, indicate values that are relative to a reference (e.g., baseline) measurement, such as a measurement taken under comparable conditions or a measurement in a control described herein.

[0126] Further, to the extent that the term "includes" is used in either the detailed description or the claims, such term is intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in a claim.

[0127] The term “may” as used herein is interpreted as being optional. Where a feature is referred to as “may be” present, said feature is optionally present.

[0128] As used herein, “microRNA” or “miRNA” refers to a small, non-coding RNA molecule, typically around 20-22 nucleotides in length, that plays a crucial role in regulating gene expression. miRNAs are involved in the post-transcriptional regulation of genes by binding to complementary sequences on messenger RNA (mRNA) molecules. This binding typically results in either the degradation of the mRNA or the inhibition of its translation into a protein. Synthetic microRNAs refers to artificially designed and synthesized miRNA. Synthetic miRNAs are usually designed to mimic the action of natural miRNAs or to serve as specific inhibitors of endogenous miRNAs. They can be used to regulate gene expression by either promoting or inhibiting the action of target genes. As used herein “microRNA response element” or “MRE” refers to typically short sequences that are complementary to the seed region / sequence of a miRNA. They can be synthetic, therein referred to as “synthetic microRNA response element” or “sMRE”.

[0129] A sMRE is a synthetic construct comprising one or more (typically 1-20) MREs miRNA binding sites specifically designed for binding to microRNAs (miRNAs) of interest. sMRE can be located on positive and / or negative strand of saRNA construct. These binding sites can be strategically interspersed with spacer sequences, which can be placed between and / or surrounding the miRNA binding sites. The spacer sequences are engineered to impart defined structural properties to the sMRE, potentially influencing factors such as miRNA accessibility, binding affinity, and stability. Together, the miRNA binding sites and spacer sequences form an optimized recognition element that enables precise interaction with target miRNAs.

[0130] MicroRNAs (miRNAs) bind to synthetic microRNA response elements (sMREs) through a process similar to the way in which they bind to natural target mRNAs. The synthetic MREs are designed sequences that mimic the natural binding sites for miRNAs, they can be (although are not exclusively) located in the 3' untranslated region (UTR) of target mRNAs. miRNAs binds to synthetic MREs via basepairing between the miRNA and the complementary sequence in the MRE. The binding may be “partial binding” or “partially complementary”, wherein the miRNA does not fully base-pair with the MRE but does so sufficiently to mediate a functional effect such as degradation of the saRNA upon recruitment of the RNA-induced silencing complex (RISC) to the site. As an example, in the case of sMREs, if the sMRE is part of a reporter construct (such as GFP), binding of the miRNA to the sMRE will typically result in decreased reporter expression (e.g., reduced GFP expression).

[0131] As used herein, the term “selection stimulus” refers to an external stimulus used to select a cell type preferentially. An example of such a stimulus is a “negative stimulus” which refers to a stimulus used to inhibit or suppress a biological process or signalling pathway. An example of a negative stimulus in the context of cell survival would be a drug, e.g. an antibiotic, including but not limited to neomycin or puromycin. Wherein resistance to said negative stimulus would be conferred by a drug resistance gene, e.g. an antibiotic resistance gene, such as a neomycin resistance (NeoR) gene or a puromycin resistance (PuroR) gene, or as described elsewhere herein.

[0132] As used herein, “non-structural proteins” or “NSP” typically refer to proteins produced by viruses that are used for replication and survival but do not form part of the viral particle itself. As viral proteins NSPs are typically involved in the replication of the viral genome. As an example, alphaviruses have non- structural proteins (nsP1 , nsP2, nsP3, and nsP4) that are involved in viral replication and immune evasion. Likewise, flavivirus have non-structural proteins also (NS1 , NS2A, NS2B, NS3, NS4A, NS4B, NS5). These viral non-structural proteins form the replication complex that is used for synthesizing the viral RNA genome, producing subgenomic RNAs, and interacting with host cell machinery to ensure viral replication and assembly. As used herein, "nucleobase complementarity" or "complementarity" when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. In some embodiments, complementary nucleobase means a nucleobase of a microRNA that is capable of base pairing with a nucleobase of its target sequence sMRE. For example, if a nucleobase at a certain position is capable of hydrogen bonding with a nucleobase at a certain position of a target sequence, then the position of hydrogen bonding between the microRNA and sMRE is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

[0133] As used herein, "nucleotide" means a nucleoside further comprising a phosphate linking group.

[0134] As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides.

[0135] As used herein, "percent identity" means the number of nucleobases in a first nucleic acid that are the same (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0136] As used herein, a "polypeptide" is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides may optionally include "non-natural" amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain.

[0137] As used herein, the term "protein" refers to a molecule comprising a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a "protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, nonnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0138] As used herein, “saRNA,” and “self-amplifying RNA,” refer to RNA with the ability to replicate itself. These RNA molecules carry the genetic instructions for the synthesis of both the RNA itself and additional copies of it. Typically, self-amplifying RNA includes the replication elements of a virus or viruses, for example from alphaviruses or other positive-sense RNA viruses, that has been modified for use in research or therapeutic applications. In this context, the structural viral polypeptides can be substituted with one or more nucleotide sequences encoding, for example, polypeptides of interest. The saRNA elements may be directly translated to the protein of interest (e.g., a viral protein for vaccination) after delivery to the cell, but also may replicate itself, leading to a greater amount of RNA and protein production.

[0139] Alphavirus-based saRNAs are positive stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or RNA-dependent RNA polymerase). The replicase is translated as a polyprotein which replicates genomic negative-strand copies from the positive-strand delivered RNA. Over time, replicase polyprotein is cleaved, allowing negative-strand transcripts to be used for replication of further copies of the positive-stranded parent RNA and also to replicate a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript can lead to in situ expression of the desired gene (the transgene) product in the cell. Alphaviruses are a group of viruses belonging to the Togaviridae family, including but not limited to Chikungunya virus, Sindbis virus, Semliki forest virus and Venezuelan equine encephalitis virus.

[0140] A self-amplifying RNA (saRNA) may have the structure of:

[0141] 5’ cap — 5' UTR — coding region for one or more non-structural proteins — a subgenomic promoter (SGP) — a transgene — 3' UTR -» a 3’ conserved sequence element (CSE) (see for example, Figure 1 A - wherein one or more sMREs may be located throughout the saRNA).

[0142] Other potential saRNA structures include:

[0143] 5’ cap — 5' UTR -» a 5’ conserved sequence element (CSE) — coding region for one or more non- structural proteins — a subgenomic promoter — a transgene — 3' UTR a 3’ conserved sequence element (CSE). Wherein one or more sMREs are located throughout the saRNA. See for example, Figure 1 A - wherein one or more sMREs may be located throughout the saRNA.

[0144] The above structures are not limiting. Further structures are permitted. As used herein “a transgene” refers to one or more transgenes. It is preferred that the transgene is downstream of the subgenomic promoter, if present.

[0145] A saRNA vehicle allows for replication intracellularly, such that cell divisions can be maintained during cell therapy manufacture. saRNA does not integrate into the cell’s genome thereby avoiding insertional mutagenesis and regulatory concerns. saRNA can allow for the delivery of a transgene, that induces saRNA degradation such that no or very little trace is detectable in the final product (e.g. the resulting cells). A small molecule can be used to induce saRNA degradation such that no or very little trace is detectable in the final product (e.g. the resulting cells).

[0146] The combination of saRNA and miRNA ensures prolonged expression and selectivity, whereby the saRNA ensures prolonged expression and the miRNA ensures selectivity. In an example of the present invention, the saRNA may include a transgene that is a drug resistance gene. If the sMRE of the saRNA is complementary to a microRNA within a particular cell, sMRE-microRNA binding will induce saRNA degradation, such that the particular cell does not have drug resistance, e.g., antibiotic resistance. As such, upon exposure to said drug, only the cells wherein the saRNA is not degraded will survive.

[0147] As used herein, the term “sensitising modifications” or "sensitising mutations" refers to intentional alterations in the nucleotide sequences encoding viral proteins, regulatory regions or addition of completely new functional RNA motifs in the saRNA constructs. These modifications result in the attenuation or modification of viral replication and expression functions without completely abolishing the functionality of the viral components. Sensitising modifications may affect the viral non-structural proteins (nsPs), untranslated regions (UTRs), polyprotein cleavage sites, subgenomic promoters, and other elements critical for the assembly and function of the RNA replication complex. The sensitising mutations described herein can be introduced individually or in combination into saRNA constructs. The selection of specific mutations allows for tailored modulation of the persistence, replication and expression profiles of the saRNA, depending on the desired application.

[0148] As used herein, the term “subgenomic promoter” refers to the promoter that initiates replication of subgenomic RNA (sgRNA) produced from a viral genome. The viral genome may be used as a template to generate both full-length genomic RNA and shorter subgenomic RNA (sgRNA). sgRNAs typically code for the viral proteins necessary for assembling new virions, playing a critical role in the viral life cycle. A subgenomic promoter may direct expression of the transgene. Optionally, the transgene sequence may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0149] As used herein, the terms "target cell" refers to any cell, tissue, or organism.

[0150] As used herein, "target sequence" means a sequence to which an entity (e.g. a compound, or another sequence) is intended to hybridize to result in a desired activity. microRNAs have sufficient complementarity to their target sMRE sequences to allow hybridization under physiological conditions.

[0151] As used herein, the term “transgene” refers to a gene introduced into the saRNA / construct, typically through genetic engineering techniques such as molecular cloning or gene synthesis. The transgene can be from the same species (cisgenic) or from a different species (transgenic). Transgenes can be exogenous, endogenous or reporter transgenes. As used herein, “untranslated region” or “UTR” refers to segments of the oligonucleotide that are not translated into protein. UTRs may be located at one or both ends e.g. at the 5’ end and / or the 3’ end. Although UTRs do not encode proteins, they play crucial roles in regulating mRNA stability, translation, and localization. A 5’ UTR may contain regulatory elements that can influence the initiation of translation, such as ribosome binding sites, 5’ cap structures which can help stabilize the oligonucleotide and facilitate translation initiation and / or regulatory sequences that allow translation to occur, such as internal ribosome entry sites (IRES). A 3’ UTR may contain sequences that affect mRNA stability and degradation, may be involved in regulating translation efficiency and localisation of the oligonucleotide in the cell and / or binding sites for microRNAs and RNA-binding proteins. The 5’ UTR may include a conserved sequence element (CSE). The 3’ UTR may include a conserved sequence element (CSE). The 5’ and 3’ UTRs may include a conserved sequence element (CSE).

[0152] Brief Description of the Drawings

[0153] The present invention will now be further described with reference to the following examples and drawings which are provided for the purposes of illustration and are not to be construed as being limitations on the invention. Reference to the accompanying drawings, in which:

[0154] Figure 1 - Graphical representation of an exemplary saRNA molecule of the invention (Figure 1A), and an exemplary cell selection protocol (Figure 1 B).

[0155] Figure 2 - Graphical representation of efficient and modular assembly of saRNA DNA templates. (A) The Golden Gate assembly process, where DNA parts with Bsal restriction sites and compatible overhangs are inserted into a receiver vector containing a T7 promoter, origin of replication (Ori), and an antibiotic selection marker. This method allows for the cloning of up to 24 parts, with the option to include inert connectors between any two positions, thereby creating a large design space for constructing complex genetic assemblies. (B) An example of resulting 1 1 -part assembled DNA template for self-amplifying RNA (saRNA) includes a 5' conserved sequence element (CSE), non-structural proteins (nsP), a subgenomic promoter (SGP), reporter genes (GFP, gLuc), internal ribosome entry sites (IRES), an antibiotic resistance gene (PuroR), untranslated regions (UTRs), and a polyadenylation (polyA) tail. The template can be linearized by specific restriction enzymes.

[0156] Figure 3 - In vitro transcription (IVT) of saRNA. (A) DNA plasmids containing the template(s) for self-amplifying RNA (saRNA) are depicted with a T7 promoter driving transcription. The plasmid is linearized after the polyA tail to allow proper transcription. Following linearization, T7 RNA polymerase transcribes the saRNA, producing the RNA molecules. (B) Gel electrophoresis of in vitro transcribed saRNA using a RiboRuler High Range RNA ladder for size comparison. Lanes #1-#5 show distinct bands corresponding manufactured saRNA molecules, indicating successful transcription of the expected RNA sizes (11 kb). Figure 4 - saRNA transfection into KOLF iPSC cells. (A) Images of KOLF induced pluripotent stem cells (iPSCs) on Day 2 post-transfection with saRNA-GFP, showing phase contrast (left), GFP fluorescence (middle), and an overlay of phase contrast and GFP fluorescence (right). (B) Flow cytometry analysis on Day 3 of non-transfected (left) and saRNA-GFP transfected KOLF cells (right). Flow cytometry dot plots display GFP expression levels, with corresponding histograms showing the fluorescence intensity distribution for each condition.

[0157] Figure 5 - HEK293 and KOLF iPSC sequencing and miRNA discovery. Volcano plot showing miRNAs that were differentially expressed between iPSC and HEK293 cells, and were expressed at least 100 cpm in one of these two cell types.

[0158] Figure 6 - Puromycin dose response in HEK293ft and KOLF iPS cells. (A-B) Graph showing the relative cell viability, as measured using MTT assay, of saRNA-transfected and nontransfected HEK293 (A) or iPSC (B) cells treated with increasing concentrations of puromycin (0-10 pg / mL). Values are presented as percentage of viability normalized to non-transfected cells. (C) Phase contrast images of non-transfected and saRNA-transfected iPSCs on Days 1 and 3 after treatment with 1 pg / mL puromycin. The top panels show non-transfected iPSCs, and the bottom panels show saRNA-transfected iPSCs at the indicated time points.

[0159] Figure 7 - Cell-specific expression of sRNA-sMRE constructs. (A) Plasmid map of PLU-D-076, highlighting the synthetic miRNA response element (sMRE) for miRNA-92a-3p. The zoomed-in region shows the detailed sequence structure of the sMRE. (B) Flow cytometry data from Day 1 post-transfection, showing GFP-positive cell populations for different sMRE constructs in HEK293ft cells (left column) and iPSCs (right column), with miRNA-binding sites for miR-92a- 3p, miR-302b-3p, and miR-196b-5p. (C-D) Relationship between saRNA-GFP expression and corresponding miRNA levels in (C) HEK293 cells on Day 2 or in (D) iPSC cells on Day 3, plotted as the percentage of GFP-positive cells against miRNA expression levels (Iog2). Simple linear regression analysis was performed (black line).

[0160] Figure 8 - Targeted iPSC elimination using saRNA-sMRE-PuroR constructs. (A) Phase contrast images of KOLF iPSCs (left column) and HEK293FT cells (right column) on day 3 posttransfection with saRNA constructs and treatment containing different miRNA response elements (MREs): PLU-R-76 (miR-92a-3p), PLU-R-85 (miR-196b-5p), and PLU-R-94 (miR- 302d-3p). Cells were treated with 1 pg / mL puromycin from Day 1 post-transfection. (B) Bar graph showing flow cytometry data for enrichment of saRNA-GFP-positive KOLF iPS cells lacking specific miRNAs over 4 days of puromycin selection (patterned bar) compared to non- puromycin-treated cells (black bar). The corresponding miRNA expression levels for each construct (PLU-R-82, PLU-R-85, and PLU-R-88) are indicated. Figure 9 - saRNA and Puromycin effects on iPSC transcriptome. (A) Volcano plot showing differentially expressed genes between KOLF iPSC transfected with saRNA vs non-transfected (B) Volcano plot showing no differentially expressed genes between saRNA transfected KOLF iPSC treated with 6 ug / ml puromycin for 2 days, vs. untreated.

[0161] Figure 10 - Effects of saRNA transfection on iPS cell growth and viability. Graph showing the number of live cells / cm2(left y-axis) and cell viability (right y-axis) in iPSC cultures transfected with saRNA at different concentrations. Transfection was performed on Day 0 (indicated by the red dashed line). Conditions include control groups with 50k and 20k non-transfected cells (black and grey lines), as well as saRNA transfected groups using LipoMAX (1 pg and 2 pg, triangle symbols) and jetMESS (1 pg and 2 pg, dashed line and diamond symbol) transfection reagents. Cell counts and viability were measured at two time points post-transfection to monitor expansion and health of the transfected cells.

[0162] Figure 1 1 - Methods for depleting or enriching specific cell types using self-amplifying RNA (saRNA) molecules containing synthetic microRNA (miRNA) responsive elements (sMREs). Panels (A-D) show representative flow-cytometry plots of HEK293 and induced pluripotent stem cell (iPSC) co-cultures transfected with different saRNA-sMRE constructs encoding GFP and an antibiotic-resistance selection marker. Constructs include R-006 (control, lacking sMREs), R-130 (HEK-specific depletion), and R-091 (iPSC-specific depletion via miR-302b target sites). Panel (E) quantifies cell-type survival after puromycin selection, demonstrating selective enrichment of iPSC (Tra-1-60+ / SSEA+) or HEK cells depending on sMRE design. Panel (F) shows selective enrichment of endodermal (CD117+) versus mesodermal (CD56+) cells following differentiation and selection, consistent with sMRE-guided degradation of saRNA altering the relative abundance of distinct cell types upon application of a selection stimulus.

[0163] Figure 12 - Lineage-selective elimination of differentiating iPSCs using saRNA-sMRE constructs. Panels (A) show representative brightfield images of iPSCs transfected with control (R-006) or lineage-targeting constructs (PLU-R-130, R-280, R-315) following induction toward endoderm, mesoderm, or ectoderm under puromycin selection. R-280 selectively eliminates endodermal cells, R-130 removes mesodermal derivatives, and R-315 targets ectodermal populations, while the control R-006 shows normal differentiation across all lineages. Panels (B) show flow cytometry validation of lineage differentiation and construct specificity. R-006 cultures display efficient endoderm (CD1 177CD56-) and mesoderm (CD56+) induction. R-130 differentiates toward endoderm (CD117+ / CD56“), but no viable mesodermal cells are recovered, confirming selective elimination. Collectively, the drawings demonstrate that saRNA- sMRE constructs enable miRNA-guided, lineage-specific cell removal to purify desired differentiated cell types. Figure 13 - Evaluation of sMRE design parameters within saRNA constructs. This figure includes constructs containing one to two tandem miR-302b binding sites (R-128, R-147) (A-C), constructs combining distinct miRNA targets such as miR-1 Oa and miR-196b (R-115) (D-F), and variants placing the sMRE sequence in different positions within the 5' UTR, subgenomic promoter (SGP), or 3' UTR regions (e.g., R-091 , R-127, R-253) (G-l). Additional constructs compare full and partial complementarity between sMRE-302b and its cognate miRNA (R-091 vs R-152) (J-L). Imaging and flow-cytometry data in HEK293 and iPSC cells show the resulting differences in GFP expression patterns. Collectively, the drawings depict how variation in binding-site number, sequence complementarity, miRNA combinations, and sMRE position influences miRNA-dependent regulation and can be used singly or in combination to adjust the behaviour of saRNA-sMRE constructs across different cell types and states.

[0164] Figure 14 - The relationship between RNA secondary structure and the accessibility of sMREs within saRNA constructs. This figure includes predicted folding maps for constructs R-091 , R- 092, and R-093, each containing four miR-302b sMREs positioned in distinct structural contexts. In these models, sMREs located within single-stranded loop regions appear more exposed, whereas those embedded in double-stranded stem regions show limited accessibility. Corresponding flow-cytometry data (A-B) obtained from HEK293 and iPSC cultures display differences in GFP expression consistent with the predicted accessibility of the sMRE sites (C- D). Collectively, the drawings present the comparative structural configurations and associated expression outcomes, illustrating how local RNA folding influences miRNA-dependent regulation within the saRNA-sMRE framework.

[0165] Figure 15 - The effect of 5' untranslated region (UTR) secondary structure on saRNA replication and stability as reported by GFP expression levels. This figure includes constructs R-161 (control), R-160, R-155, and R-157, which differ by engineered disruption of stem-loop regions SL1-SL4 within the 5' UTR (A). Predicted RNA folding diagrams (B) and corresponding flowcytometry data collected on day 21 following transfection and selection (C) show variations in GFP signal intensity reflecting changes in effective saRNA output, which is a direct reporter of the balance between RNA replication efficiency and RNA stability. Panel (D) presents RT-qPCR analysis comparing the non-modified construct R-006 with a 5' UTR-disrupted variant R-130 in iPSC cultures. In these constructs, the 5'UTRs were modified by altering the structure of stemloop 1 (SL1): R-160 contains a smaller and weaker loop; R-155 includes a longer and more complex SL1 that largely preserves the conformation of SL2-SL4; and R-157 incorporates broader structural perturbations across multiple loops. Together, the drawings depict how targeted modification of 5' UTR structural elements influences saRNA replication dynamics and transcript persistence, providing examples of sensitising modifications within regions such as UTRs and CSEs. Figure 16 - Decoupling of the subgenomic promoter (SGP) from the non-structural protein (nsP) coding region within saRNA constructs to enable modular control of replication and transgene expression. Schematic representations of constructs R-006 (control) and modified variants such as R-162, R-188, R-192, R-223, and R-238, representing decoupled, mutated, truncated, and rescued SGP (i.e. attaching deleted SGP sequence back to truncated SGP) configurations. In these designs, separation of the SGP from the nsP coding sequence allows insertion of sMRE upstream of the SGP, thereby restricting miRNA-mediated regulation primarily to genomic RNA copies while leaving subgenomic transcripts unaffected. This arrangement decreases the number of RNA molecules targeted by miRNAs, allowing higher transgene expression levels while maintaining overall system responsiveness to miRNA control. Panel shows flow-cytometry plots from iPSC cultures collected at days 1 and 3 following transfection, comparing GFP- positive cell fractions and mean fluorescence intensities (MFI) among constructs with different SGP configurations. Constructs with decoupled or mutated SGPs demonstrate reduced GFP- positive cell populations and mean fluorescence intensity compared to the control, while SGP rescue restores expression. Variations in signal intensity correspond to differences in promoter integrity and positioning, illustrating how structural separation of the SGP and nsP regions influences replicase output and transgene expression within the saRNA framework. These SGP modifications represent examples of sensitising modifications that adjust replication and expression dynamics while maintaining miRNA-dependent control.

[0166] Figure 17 - The incorporation of RNA stability-regulating elements, specifically AU-rich elements (AREs),with saRNA constructs to modulate transcript persistence and expression balance. Panels (A-C) include schematic representations of constructs R-247, R-248, and R- 249, each containing 5*, 10*, or 15* tandem ARE repeats positioned downstream of the transgene, compared with R-006 (control) lacking added elements. Flow-cytometry plots and quantitative mean fluorescence intensity (MFI) analyses obtained after transfection and selection (9 days) display changes in GFP expression patterns associated with inserted ARE repeats. The drawings show that the introduction of ARE motifs reduces saRNA stability while maintaining sufficient transgene expression to support selective cell survival in the absence of targeting microRNAs, reflecting the balance between RNA turnover and functional output. Panels (D-E) compare constructs R-162 (control), R-344, R-232, R-211 , and R-345 to evaluate the combined effects of sMREs and ARE sensitisation. Schematic representations and flowcytometry plots (D) show that constructs containing multiple distinct sMREs (R-211) or a single sMRE combined with an ARE (R-345) display enhanced miRNA-dependent degradation, evidenced by reduced GFP-positive fractions and lower MFI values relative to single-sMRE or control constructs (Day 1 post-transfection). Quantification in panel (E) summarizes changes in GFP-positive cells between Day 1 and Day 3, distinguishing constructs with no effect on GFP levels in which saRNA replication and stability dynamics dominate - from those exhibiting low effect (i.e. fewer cells get transfected) and high effect (i.e. drop in GFP signal over time) sMRE activity driven by active miRNA regulation. Together, the drawings illustrate how ARE motifs and sMRE configurations function as sensitising modifications that fine-tune RNA stability and regulatory responsiveness within the saRNA framework.

[0167] Figure 18 - The examples of saRNA constructs incorporating sMREs and transgenes encoding lineage-specifying transcription factors with selectable markers. Panel (A) shows schematic representations of constructs R-195, R-335 and R-321 , where R-195 encodes the human NGN2 transcription factor together with GFP, and R-335, R-321 encode MyoD1 and other transgenes together with an HSV-TK cassette and synthetic miRNA-responsive elements (sMRE-1 , sMRE- 133b, and sMRE-206) complementary to microRNAs enriched in myogenic cells. Panel (B) presents brightfield and fluorescence images of iPSCs transfected with R-195 showing GFP expression and neuronal morphology on day 5. Panel (C) shows brightfield images of cultures transfected with R-335, comparing untreated and 10 pM ganciclovir (GCV)-treated cells on day 6. Panel (D) displays immunocytochemistry images for myosin heavy chain (MyHC) in R-321 - transfected cultures with and without 10 pM GCV treatment on day 6. Panel (E) shows quantitative image analysis of MYHC-positive cells under the indicated conditions. Together, these drawings demonstrate directed cell fate specification achieved through saRNA-delivered transcription factors, with the option to combine this approach with sMRE-mediated selection to selectively enrich the population for the intended cell type.

[0168] Figure 19 - Strategies for extending miRNA responsiveness of saRNA constructs to contexts where target miRNAs are expressed at low abundance. Panels (A-B) show representative flowcytometry plots and degradation-activity quantification for constructs such as R-006, R-088, and R-186, comparing HEK293 and iPSC populations at Days 1 and 3. Constructs containing sMREs complementary to moderately expressed miRNAs (miR-205) exhibit time-dependent decreases in GFP-positive cell fractions and mean fluorescence intensity (MFI) in the miRNA- rich cell type, confirming active miRNA-mediated degradation. Panels (C-E) extend this analysis to additional constructs (R-113, R-232, R-344, R-345, and R-211), showing that incorporation of AREs or multiple distinct sMREs enhances responsiveness and produces regulatory activity within the sub-2,000 counts per million (cpm) miRNA expression range (miR- 200c). Representative plots (D) confirm that constructs such as R-232 exhibit preferential degradation in iPSCs relative to HEK293 cells (based on GFP levels), consistent with miR-200c activity, while fluorescence-microscopy saRNA-GFP images (E) illustrate cell-type-specific survival under puromycin selection corresponding to targeted miRNA abundance. The data demonstrate that combinatorial miRNA targeting and sensitising modifications expand the effective range of miRNA-dependent regulation. Collectively, the drawings demonstrate that through the use of sensitising modifications, synergistic sMRE configurations, and related design strategies shown in preceding figures, even low-abundance miRNAs can be effectively harnessed to achieve precise cell-type and state-specific regulation within the saRNA-sMRE platform. Figure 20 - microRNA expression profiles in HEK293 and iPSC cells, expressed as counts per million (cpm). Panel (A) shows a comparative table listing representative miRNAs with differential abundance between the two cell types, including examples such as miR-302b, miR- 10a, miR-196b, and miR-200c. Panels (B) and (C) present summary plots of all detected miRNAs, illustrating overall and filtered subsets (> 10 cpm) with filtered median expression levels of approximately 135-147 cpm in both cell types. The drawings illustrate that a broad range of miRNAs are expressed at levels which, when used in combination, could achieve total expression values corresponding to those demonstrated to be functionally selective in saRNA- sMRE constructs, providing support for broad applicability of the approach to cell-type- and state-specific saRNA construct design.

[0169] Figure 21 - Demonstration of functional regulation using a sMRE positioned on the negative strand of a saRNA. Panels (A) show representative flow cytometry plots of iPSC and HEK293 cells transfected with control saRNA (R-006) or construct R-110 containing an sMRE complementary to miR-302b positioned on the negative (antisense) strand. In iPSCs, R-110 exhibits strong reduction of GFP-positive cells compared to the control, while GFP expression is maintained in HEK293 cells, demonstrating strand-specific miRNA-dependent degradation activity. Panel (B) quantifies the relative GFP expression ratios between HEK and iPSC cultures at day 1 and day 3 post-transfection, normalized to the control construct R-006. The observed repression in iPSCs confirms that miRNA targeting can occur through complementary elements positioned on the antisense strand of the saRNA molecule. Panel (C) presents representative brightfield images of iPSC and HEK293 cultures transfected with R-110 under puromycin selection at day 7, showing selective survival and maintenance of HEK293 cells, consistent with the flow cytometry results. Collectively, the drawings demonstrate that functional miRNA- dependent regulation can be achieved via antisense-oriented sMREs, extending the design flexibility of saRNA-sMRE constructs.

[0170] Examples

[0171] HEK293FT and iPSC cell culture

[0172] Human embryonic kidney 293 cells (HEK293FT, also called HEK293) (Cat. No. R70007; ThermoFisher) were cultured in a humidified incubator at 37°C and 5%CO2 on standard tissue culture treated flasks and plates in DMEM medium (Cat. No. 11574456) supplemented with 10% FBS (Thermofisher, Cat. No. 17563595), 0.1 mM NEAA (Thermofisher, Cat. No. 12084947), 1 mM Sodium Pyruvate (Thermofisher, Cat. No. 1 1530396) and 1 % Pen / Strep (11548876). Cells were split every few days before reaching full confluency using Accutase (Thermofisher, Cat. No. 11348181) and / or mechanical dissociation using a pipette.

[0173] Human induced pluripotent cells (iPSC) (KOLF2.1J and iPSC SK003.2) were obtained from the Jackson Laboratory (JIPSC1000) and Reprocell Inc. (RCRP011 N) respectively. iPSC cells were cultured in a humidified incubator at 37°C and 5%CO2 on Vitronectin (Cat. No. 15661215) coated plates in StemFlex culture medium (Thermofisher, Cat. No. 15627578) with or without the addition of Penicillin / Streptomycin (Thermofisher, Cat. No. 11548876). Cell media was changed every 1-2 days depending on confluency and state of culture. Cells were passaged as needed, avoiding cell overgrowth. To passage the cells, the culture medium was removed and TripLE (Cat. No.12563011 , ThermoFisher) was added to cover the cells for 3-5 minutes at room temperature. TripLE was then aspirated (without dislodging the cells from the plate). Cells were washed off the plate by pipetting with StemFlex media containing RevitaCell supplement (Cat. No. A2644501) or Rock-inhibitor (Cat. No. 15580821). The required amount of cell suspension was added to coated cell culture plasticware, containing StemFlex culture media supplemented with RevitaCell.

[0174] Cells were selected by the addition of Puromycin (Cat. No. 12122530, 10mg / mL) to the cell culture medium at 1 pg / ml or the otherwise indicated concentration. Puromycin was stored at -20°C for up to 12 months or at 4°C for up to 2 months. Generally, puromycin-containing cell culture media was made up on the day of use or a few days in advance. During the first days of cell selection, media was changed more frequently (daily) to remove dying cells and cell debris.

[0175] Cell transfection

[0176] RNA can be transfected into cells with a variety of different methods. We have successfully used lipofection with different reagents, electroporation (nucleofection, Lonza 4D) and lipid nano-particles (LNPs). Cells need to be at 50 - 70% confluence on the day of transfection.

[0177] Lipfectamine MessengerMax: The following describes the transfection with Lipofectamine MessengerMax reagent (Cat. No. LMRNA003; ThermoFisher). For cells grown in 12-well plates, 1 pg of RNA was mixed with 50 pL OptiMEM (Cat. No. 31985062; ThermoFisher). 3 pL of MessengerMax Reagent was mixed with 50 pL OptiMEM and immediately added to RNA-OptiMEM mixture (incubation of the OptiMEM-MessengerMax mixture before mixing with the RNA solution reduces transfection efficiency). Solutions were gently mixed by flicking the tube slightly and incubated at room temperature for 5 minutes. During incubation, cell culture media was aspirated from the cells and replaced with 500pl OptiMEM per well. The RNA-MessengerMax transfection mixture was added drop-wise to the well and the plate was shaken briefly to ensure distribution of the mixture throughout the well. Cells were then returned to the incubator. After 4-6 hours, the OptiMEM containing the Lipofection mixture was aspirated and normal culture media was added to the cells, which were then returned to the incubator.

[0178] JetMessenger: The following describes the transfection protocol using jetMESSENGER® reagent (Cat. No. 101000056; Polypus). For cells grown in 12-well plates, 1 pg of RNA was diluted in 100 pL of jetMESSENGER® RNA buffer. The solution was mixed gently and spun down briefly. Next, 2 pL of jetMESSENGER® reagent was added to the RNA solution (RNA / Reagent ratio 1 :2). The mixture was gently mixed by flicking the tube, spun down, and incubated at room temperature for 10 minutes. After incubation, the RNA-jetMESSENGER® transfection mixture was added dropwise to the well containing cells at 60-80% confluency, and the plate was briefly shaken to ensure even distribution of the mixture throughout the well. Cells were returned to the incubator, and transfection was allowed to proceed until further analysis.

[0179] Flow

[0180] Cells for flow cytometry were dissociated using either Accutase or TripLE and resuspended in culture medium. When appropriate, live / dead staining was done by adding 7AAD dye (Cat. No. A1310, ThermoFisher). Samples were analyzed using BD FACSverse machine or BD LSRFortessa™ Cell Analyzer. Negative control (non-treated cells) were used to set the gating strategy. Samples used for comparisons were analysed at the same laser voltage and 10,000 single events were captured per sample.

[0181] Live cells were stained for the cell surface markers TRA-1-60 (BioLegend 330610), SSEA-5 (Miltenyi Biotec 130-106-663), CD56 (Cat. No 318310; BioLegend) and CD117 (Cat. No 313238; BioLegend). Cells were dissociated as described before, then centrifuged (300g, 4 minutes), media was aspirated and cell pellets were resuspended into 10Oul of PBS containing the desired antibodies. Samples were incubated for 30minutes at room temperature in darkness, centrifuged (300g, 4 minutes) and resuspended in 10Oul PBS. All antibodies were used at the manufacturer’s recommended dilution. Analysis was then completed as described above.

[0182] Immunocytochemistry and imaging

[0183] Cells were prepared for immunofluorescence staining using a standard permeabilisation and blocking protocol. A permeabilisation solution (PBST) was prepared consisting of phosphate-buffered saline (PBS) containing 0.1 % Triton X-100, and a blocking solution was prepared consisting of PBS containing 5% bovine serum albumin (BSA). Cells were first washed once in PBS, then incubated with 0.5 mL of PBST for 20 minutes at room temperature to permeabilise cellular membranes, followed by incubation in blocking solution for 1 hour to reduce non-specific antibody binding. Primary antibodies were diluted in blocking solution, and cells were incubated overnight at 4°C in the primary antibody solutions. The following morning, cells were washed three times in PBST before incubation with secondary antibodies diluted in blocking solution for 1 hour at room temperature in the dark. Cells were then washed twice in PBS. Nuclear counterstaining was performed by incubating the cells with 4',6-diamidino-2-phenylindole (DAPI) diluted in PBS to a final concentration of 1 pg / mL for 5 minutes in the dark at room temperature, followed by one wash in PBS. After staining, cells were kept in PBS for imaging. Fluorescence images were acquired using an Operetta CLS high-content analysis system operated in non-confocal mode under identical exposure settings for all experimental conditions.

[0184] The following antibodies were used for immunofluorescence staining and analysis. Primary antibodies included mouse anti-human myosin heavy chain (Cat. No. MAB4470; R&D Systems) used at 5 pg / mL. Secondary antibodies donkey anti-mouse AF488 (Cat. No. ab150105; Abeam) antibody was used at a 1 :500 dilution. RT-qPCR

[0185] RT-qPCR was performed using the Cells-to-CT™ 1-Step TaqMan® Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were lysed directly without RNA purification by adding lysis buffer, incubating for 5 minutes at room temperature, and stopping the reaction with stop solution. Lysates were mixed with the TaqMan® 1-Step qRT-PCR Mix and specific TaqMan® Gene Expression Assays in a total volume of 20 pL per reaction, using 1 pL of lysate as input. Amplification was carried out on a real-time PCR instrument with the following cycling conditions: 50 °C for 5 minutes for reverse transcription, 95 °C for 20 seconds for enzyme activation and initial denaturation, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. Relative expression levels were determined using the AACt method, normalizing to housekeeping GAPDH control. The following TaqMan assays were used: GFP assay (Mr04329676_mr, #4453320), GAPDH assay (Hs02786624_g1 , #4453320).

[0186] Example 1 : Efficient and modular assembly of DNA templates for saRNA manufacturing (Figure 2)

[0187] The individual plasmid parts were synthesized and cloned into GW-pUC-Amp vectors by Genewiz / Azenta. Any parts containing Bsal or Esp3l restriction sites were modified, or ‘domesticated,’ to remove these sites without altering the underlying codon usage. For instance, nine silent mutations were introduced into the nsP1234 coding sequence to eliminate these restriction sites. These parts form a foundational library using which very large variety of DNA plasmids containing saRNA templates can be built. Detailed sequences of the parts used in the assemblies are represented by SEQ ID NOs: 41 to 84.

[0188] DNA plasmids containing templates for saRNA were constructed using a Golden Gate (GG) based cloning approach. The cloning reactions were prepared by assembling a mixture that included 80 ng of each plasmid part, 1 pL of Bsal-HFv2 enzyme (Cat. No. R3733L; NEB), 1 pL of T4 DNA Ligase (Cat. No. M0202M; NEB), 2.5 pL of T4 DNA Ligase 10X Buffer (Cat. No. M0202M; NEB), and nuclease-free water to bring the total volume to 25 pL. The reactions were assembled in PCR tubes and subjected to a cycling protocol consisting of 60 cycles of 5 minutes at 37°C, followed by 5 minutes at 16°C, and a final incubation for 5 minutes at 60°C. After the reaction reach completion, the samples were stored at 4°C until they were ready for transformation.

[0189] For propagation of the successfully assembled DNA plasmids, NEB® Stable Competent E. coli (Cat. No. C3040I; NEB) or One Shot™ Maehl ™ T1 Phage-Resistant Chemically Competent E. coli (Cat. No. C862003; ThermoFisher) cells were used. The manufacturer's protocols were followed to transform these competent cells. Once the transformations were completed, colonies harboring the correct DNA plasmids were identified by Nanopore sequencing.

[0190] An example of a successful 11 -part assembly is shown in Figure 2A. The resulting DNA construct contain coding sequences for the nsp1234 polyprotein based on Venezuelan equine encephalitis (VEE) genome (strain TC-83), eGFP, Luciferase, and puromycin resistance proteins. Additionally, the construct has T7 promoter, 26S subgenomic promoter (SGP), several internal ribosome entry sites (IRES), VEEV conserved sequence elements (CSE), untranslated regions (UTRs), a polyadenylation sequence of 90 nucleotides and linearization enzyme binding sites. The VEEV conserved sequence elements (CSE) play a critical role in the viral replication cycle by ensuring proper RNA packaging and genome stability. Adding additional parts and changing layouts are possible using this modular assembly.

[0191] Example 2: In vitro transcription (IVT) of saRNA constructs (Figure 3).

[0192] In vitro transcription (IVT) was utilized to produce RNA molecules from a linearized DNA template in a cell-free system, enabling the generation of large quantities of RNA with a defined sequence (Fig. 3A).

[0193] To prepare the DNA template, plasmids were linearized using either Esp3l, Bsal-HFv2, Xbal or Mlul-HF restriction enzymes (Cat. No. R3198S; NEB), following the manufacturer’s instructions for a 2-hour incubation. Post-digestion, the linearized DNA was purified using the ReliaPrep™ DNA Clean-Up and Concentration System (Cat. No. A2891 ; Promega). DNA concentration was measured with a Nanodrop spectrophotometer, and successful linearization was confirmed via agarose gel electrophoresis.

[0194] For RNA synthesis, the linearized DNA template was transcribed using the RiboMAX™ Large Scale RNA Production System (Cat. No. P1300; Promega) and Trilink CleanCap Reagent AG (Cat. No. N- 7113-10; Trilink). A 50 pL reaction was prepared, consisting of 10 pL T7 5X buffer, 7.5 mM of each deoxynucleotide (dNTP) and CleanCap AG reagent, 5 pg of linearized DNA, and 5 pL of T7 enzyme mix. The reaction was brought to the final volume with nuclease-free water. Reactions were incubated at 37°C for 2 hours, followed by a 15-minute DNase I treatment at 37°C to degrade remaining DNA.

[0195] The resulting RNA was purified using the Monarch® RNA Cleanup Kit (Cat. No. T2040S; NEB), quantified using Nanodrop, and its integrity was assessed by agarose gel electrophoresis. The agarose gel confirmed the successful generation of 11 kb RNA constructs (Fig. 3B), demonstrating the effectiveness of the IVT process.

[0196] Example 3: saRNA Delivery into iPSC KOLF Cells Using Lipofectamine-Based Transfection (Figure 4). iPSC KOLF cells were cultured under standard conditions. One day prior to transfection, cells were seeded at approximately 50% confluence, targeting 50-70% confluence on the day of transfection to optimize conditions for efficient saRNA delivery.

[0197] On the day of transfection, saRNA constructs encoding either GFP-E3L-PuroR (Simplicon from Merck #SCR725; Fig. 4A) or GFP alone (VectorBuilder psaRVac3.1-EGFP; Fig. 4B) were introduced into the cells using Lipofectamine-based transfection, following the protocol outlined in the “Cell Transfection” section of Materials and Methods.

[0198] Two days post-transfection, the cells were imaged, revealing a significant proportion of GFP-positive cells, indicating successful transfection (Fig. 4A). To further assess transfection efficiency, flow cytometry was performed on Day 3 (details provided in the “Flow Cytometry” section of Methods). The analysis showed that over 95% of the cells expressed GFP, with more than 73% of the population displaying very bright GFP fluorescence (Fig. 4B).

[0199] These results demonstrate that saRNA-GFP constructs can be efficiently delivered into iPSC KOLF cells using Lipofectamine-based transfection reagents, achieving high levels of GFP expression across the cell population.

[0200] Example 4: Discovery of cell-type specific miRNAs in HEK293 and iPSC (Figure 5).

[0201] HEK and iPSC were cultured and sent for small RNA sequencing by GeneWIz / Azenta. In total 3 replicates of HEK293 cells and 4 replicates of iPSC were sequenced and analysed.

[0202] Limma Voom was used to calculate differentially expressed miRNAs between HEK and iPSC, defined as those with at least 2-fold difference in miRNA expression levels between any two cell types and FDR < 0.05.

[0203] From the many differentially expressed miRNAs, 6 miRNAs were chosen that were exemplary of the full range of baseline and differential miRNA expression between the cell lines: 302b, 302d, 92a, 10a, 205, 196b, see Table 2 below:

[0204] Table 2: miRNAs

[0205] The miRNAs that were found to be significantly upregulated in HEK and expressed above 100 cpm in HEK are listed below. hsa-miR-218-5p, hsa-let-7g-5p, hsa-miR-10b-5p, hsa-let-7f-5p, hsa-miR-99a-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-miR-181 a-5p, hsa-miR-10a-5p, hsa-let-7e-5p, hsa-miR-181 b-5p, hsa-miR- 125b-5p, hsa-let-7b-5p, hsa-miR-196b-5p, hsa-miR-26b-5p, hsa-let-7i-5p, hsa-miR-196a-5p, hsa-miR-215-5p, hsa-miR-615-3p, hsa-miR-29b-2-3p, hsa-miR-29b-1-3p, hsa-miR-330-3p, hsa-miR-500a-5p, hsa-miR-146b-5p, hsa-miR-362-5p, hsa-miR-502-3p, hsa-miR-582-3p, hsa- miR-1180-3p, NovelmiRNA-44, NovelmiRNA-13, hsa-miR-34a-5p, hsa-miR-484, hsa-miR-421 , hsa-miR-660-5p, hsa-miR-339-5p, hsa-miR-548b-5p, NovelmiRNA-470, hsa-miR-194-5p, hsa- miR-16-5p, hsa-miR-181d-5p, hsa-miR-30b-5p, hsa-miR-15b-5p, hsa-miR-7-5p, hsa-miR-31- 5p, hsa-miR-618, hsa-miR-7706, hsa-miR-3613-5p, hsa-miR-222-3p The miRNAs that were found to be significantly upregulated in iPSC and expressed above 100 cpm in iPSC hsa-miR-200c-3p, hsa-miR-92b-3p, hsa-miR-512-3p, hsa-miR-302d-3p, hsa-miR-130a-3p, hsa-miR-335-3p, hsa-miR-520f-3p, hsa-miR-302b-3p, hsa-miR-135b-5p, hsa-miR-302a-5p, hsa-miR-526b-5p, hsa-miR-517b-3p, hsa-miR-517-3p, hsa-miR-363-3p, hsa-miR-516a-5p, hsa-miR-20b-5p, hsa-miR-1323, hsa-miR-519b-5p, hsa-miR-523-5p, hsa-miR-519c-5p, hsa- miR-526a-3p, hsa-miR-519a-5p, hsa-miR-518c-3p, hsa-miR-187-3p, hsa-miR-520e-3p, hsa- miR-1283, hsa-miR-525-5p, hsa-miR-183-5p, hsa-miR-373-3p, hsa-miR-519b-3p, hsa-miR- 518f-5p, hsa-miR-100-5p, hsa-miR-523-3p, hsa-miR-518b, hsa-miR-519d-3p, hsa-miR-135a- 5p, hsa-miR-520a-5p, hsa-miR-182-5p, hsa-miR-204-5p, hsa-miR-219a-3p, hsa-miR-520b-3p, hsa-miR-155-5p, hsa-miR-520g-3p, hsa-miR-203a-3p, hsa-miR-96-5p, hsa-miR-518d-5p, hsa- miR-526a-5p, hsa-miR-520c-5p, hsa-miR-520g-5p, hsa-miR-486-5p, hsa-miR-95-3p, hsa-miR- 21 -5p, hsa-miR-106a-5p, hsa-miR-372-3p, hsa-miR-1298-5p, hsa-miR-200a-3p, hsa-miR-498- 5p, hsa-miR-429, hsa-miR-191-5p, hsa-miR-92a-2-3p, hsa-miR-92a-3p, hsa-miR-767-5p, hsa- miR-124-3p, hsa-miR-124-5p, hsa-miR-92a-1-3p, hsa-miR-199b-5p, hsa-miR-105-5p, hsa- miR-516b-5p, hsa-miR-345-5p, hsa-miR-371 a-5p, hsa-miR-200b-3p, hsa-miR-1-3p, hsa-miR- 874-3p, hsa-miR-130b-5p, hsa-miR-425-5p, hsa-miR-127-3p, hsa-miR-515-5p, hsa-miR-504- 5p, hsa-miR-455-5p, hsa-miR-381-3p, hsa-miR-205-5p, hsa-miR-146a-5p, hsa-miR-574-3p, hsa-miR-452-5p, hsa-miR-296-3p, hsa-miR-148a-3p, hsa-miR-224-5p

[0206] Example 5: Selection of saRNA-GFP-PuroR transfected cells using Puromycin in HEK293ft and iPS cells (Figure 6).

[0207] HEK293ft and KOLF iPSC were cultured using standard methods. Some of HEK293ft and KOLF iPSC cells were transfected with saRNA containing GFP-E3L-PuroR (Cat. No. SCR725; Merck) and put under 1 ug / mL Puromycin selection until all cells were GFP positive and thus also Puromycin resistant.

[0208] On the day of the experiment, a 96 well-plate was half seeded with non-transfected HEK293ft and another half seeded with HEK293ft that were transfected with saRNA-GFP-PuroR and selected with Puromycin. The next day, the following Puromycin doses were added onto cells in guadruplicates: 0, 0.25, 0.5, 0.75, 1 , 1 .5, 2, 6, 10 pg / ml. Cells were incubated for two days, followed by MTT assay (Cat. No. 10124622). Briefly, 10 pL of MTT solution was added to each well containing cells and medium, as well as to a negative control well containing only medium. Following incubation for 2-5 hours at 37°C, all of medium was removed from the wells, and 50 pL of DMSO was added to each well. After mixing, the samples were incubated for an additional 10 minutes at 37°C, mixed again, and the absorbance was read at 540 nm (Fig.6A-B).

[0209] In a separate experiment, iPSCs and HEK cells were transfected with saRNA containing GFP-E3L- PuroR as described above. Two days after transfection, the transfected and non-transfected cells were then subjected to Puromycin at different doses and images were taken each day for three days to monitor cell survival and confluency (Fig.6A-C). Data shows that non-transfected iPSC KOLF cells are extremely sensitive to Puromycin as even at the lowest tested concentration of 0.25ug / mL, complete cell elimination was demonstrated. On the other hand, non-transfected HEK293FT cells are more tolerant, demonstrating viable cells even at 0.5ug / mL of Puromycin. Neither of the cells can survive 1 ug / mL Puromycin for 3 days. Transfection of saRNA- GFP-E3L-PuroR into these cells gave them a resistance to lethal effects of Puromycin even at highest concentrations of 10ug / mL.

[0210] Example 6: Cell-type specific expression of saRNA-GFP-sMRE in iPSC, HEK293ft (Figure 7)

[0211] The following constructs were manufactured, each containing 4-repeats of fully complementary miRNA responsive elements (sMRE) in their 3’ UTR: PLU-R-076 (sMRE-92a-3p), PLU-R-082 (sMRE-10a-5p), PLU-R-085 (sMRE-196b-5p), PLU-R-088 (sMRE-205-5p), PLU-R-091 (sMRE-302b-3p), PLU-R-094 (sMRE-302d-3p) (Fig.7A).

[0212] To test these constructs, the following steps were taken. Cells were cultured, one day before transfection, cells were seeded at a confluence of around 50%, aiming to achieve 50-70% confluence on the day of transfection. On the day of transfection, the following constructs were delivered into either iPSC KOLF or HEK293FT (HEK) cells using Lipofectamine Messenger Max (see “Cell Transfection” for details). After transfection, cells were maintained in a 37°C incubator for 1-3 days. Flow cytometry was done on day 1 for both cell lines (Fig.7B) and then also on day 2 for HEK293ft and day 3 for iPSC KOLF to quantify GFP positive cells (Fig.7C-7D).

[0213] PLU-R-091 containing hsa-miR-302b-3p binding sites in the sMRE showed a drastic difference in % GFP positive cells between HEK and iPSCs on day 1 , returning 59% and 0.2% respectively (Fig. 7B). This clearly demonstrates cell type specific saRNA transgene suppression caused by the very high levels of hsa-miR-302b-3p in iPSC as opposed to the very low levels in HEK (Fig. 20). When looking at day two and day three flow cytometry results from 6 saRNAs containing sMREs that bind miRNAs with differing levels of expression between HEK and iPSCs, we can see that there is a clear inverse relationship between the logarithm of the expression level of a miRNA in counts per million (cpm) and the % GFP positive cells arising after transfection with the saRNA that contains the corresponding sMRE (Fig. 7C,D). For miRNAs expressed up to 100cpm (Log2 value of 6.6) there seems to be no activity. At values above 100 cpm suppression activity begins and increases until saturating around a cpm of 32768 (Log2 value of 15) where there are very little or no GFP positive cells, depending on the cell line and specific construct. This implies that each 2-fold increase in miRNA expression level above 100 cpm can lead to a roughly 10% suppression of transgene activity from corresponding saRNA-sMRE constructs (Fig. 7C,D).

[0214] Overall, the data shows that miRNA-controlled expression of transgenes can be achieved by introducing synthetic miRNA responsive elements (sMRE) in the saRNA constructs tested. With the use of cell-type specific microRNAs, cell-type specific expression of transgene can be achieved (Fig.7B). Example 7: Selective Elimination of iPSCs with Specific miRNA Expression Using saRNA-GFP-PuroR- sMRE Constructs and Puromycin Selection (Figure 8)

[0215] To demonstrate cell-type-specific elimination based on miRNA expression, saRNA constructs (PLU-R- 076, PLU-R-085, and PLU-R-094) containing unigue sMREs (sMRE-92a-3p, sMRE-196b-5p, and sMRE-302d-3p, respectively) were transfected into HEK293FT and KOLF iPSC (see ‘Materials and Methods’ for lipofectamine MessengerMax transfection details). One day after transfection, 1 pg / mL of puromycin was added to the cells. Two days later, the cells were imaged, revealing cell confluence and morphology in KOLF iPSCs and HEK293FT cells (Fig. 8A). The results showed that the constructs acted differently depending on the pre-existing miRNA levels in each cell type. miR-92a-3p levels were high in both cell lines, resulting in dead iPSCs and dead or dying HEK293FT cells (Fig. 8A, top panel). Most notably, PLU-R-94 caused a significant reduction in iPSC viability due to the high miR-302d-3p levels in these cells, while this effect was not observed in HEK293FT cells, which have low miR-302d-3p levels (Fig. 8A, bottom panel). This highlights the cell-specific interaction between PLU-R-94 and miR-302d- 3p in iPSCs.

[0216] Overall, these findings demonstrate that mid-to-high miRNA expression levels correlate with the degradation and loss of the saRNA-GFP-PuroR-sMRE construct in HEK293FT and iPSCs. In contrast, in cells with low miRNA expression, the saRNA-GFP-PuroR-sMRE constructs remain functional, allowing for puromycin resistance and GFP enrichment. By harnessing this cell-type selectivity and pairing it with antibiotic selection, specific cell populations can be enriched in culture, selectively eliminating cells with high miRNA expression while preserving those with low miRNA levels (Fig. 8B).

[0217] Example 8: Effects of saRNA on cell transcriptome (Figure 9)

[0218] KOLF iPSC cells were transfected with an equimolar pool of saRNA constructs, specifically PLU-R-001 , PLU-R-002, and PLU-R-005, using standard Lipofectamine MessengerMax transfection protocols (see Methods and Materials for details). One day post-transfection, cells were divided into two groups: one group was treated with 6 pg / mL puromycin, while the other group was left untreated. On day 3, cell pellets from both groups were collected, washed with PBS, and frozen at -80°C. The frozen pellets were then shipped on dry ice to Genewiz / Azenta for bulk RNA sequencing. Ribosomal RNA was depleted from the samples, and 20 million reads per sample were obtained for analysis.

[0219] Raw read counts from sequencing were combined into a matrix, and sample metadata was assigned based on cell type, transfection status, and puromycin concentration. Low-expression genes were filtered out (cpm < 1) and counts were Iog2 normalised. Differential expression analysis was conducted using the edgeR and Limma / Voom packages. Contrasts were set between 2 x transfected KOLF and 2 x non-transfected KOLF samples, and between 2 x transfected KOLF samples treated with 6ug / ml puromycin and 2 x untreated transfected KOLF samples. Differentially expressed genes were visualized using a volcano plot. In saRNA transfected cells, several genes were significantly upregulated, including but not limited to ISG15, IFIT1 , IFIT3, RIGI, and 0AS2, which are commonly associated with antiviral and immune responses (Fig. 9A). This suggests that the saRNA constructs triggered an innate immune response in some KOLF iPSCs. In contrast, the treatment with 6 pg / mL puromycin did not result in significant changes to the cell transcriptome, as shown in Figure 9B. Despite this relatively high dose, the effect of puromycin on gene expression was minimal and non-significant, indicating that puromycin primarily acts as a selection agent without broadly affecting transcription.

[0220] Example 9: saRNA Transfection Has Minimal Impact on iPSC Growth and Viability (Figure 10)

[0221] RCBil 0 iPSC cells were seeded at two different densities, 50k cells / cm2and 20k cells / cm2. The following day, the cells were either left non-transfected as controls or transfected with 1 pg or 2 pg of saRNA using two different transfection reagents: Lipofectamine MessengerMax and JetMessenger, following the protocols described in the "Materials and Methods" section. On day 2, the cells were split to maintain optimal growth conditions, and cell viability and live cell counts were monitored over a five-day period using the NucleoCounter® (NC-200™, ChemoMetec).

[0222] The results show that both non-transfected control cells and saRNA-transfected cells grew at similar rates throughout the five-day period, regardless of the saRNA dose (1 pg or 2 pg) or the transfection reagent used. By day 5, all conditions resulted in comparable live cell densities, indicating that saRNA transfection did not negatively impact cell proliferation. Furthermore, cell viability remained consistently high (80-90%) in all groups, demonstrating that the transfection process did not affect cell health or growth (Figure 10).

[0223] These findings suggest that saRNA delivery using Lipofectamine MessengerMax and JetMessenger is well-tolerated by iPSCs and does not impair cell proliferation or viability, even at varying cell densities and saRNA concentrations.

[0224] Example 10: Method of altering the proportion of cell types in a mixed population using saRNA molecules comprising sMREs and a selection stimulus (Figure 11)

[0225] To investigate selective enrichment or depletion of specific cell types using saRNA molecules containing sMREs, HEK293FT (HEK) and iPSC co-cultures were used (Fig 1 1. A-E). HEK and iPSC cells were seeded together and transfected with the respective saRNA constructs expressing GFP and puromycin resistance (PuroR) and containing sMREs for various miRNAs. After transfection, puromycin selection was applied for two days (Fig. 11 B-E). Subseguently, cells were analyzed by flow cytometry (FACS) using SSEA-5 and Tra-1-60 as pluripotency markers to assess the relative survival and depletion of specific cell types and GFP to validate transfection. In HEK / iPSC co-cultures, the control construct R- 006 (with no sMRE) resulted in mixed survival of both cell types (Fig. 1 1 B,E). The R-091 construct, containing the iPSC-specific miR-302b-3p sMRE, led to depletion of iPSCs (Fig. 11 C,E), whereas R- 130, containing the HEK and mesoderm-specific miR-10a-5p sMRE, selectively depleted HEK cells (Fig. 11 D,E). We also tested the ability of saRNA with sMRE to enrich specific cell types during and after differentiation. iPSCs were stably transfected with either R-006 or R-130 constructs and cultured under continuous puromycin selection for several days. saRNA-expressing iPSCs were then differentiated for four days toward mesoderm or endoderm lineages using the STEMdiff™ Mesoderm Induction Medium (Cat. 05221) or STEMdiff™ Definitive Endoderm Kit (Cat. 051 11) from Stemcell Technologies. Following differentiation, the mesodermal and endodermal cell populations were dissociated with TrypLE (~4 min), counted, and mixed at a 1 :1 ratio (200,000 cells of each population per well in a 12-well plate). The mixed cultures were maintained in Essential 8 medium supplemented with Activin A (20 ng / mL) and FGF (20 ng / mL). After seeding mixed cell populations, puromycin selection was applied, and the cell populations were analyzed by FACS on day 5 using CD56 and CD117 markers to assess lineagespecific enrichment. The control R-006 again yielded mixed survival, while R-130 conferred a pronounced enrichment of endodermal cells after puromycin selection (Fig. 11 F).

[0226] These results demonstrate that the relative proportions of a mixed population of cells can be selectively altered with the use of cell type-specific sMREs within saRNA constructs combined with a selection mechanism. Additionally, they demonstrate that the saRNAs can be delivered both after or before cell mixing and / or cell differentiation.

[0227] Example 11 : miRNA discovery and selective elimination of iPSC derived Definitive Endoderm (DE), Mesoderm and Ectoderm using saRNA-PuroR-sMRE (Figure 12)

[0228] To discover novel miRNA targets that are specific to the three main developmental lineages, we first created endoderm and mesoderm cells using the STEMdiff™ Definitive Endoderm Kit (Cat. No. 05110) alongside the STEMdiff™ Mesoderm Induction Medium (Cat. No. 05221). Endoderm and mesoderm differentiation samples were also subject to flow cytometry sorting on the BD FACS Aria system in triplicate using the markers (CD117 and CD184 high in endoderm; CD56 high for mesoderm) and capping the maximum number of sorted events per sample to 500,000. Sorted samples were stored in RNAIater buffer and frozen, before shipping to Genewiz for small RNA seguencing.

[0229] For the ectodermal lineage, smalIRNA-seg counts data from D14 iPSC derived Neurons was downloaded from the public GEO database under accession GSE65367. Average counts per million (cpm) was calculated as the mean of the cpm (using the EdgeR package) of the two control D14 neuron samples.

[0230] Two endoderm and two mesoderm and four impurity (including iPSC) smalIRNA-seg samples were processed for seguencing. 686 reliably expressed miRNAs were analysed further. The Limma Voom R package was used to normalise, model and identify differentially expressed miRNAs between the sorted populations of definitive endoderm and mesoderm and “impurities” containing iPSC as those with at least 2-fold difference in miRNA expression levels between any two cell types. The miRNAs that were found to be significantly upregulated in mesoderm and expressed above 100 cpm in mesoderm are listed below.

[0231] • hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-615-3p, hsa-miR-27b-3p, hsa-miR-196a-5p, hsa- miR-196b-5p, hsa-miR-335-3p, hsa-miR-363-3p, hsa-miR-155-5p, hsa-miR-143-3p, hsa-miR- 146b-3p, hsa-miR-96-5p, hsa-miR-24-2-3p, hsa-miR-24-1-3p, hsa-miR-24-3p, hsa-miR-23b- 3p, hsa-miR-199b-3p, hsa-miR-5100, hsa-miR-4473, hsa-miR-345-5p, NovelmiRNA-605

[0232] The miRNAs that were found to be significantly upregulated in endoderm and expressed above 100 cpm in endoderm are listed below:

[0233] • hsa-miR-148a-3p, hsa-miR-302a-5p, hsa-miR-375-3p, hsa-miR-1260b, hsa-miR-1260a, hsa- miR-472, sa-miR-148b-3p, hsa-miR-1263, hsa-miR-1247-3p, hsa-miR-378-3p, hsa-miR-106b- 3p, hsa-miR-28-3p, hsa-miR-200c-3p, hsa-miR-200b-3p, hsa-miR-140-3p, hsa-miR-127-3p, hsa-miR-489-3p, hsa-miR-379-5p, hsa-miR-323a-3p, hsa-miR-409-3p, hsa-miR-2113, hsa- miR-548au-3p, hsa-miR-3158-3p, novelmiRNA-529, novelmiRNA-472

[0234] Literature review was performed to identify miRNAs for ectoderm (neural progenitors) and the list was filtered using our in-house and public smalIRNA-seq data. sMREs that are targeted by specific miRNAs, namely hsa-miR-375-3p, hsa-miR-10a-5p, hsa-miR-10b- 5p, hsa-miR-196a-5p, hsa-miR-218-5p, hsa-miR-219a-5p were synthesized by Genewiz / Azenta and cloned into saRNA templates as described previously. Following in vitro transcription (IVT) to manufacture these molecules, iPSC cells were transfected with the saRNA constructs on day 0. From day 1 , the cells were continuously treated with 0.5 pg / mL puromycin, and cell differentiation was initiated once cell growth had stabilised (usually day 3-5). On days 5-10 post differentiation induction, images were taken to assess cellular survival.

[0235] The following protocols were used for differentiations: endoderm and mesoderm cells using the STEMdiff™ Definitive Endoderm Kit (Cat. No. 05110) alongside the STEMdiff™ Mesoderm Induction Medium (Cat. No. 05221) as per the manufacturer’s instructions. For ectoderm differentiation, dual smad inhibition protocol was used (cell cultured in E6 medium supplemented with 10uM SB431542 + 500nM LDN193189).

[0236] The results showed that the saRNA-sMRE constructs effectively and specifically eliminated each differentiating cell lineage (mesoderm, endoderm and ectoderm) enabling targeted purification of a desired differentiated cell product (Fig. 12). This approach demonstrated the ability of saRNA-sMRE to enhance the efficiency of cell purification during the differentiation process. Example 12: Optimization of sMRE design for controlling functional responsiveness and activity of saRNA-sMRE constructs (Figure 13)

[0237] To better control the functional responsiveness of synthetic miRNA recognition elements (sMREs), we designed and tested a panel of saRNA-GFP-sMRE variants in HEK293 and iPSC cultures (Fig. 13). Each variant incorporated different sMRE configurations to systematically evaluate the effects of miRNA binding site copy number, combinatorial targeting, positional context, and seguence complementarity on construct activity. Following transfection with the saRNA-GFP-sMRE variant, GFP fluorescence was guantified by imaging and flow cytometry at days 1 and 3 post-transfection, and GFP+cell fractions were normalized to control constructs lacking sMREs (R-006).

[0238] Variants R-128 and R-147 contained one or two copies of miR-302b-3p target sites respectively (Fig. 13A). Even a single miRNA binding site was sufficient to elicit strong repression of saRNA expression in iPS cells expressing the corresponding miRNA (Fig. 13B-C).

[0239] R-082, R-085, and R-115 included combinatorial sMREs designed to target distinct miRNAs, specifically miR-10a-5p and miR-196b-5p (Fig. 13D). Constructs containing dual sMREs targeting distinct miRNAs exhibited synergistic or additive effects, resulting in enhanced degradation of the saRNA (Fig. 13E-F).

[0240] To evaluate positional influences, sMREs were placed in different regions of the saRNA molecule, including the 5' untranslated region (UTR), a pre-subgenomic promoter (pre-SGP) position, or the 3'UTR (constructs R-091 , R-253, and R-127 in Fig. 13G). sMREs maintained functional activity when positioned in various locations along the saRNA molecule, indicating that regulatory tuning can be achieved independently of insertion site (Fig. 13H-I).

[0241] Finally, R-091 and R-152 were compared to assess the impact of sMRE complementarity, with R-152 containing partially complementary binding sites (approximately 45% complementarity to the cognate miRNA) (Fig. 13J). Constructs with partially complementary sMREs demonstrated near-complete repression efficiency, confirming that reduced complementarity does not abolish functionality and that the sMRE system is tolerant to flexible sequence design (Fig. 13K-L).

[0242] Overall, these findings establish that sMRE parameters - including number, position, degree of complementarity, and multi-miRNA combinations - can be rationally engineered and tuned independently or in combination to achieve robust and customizable cell type-specific regulation of saRNA activity.

[0243] Example 13: Influence of RNA secondary structure and accessibility on sMRE functionality in saRNA constructs (Figure 14)

[0244] To investigate the influence of RNA secondary structure and accessibility on sMRE functionality within saRNA constructs, we transfected HEK and iPSCs with a series of saRNA-GFP-sMRE variants containing four miR-302b-3p embedded in distinct RNA structural contexts within the 3' untranslated region (UTR) of the saRNA (Fig. 14). Each construct was designed to systematically vary the local RNA folding environment around the sMRE to assess how structure-driven accessibility impacts miRNA- mediated repression. For miR-302b-3p, three configurations were tested: R-091 , featuring a canonical unstructured context of four binding sites in a row; R-092, in which the sMRE was embedded within a predicted double-stranded stem; and R-093, with the sMRE forced into a single-stranded loop region by flanking each binding site with a fully complementary stem (Fig. 14A). In parallel, analogous constructs were created for miR-92a: R-076 (canonical), R-077 (stem-embedded), and R-078 (loop-embedded) (Fig. 14B).

[0245] Predicted RNA secondary structures were modelled computationally using MXFold2 to estimate the relative accessibility of each sMRE configuration. Constructs were transfected into HEK and iPSCs, and GFP fluorescence was measured by flow cytometry at days 1 and 3 post-transfection, with GFP+cell fractions normalized to a control construct lacking sMREs (R-006). RNA structure predictions revealed that both the canonical and loop-embedded sMREs were highly accessible, with over 80% of the miRNA binding sequence predicted to be single-stranded and accessible for miRNA binding (Fig. 14A). Correspondingly, these constructs exhibited strong repression of GFP expression, indicating efficient miRNA-mediated degradation of the saRNA (Fig. 14A,C,D). In contrast, stem-embedded sMREs displayed lower accessibility and substantially reduced repression activity (Fig. 14A,C,D). This finding was repeated with constructs R-076-78 in HEK cells (Fig. 14B).

[0246] Together, these results demonstrate that local RNA folding and secondary structure play a critical role in determining sMRE accessibility and, consequently, the efficiency of miRNA-directed degradation. Engineering sMREs to be highly accessible, for example by using stem-loops or open-unstructured contexts, enhances their responsiveness, providing a tunable structural design parameter for optimizing saRNA-sMRE performance.

[0247] Example 14: Regulation of saRNA replication and responsiveness to degradation through 5'UTR engineering as a sensitizing modification (Figure 15)

[0248] We engineered a set of saRNA-GFP constructs with modified 5'UTRs and assessed their expression levels in iPSCs (Fig. 15A-C). The constructs included a control (R-161) containing an intact 5'UTR, as well as variants R-160, R-155, and R-157, in which specific stem-loop structures (SL1-SL4) were disrupted to varying degrees, visualised using Geneious Andronescu 2007 RNA energy model (Fig. 15A-B). iPSCs were then transfected with each construct and cultured continuously under puromycin selection for up to 21 days. Flow cytometry was used to assess GFP fluorescence intensity as a readout of saRNA expression. Disruption of the SL1-SL4 stem-loops in the 5'UTR resulted in markedly reduced GFP mean fluorescence intensity (MFI) compared to the control construct (Fig. 15C). Cells harboring the disrupted constructs maintained viability and GFP expression under puromycin selection for more than 20 days, indicating that the modified saRNAs were capable of sustained replication at reduced efficiency. This suggested that perturbation of the 5'UTR secondary structure dampened saRNA amplification but did not abolish replication entirely, leading to a lower stable GFP expression level likely due to a lower RNA copy number.

[0249] In a separate experiment, RT-qPCR was used to quantify levels of GFP mRNA levels from construct R- 130 which also contained a heavily disrupted SL1 . This revealed faster decay of RNA transcripts relative to R-006, consistent with reduced RNA replication efficiency and / or decreased transcript stability (Fig. 15D-E).

[0250] These findings demonstrate that engineering the 5'UTR secondary structure provides a means to finetune the balance between saRNA replication and copy number. By selectively disrupting conserved stem-loop elements, the saRNA copy number can be reduced, enabling more sensitive tunable control over saRNA-sMRE construct behavior in the presence of miRNAs.

[0251] Example 15: Engineering of saRNA constructs through decoupling of the subqenomic promoter (SGP) from nonstructural protein (nsP) coding regions as a sensitizing modification enabling independent regulation and selective placement of regulatory elements (Figure 16)

[0252] In order to increase our control over the transgene expression levels independent of saRNA replication stability, we engineered a series of saRNA constructs in which the sub-genomic promoter (SGP) was selectively modified, decoupled, or truncated relative to the nonstructural protein (nsP) coding region the SGP is normally partly embedded in (Fig. 16). iPS cells were transfected with these constructs, which included an intact wild-type control (R-006) and a panel of variants: R-162 and R-223 (decoupled SGPs), R-188 (mutated SGP), R-192 (truncated SGP), and R-238 (rescued SGP restoring promoter functionality).

[0253] Following transfection, GFP expression was quantified by flow cytometry at days 1 and 3, measuring both the proportion of GFP-positive cells and the mean fluorescence intensity (MFI) within the GFP- positive population. Constructs featuring decoupled SGPs (R-162 and R-223) displayed GFP-positive cell fractions comparable to the intact control (R-006), indicating that RNA replication and initial transcript synthesis were preserved (Fig. 16). However, these variants exhibited a roughly fourfold reduction in GFP MFI, suggesting a pronounced decrease in subgenomic promoter-driven expression levels and a lower abundance of subgenomic RNA transcripts. In contrast, constructs carrying mutated (R-188) or truncated (R-192) SGP sequences showed minimal to no detectable GFP expression, confirming that promoter integrity is essential for functional expression of the reporter gene. Notably, the rescued variant R-238 restored activity nearly to control levels, validating that the observed attenuation in other variants stemmed specifically from SGP disruption rather than unrelated structural effects (Fig. 16).

[0254] Together, these findings demonstrate that decoupling the SGP from the nsP coding region provides a powerful means to independently regulate replication and transgene expression within the saRNA system. By relocating the SGP, it becomes possible to fine-tune promoter-driven expression while maintaining core replication competency. Moreover, this decoupling architecture permits the upstream insertion of regulatory elements such as sMREs, thereby restricting miRNA-mediated degradation to genomic RNA and enhancing the modularity, flexibility, sensitivity and precision of saRNA-sMRE construct design.

[0255] Example 16: Regulation of saRNA stability and abundance through AU-rich element (ARE) insertion as a sensitizing modification enabling more sensitive miRNA-based regulation (Figure 17)

[0256] To investigate how RNA stability can be modulated to enhance the sensitivity of saRNA constructs to miRNA-mediated regulation, we engineered and tested a series of saRNAs containing AU-rich elements (AREs) alone or in combination with sMREs (Fig. 17). AREs are known cis-acting destabilizing motifs that promote transcript decay, and their incorporation was hypothesized to sensitize saRNA molecules to miRNA-guided degradation, thereby enabling more tunable regulation of RNA abundance. HEK and iPSCs were transfected with saRNA-GFP constructs containing increasing numbers of ARE repeats or combinations of AREs and sMREs. iPSCs were maintained under puromycin selection for 10 days (Fig. 17B,C). Flow cytometry measurements were taken at days 1 , 3 and 10 post-transfection (Fig. 17B-E) The first set of constructs (R-247, R-248, and R-249) contained 5*, 10*, and 15* ARE repeats, respectively, inserted downstream of the GFP. Flow cytometry analysis revealed a decrease in GFP mean fluorescence intensity with any number of ARE copies (Fig. 17B,C). Despite this reduction in expression, the transfected iPSCs remained viable and maintained stable populations under puromycin selection for over 10 days, indicating that the reduced GFP signal reflected lower RNA abundance rather than cytotoxicity. These results confirm that the inclusion of ARE motifs effectively decreases saRNA stability and steady-state expression levels in a controlled, repeat-dependent manner.

[0257] To assess whether ARE-mediated destabilization could enhance miRNA responsiveness, we next tested constructs combining sMREs with AREs. The construct R-211 contained three distinct sMREs targeting different miRNAs, serving as a benchmark for strong miRNA-driven degradation. R-344 carried a single sMRE targeting the lowly expressed miRNA miR-200c-3p and showed minimal repression in isolation. Moving the sMRE upstream of the SGP (as seen in R-232) mildly increased the repression. Remarkably, the addition of 5 AREs as seen in the R-345 construct transformed the weakly responsive single sMRE designs (R-344, R-232) into a highly responsive construct, producing repression effects comparable to those achieved by R-211 (Fig. 17D-E).

[0258] These results demonstrate that AU-rich elements and sMREs placed upstream or downstream of the SGP can act synergistically to control RNA stability and fine-tune saRNA levels in response to lowly expressed miRNAs. These sensitizing modifications enable more responsive and precise miRNA-based regulation, expanding the versatility of saRNA-sMRE design for selective, cell-type-specific gene expression control. Example 17: Simultaneous induction and selective purification of differentiated lineages using saRNA- sMRE constructs encoding lineage-defining transcription factors and selection mechanisms (Figure 18) To demonstrate the dual capability of engineered saRNA-sMRE constructs to simultaneously induce differentiation and purify specific differentiated lineages, we designed saRNA molecules encoding lineage-defining transcription factors combined with sMRE-linked selection mechanisms (Fig. 18A). Human iPSCs were transfected with these constructs and maintained under standard pluripotent culture conditions. For neuronal induction, construct R-195 was created, encoding the proneural transcription factor NGN2 as well as E3L, GFP and PuroR. Within five days of transfection, R-195-transfected iPSCs displayed neuron-like morphologies characterized by elongated processes and clustered networks, accompanied by strong GFP fluorescence (Fig. 18B). These phenotypic changes were consistent with rapid and efficient NGN2-driven neuronal differentiation, confirming that saRNA-mediated expression of lineage-specifying transcription factors can trigger direct conversion of iPSCs into neuronal lineages even overcoming pluripotent maintenance media.

[0259] To extend this approach to myogenic differentiation and test selective purification, we generated constructs R-335 and R-321 encoding human MY0D1 (hMyoDI) alongside a thymidine kinase (TK) selection module, E3L, PuroR and sMREs. R-335 also included an iRFP reporter gene. Both constructs incorporated sMREs complementary to myocyte expressed miRNAs (miR-1-3p, miR-133b-3p, and miR- 206-3p). Upon transfection, both constructs created myosin heavy chain (MyHC) positive cells, however the vast majority were non-MyHC positive cells suggesting inefficient conversion and differentiation. When 10 pM ganciclovir (GCV) was applied to cultures transfected with R-321 and R-335, non-myogenic cells expressing active HSV-TK underwent selective elimination, resulting in a striking enrichment of MYHC-positive myocytes (Fig. 18C-E). This confirmed that the saRNA-TK-sMRE system could be leveraged for post-differentiation purification, effectively coupling lineage induction with selective cell removal.

[0260] The enrichment in Fig. 18E was quantified via image analysis. For each well 49 images were captured in a 7x7 grid using the Operetta automated imaging system. Immunocytochemistry (ICC) images were quantified in Imaged using a custom macro. In brief, DAPI stained images were rolling ball background corrected with a grid size of 30 pixels. They were then converted to 8-bit and thresholded using the ‘Moments’ function, before being converted to a mask and applying the watershed function. Individual nuclei were annotated using ‘Analyse particles’ with a size range of 25-1000 pixels. The non DAPI images (i.e. MyHC) were converted to a mask using a raw threshold value of 14000. Each DAPI annotated nucleus with a mean MyHC value > 1 across its area was annotated to be MyHC positive. The mean and standard deviation of the fraction of DAPI nuclei that are MyHC positive across the 49 images in each well are reported.

[0261] Together, these findings show that saRNA-based delivery of lineage-defining transcription factors enables rapid and efficient differentiation of iPSCs into specific cell types, while the inclusion of sMRE- regulated, TK-linked selection modules allows for precise enrichment of the desired lineage. This combined strategy of induction plus purification establishes a powerful and modular framework for generating homogeneous, lineage-specific cell populations suitable for downstream therapeutic or research applications.

[0262] Example 18: Expansion of saRNA responsiveness to low-abundance miRNAs via synergistic sMRE configurations and sensitising modifications, enhancing specificity and broad applicability of the invention (Figure 19-20)

[0263] To demonstrate that the sensitized saRNA-sMRE configurations previously shown to reduce GFP expression could also translate into improved cell-type purification, we applied them to both HEK and iPSCs (Figs. 19-20). Cells were transfected with control or sensitized saRNA-sMRE constructs, including those containing design features such as AREs, decoupled SGPs, or multi-sMRE architectures, and analysed with FACS at days 1 and 3 or exposed to puromycin selection followed by imaging.

[0264] Consistent with earlier fluorescence data, these constructs showed cell-type specific degradation of various amounts (Fig. 19A-D). Combining multiple sensitising modifications together as in R-345 exhibited the strongest cell type specific miRNA mediated suppression of GFP expression seen using miRNAs expressed below 2000 counts per million (Fig. 19C, 20), corresponding to approximately a 15- fold difference in miRNA expression between cell-types.

[0265] Fig. 19E shows that constructs targeting individual low-abundance miRNAs (Fig. 20) produced small GFP-positive iPSC colonies, indicating that while each sMRE alone induced measurable miRNA- mediated degradation, residual saRNA activity supported survival under selection. In contrast, combining these targets within a multi-sMRE construct (R-211) completely abolished viable GFP- positive colonies in iPSC and led to huge reductions in HEK cells, demonstrating a synergistic degradation effect driven by the combined action of multiple low-abundance miRNAs on sensitized saRNA-sMRE constructs, corresponding to a 3.5-fold difference in miRNA levels between selected and non-selected cells (PLU-R-232 vs PLU-R-21 1 in iPSC).

[0266] These results demonstrate that sensitizing modifications enhance sMRE activity at the RNA level and also translate into more effective cell purification, extending the utility of the saRNA-sMRE purification system to very lowly expressed miRNAs, which form the majority of miRNAs in any given cell type.

[0267] Example 19: Negative strand targeting (Figure 21)

[0268] To assess whether sMREs positioned in the antisense orientation (i.e. targeting the negative strand of saRNA) can confer functional miRNA-dependent regulation of transgene expression, we engineered R- 110 containing the reverse complement of the miR-302b-3p binding site in the sMRE. GFP expression was guantified by flow cytometry at days 1 and 3 post-transfection in HEK and iPSCs. Robust expression from R-110 was seen in HEK cells (83% GFP positive) but was almost completely missing in iPSCs (4% GFP positive), demonstrating that negative strand miRNA targeting worked to effectively suppress saRNA based expression in a cell-type specific way (Fig. 21 A, B). Brightfield imaging was performed on day 7 under puromycin selection, demonstrating that the reduction in GFP translated to a loss of puromycin resistance and effective selective elimination of iPSCs (Fig. 21 C). These results demonstrate that sMREs designed to target the negative saRNA genome strand remain functional and capable of directing miRNA-mediated saRNA repression. This finding expands the structural and regulatory flexibility of the saRNA-sMRE platform, enabling bidirectional or antisense- embedded configurations for precise, context-specific control.

Claims

CLAIMS1. A self-amplifying RNA (saRNA) molecule comprising:(a) a region coding for a viral non-structural protein (nsP);(b) a conserved sequence element (CSE); and(c) an untranslated region; and(d) a polyadenyl sequence wherein the saRNA molecule further comprises a synthetic microRNA (miRNA) responsive element (sMRE); and wherein the saRNA molecule further comprises a transgene.

2. The self-amplifying RNA (saRNA) molecule according to claim 1 , wherein the conserved sequence element (CSE) comprises a 5’ conserved sequence element (CSE) and / or a 3’ conserved sequence element (CSE).

3. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the saRNA construct further comprises one or more RNA regulatory elements selected from the group consisting of an internal ribosome entry sequence (IRES), a ribozyme, a Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE), an AU-rich element, and an RNAse recognition sequence and splicing site.

4. The self-amplifying RNA (saRNA) molecule according to claim 3, where the one or more RNA regulatory elements comprises an IRES.

5. The self-amplifying RNA (saRNA) molecule according to claim 3 or claim 4, where the one or more RNA regulatory elements comprises an AU-rich element.

6. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the transgene encodes a 2A peptide.

7. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the sMRE is fully complementary to the miRNA sequence.

8. The self-amplifying RNA (saRNA) molecule according to any preceding claim wherein the saRNA further comprises at least one of element selected from the group consisting of a subgenomic promoter (SGP), a 5’-cap, a 5 -UTR, and a 3 -UTR or a combination thereof.

9. The self-amplifying RNA (saRNA) molecule according to claim 8, wherein the sMRE are located at the 5’ UTR, pre-SGP, post-SGP and / or 3’ UTR.7410. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the transgene is a selection marker selected from the group consisting of a drug resistance gene, a suicide gene, a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein.

11. The self-amplifying RNA (saRNA) molecule according to claim 10, wherein the drug resistance gene is an antibiotic resistance gene.

12. The self-amplifying RNA (saRNA) molecule according to claim 11 , wherein the antibiotic resistance gene is selected from the group consisting of puromycin N-acetyl-transferase (PuroR), an aminoglycoside 3'-phosphotransferase, Sh ble (BleoR), hygromycin B phosphotransferase, dihydrofolate reductase and blasticidin S deaminase, or a combination thereof.

13. The self-amplifying RNA (saRNA) molecule according to claim 12, wherein the antibiotic resistance gene is selected from the group consisting of PuroR, and BleoR, or a combination thereof.

14. The self-amplifying RNA (saRNA) molecule according to any one of claims 10 to 13, wherein the suicide gene is selected from the group consisting of Herpes simplex virus-thymidine kinase (HSV- TK), nitroreductase (NTR), cytosine deaminase (CD), carboxypeptidase G2 (CPG2), purine nucleoside phosphorylase (PNP), Granzyme, Caspase-9 (Casp9), Diphtheria Toxin A (DT-A), Fas Ligand (FasL), Ribonuclease, Bcl-2-associated X protein (Bax) and TNF-Related Apoptosis-Inducing Ligand (TRAIL), or a combination thereof.

15. The self-amplifying RNA (saRNA) molecule according to claim 14, wherein the suicide gene is Herpes simplex virus-thymidine kinase (HSV-TK).

16. The self-amplifying RNA (saRNA) molecule according to any one of claims 10-15, wherein the immunomodulator is selected from the group consisting of E3L, B18R, and Adenovirus VA RNA, or a combination thereof.

17. The self-amplifying RNA (saRNA) molecule according to claim 16, wherein the immunomodulator is E3L.

18. The self-amplifying RNA (saRNA) molecule according to any one of claims 10 to 17, wherein the fluorescent protein is selected from the group consisting of enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), iRFP670, mCherry, LSSmApple.

19. The self-amplifying RNA (saRNA) molecule according to claim 18, wherein the fluorescent protein is EGFP.

20. The self-amplifying RNA (saRNA) molecule according to claim 18 or claim 19, wherein the fluorescent protein is iRFP670.

21. The self-amplifying RNA (saRNA) molecule according to any one of claims 10 to 19, wherein the transcription factor is selected from the group consisting of ASCL1 , POU3F2 (BRN2), MYT1 L, NEUROD1 , GATA4, MEF2C, TBX5, HAND2, FOXA2, HNF4A, PDX1 , NEUROG3 (NGN3), MAFA, MYOD1 , ETV2, FOXC2, FLI1 , CEBPA, PPARG, SOX10, OLIG2, NFIA, LIN28A, SALL4, LMX1A, PITX3, NR4A2 (NURR1), NKX2-5, ISL1 , SOX17, GFI1 , POU4F2 (BRN3B), ATOH1 , GATA6, NKX6-1 , FOXP3, HOXA9, MEIS1 , TAL1 , RUNX1 , CDX2, EOMES, TCF7L2, ESRRB, NR5A2, LHX3, LHX6, PAX6, PAX7, SOX9, PROX1 , TCF21 , ARNTL (BMAL1), NEUROG2 (NGN2), HNF1 B, HOXA5, PAX3, EMX2, LHX2, PITX2, PAX4, SOX11 , IRX3, ISL2, FOXD3, MSX1 , PHOX2B, SP8, GATA3, TCF7, BCL1 1 B, NOTCH1 , IKZF1 , LEF1 , RXR, STAT5, FOXP1 , ID2, MAFB, ZBTB16 (PLZF), GATA1 , BAF60C, JMJD3, NKX2-2, RFX6, HNF1A, ARX, HES1 , GLIS3, ONECUT1 , SPI1 (PU.1), C / EBPp, KLF1 , BCL2, MYOG, HHEX, GATA2, GFI1 B, SNAI2 (Slug), NANOG, SOX7, HNF6, CITED2, HAND1 , HOXB4, ZEB1 , TCF4, HES5, SIX2, IRF8, MNX1 (HB9), HOXC8, HOXC9, PHOX2A, LMX1 B, EN1 , OTX2, TH, FOXA1 , TP63, PAX2, MITF, NRL, SIX3, CEBPG, NKX2-1 (TTF1), RUNX2, SPDEF, TBX20, and NKX3-1.

22. The self-amplifying RNA (saRNA) molecule according to claim 21 wherein the transcription factor is used to direct cells toward a particular fate.23 The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the saRNA further comprises a sensitising modification.

24. The self-amplifying RNA (saRNA) molecule according to claim 23, wherein the sensitising modification comprises:(a) modification of the secondary structure of the UTR and / or CSE;(b) incorporation of one or more RNA regulatory / stability elements;(c) modification of the genomic organization to decouple the SGP from the nsP coding sequence; and / or(c) incorporation of a combination of sMRE specific to a particular cell type and / or lineage.

25. The self-amplifying RNA (saRNA) molecule according to claim 24, wherein the sensitising modification comprises incorporation of five or more AU-rich elements (AREs).

26. The self-amplifying RNA (saRNA) molecule according to any one of claims 23 to 25, wherein the sensitising modification is present in the coding region for the nsP, the SGP and / or the CSE.

27. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the saRNA further comprises a sequence encoding a post-translational regulatory element selected from the group consisting of a degron, a ubiquitination signal, a phosphorylation site, a SUMOylation site, aprotease cleavage site, a destabilization domain, a ribozyme skipping 2A peptide, or a combination thereof.

28. The self-amplifying RNA (saRNA) molecule according to any preceding claim, wherein the saRNA is encapsulated in a lipid nanoparticle (LNP).

29. A method of altering the proportion of a first cell type within a population of cells relative to the proportion of a second cell type in the population of cells, where the population of cells is differentiated from a population of undifferentiated cells, the method comprising:(a) delivering a self-amplifying RNA (saRNA) molecule into the population of undifferentiated cells, wherein the saRNA molecule comprises a transgene encoding a selection marker selected from the group consisting of a drug resistance gene, a suicide gene, a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein, wherein each saRNA construct comprises a synthetic microRNA (miRNA) responsive element (sMRE) complementary to a miRNA present in the cells of the second cell type in the population;(b) causing the undifferentiated cells to differentiate into the population of cells;(c) allowing the sMRE to hybridize to the miRNA and thereby for the saRNA to be degraded in the cells of the second cell type in the population; and(d) applying a selection stimulus, whereby after step (d) the proportion of the first cell type in the population of cells is altered relative to the proportion of the second cell type in the population of cells.

30. The method according to claim 29, wherein the undifferentiated cells are pluripotent stem cells, optionally induced pluripotent stem cells (iPSCs).

31. The method according to claim 29 or 30, wherein the transgene encodes a transcription factor which drives differentiate into a specific cell type or specific lineage.

32. A method of altering the proportion of a first cell type within a population of cells relative to the proportion of a second cell type in the population of cells, the method comprising:(a) delivering a self-amplifying RNA (saRNA) molecule into the population of cells, wherein the saRNA molecule comprises a transgene encoding a selection marker selected from the group consisting of an antibiotic resistance gene, a suicide gene, a transcription factor, inhibitor, immunomodulator, a cell surface marker and / or a fluorescent protein, wherein each saRNA construct comprises a synthetic microRNA (miRNA) responsive element (sMRE) complementary to a miRNA present in the second cell type;(b) allowing the sMRE to hybridize to the miRNA and thereby for the saRNA to be degraded in the cells of the second cell type in the population; and(c) applying a selection stimulus whereby after step (c) the proportion of the first cell type in the population of cells is altered relative to the proportion of the second cell type in the population of cells.

33. The method according to any one of claims 29 to 32, wherein the selection marker is a drug resistance gene and the proportion of the first cell type is increased relative to the second cell type wherein the first cell type is a desired cell type.

34. The method according to claim 33, wherein the drug resistance gene is PuroR and / or BleoR.

35. The method according to any one of claims 29 to 34, wherein the selection marker is a suicide gene and the proportion of the first cell type is decreased relative to the second cell type wherein the second cell type is a desired cell type.

36. The method according to claim 35, wherein the suicide gene is HSV-TK.

37. The method according to any one of claims 29 to 36, wherein the saRNA is an saRNA according to any one of claims 1 to 28.

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