WO2025196762A1

WO2025196762A1 - Synthetic nucleic acid molecules sensing cell and/or environmental conditions, to autonomously provide a desiered cell phenotype, cells, products and uses thereof

Info

Publication number: WO2025196762A1
Application number: PCT/IL2025/050265
Authority: WO
Inventors: Nir Shani; Lior Nissim
Original assignee: Meatologic Ltd; Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Meatologic Ltd; Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2024-03-22
Filing date: 2025-03-20
Publication date: 2025-09-25
Anticipated expiration: 2026-09-22

Abstract

The present disclosure relates to methods for the production of a cell-based product based on using synthetic promoters or a cellular input-output unit thereof, configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing dynamic cellular- and/or environmental- state and/or condition, that leads to a desired phenotype of the cells. The present disclosure further provides cell-based products, in particular, animal cell-based meat products (ACBM).

Description

SYNTHETIC NUCLEIC ACID MOLECULES SENSING CELLULAR AND/OR ENVIRONMENTAL CONDITIONS, TO AUTONOMOUSLY PROVIDE A DESIERED CELL PHENOTYPE, CELLS, PRODUCTS AND USES THEREOF

FIELD OF THE INVENTION

The present disclosure relates to synthetic biology. More specifically, the present disclosure provides synthetic biology tools for autonomously controlling cell phenotype and growth in dynamic cellular- and/or environmental- state and/or conditions and uses thereof in producing cellbased products, specifically, animal cell-based meat (ACBM) products.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

[1] Wu, M.-R., Nissim, L., Stupp, D., et al. (2019) Nature communications 10:1-10.

- [2] Nissim, L„ Wu, M.-R., Pery, E„ et al. (2017) Cell 2017;171:1138-1150.

- [3] WO 2020/261277.

- [4] WO 2018/169901.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND OF THE INVENTION

Emerging technologies across the alternative protein landscape are poised to transform protein production in the coming years, by offering higher efficiency, greater consistency, and fewer harms to public health, the environment, and animals as compared with conventional meat production. Within this landscape, the animal cell-based meat (ACBM) industry is a relatively young but rapidly growing field. Despite massive funding and dozens of companies promoting the effort, the high cost of production remains a significant challenge for the ACBM industry. According to current publications, in addition to economic scalability that has not been achieved yet, development progress in various areas of ACBM is significantly lagging behind previous projections.

Culturing of mammalian cells in quantities sufficient to support industrial-scale production is complex, challenging, and requires specific conditions and signals that control and guide the cell through the different stages of the production process that includes high cell mass production, and provision of food products displaying specific tissue architecture (meat- like tissue).

The "SmartCell platform", is a biological software system based on optimization of a next generation synthetic biology technology described in Nissim et al. [l-2].Nissim et al., further provided systems for expressing transcripts regulated by an mRNA trans-splicing-based Boolean and logic gate, i.e. expression of transcripts that occur only in the simultaneous presence of two different input signals [3]. Lue et al. [4], provides synthetic promoters that are differentially modulated between certain diseased cells.

There is a need for developing a manufacturing process for ACBM production that is highly regulated, controlled and reproducible.

Moreover, to upscale ACBM production and reach price parity with livestock-based meat, there is a need to introduce novel interdisciplinary solutions.

SUMMARY OF THE INVENTION

Thus, a first aspect, the present disclosure relates to a method for the production of a cell-based product, the method comprising the step of introducing into at least one cell, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or a cellular platform thereof. The conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, optionally, during and/or associated with cell culturing and/or production of the cell-based product. More specifically, the controlled expression of at least one nucleic acid sequence of interest, results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site.

A further aspect of the present disclosure relates to a cell-based product comprising at least one cell or at least one population of the cells, or any product of interest produced by, or produced from the cells. The cell/s of the disclosed product comprise and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising the at least one nucleic acid molecule. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition. Thereby the cell/s of the disclosed cell-based product autonomously exhibits at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

A further aspect of the present disclosure relates to a method of preparing an animal cell-based meat (ACBM) product. The method comprising: (a), culturing under suitable conditions at least one source cell or at least one population of the cells. It should be understood that the source cell/s comprise and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence (specifically, a synthetic promoter), or an inputoutput (or sensor-output) unit, a nucleic acid cassette and/or a platform comprising the nucleic acid molecule. The next or additional step (b), involves processing the cells and/or at least one product produced by, or produced from the cell to prepare the food product. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition. Still further, the controlled expression of at least one of the nucleic acid sequences of interest results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

A further aspect of the present disclosure relates to an animal cell-based meat (ACBM) product comprising at least one cell or at least one population of the cells, and/or any product of interest produced by, or produced from said cells. The cell/s of the ACBM disclosed herein may comprise and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising the at least one nucleic acid molecule. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, thereby the cell of the disclosed ACBM, autonomously exhibits at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

A further aspect of the present disclosure relates to a nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions. The controlled expression of at least one of the nucleic acid sequences of interest results in at least one desired phenotype adapted to the suspension condition. It should be understood that the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. More specifically, the transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine (A) or thymine (T), S is guanin (G) or cytosine (C), and N is any nucleic acid residue.

Still further, the present disclosure provides in another aspect thereof, a nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing adherence conditions. The controlled expression of at least one of the nucleic acid sequence of interest, results in at least one desired phenotype adapted to the adherence condition. More specifically, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. Specifically, the transcription factor binding site comprising the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; or any functional fragments thereof; wherein R is adenine (A) or guanin (G), H is A, cytosine (C) or thymine (T), D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

A further aspect relates to a cellular input-output unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cell and/or environmental state and/or condition. The unit comprising: (i) at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The at least one synthetic conditional nucleic acid sequence of (i), is configured for controlling the expression of at least one nucleic acid sequence of interest of (ii), upon sensing one of:

(I), suspension conditions, accordingly, the controlled expression of at least one of the nucleic acid sequence of interest results in at least one desired phenotype adapted to the suspension condition. More specifically, the synthetic conditional nucleic acid sequence of the disclosed unit comprises at least two repeats of a transcription factor binding site, and the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

Alternatively, or additionally, (II), adherence conditions, in such case the controlled expression of at least one nucleic acid sequence of interest results in at least one desired phenotype adapted to the adherence condition. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. More specifically, the transcription factor binding site comprising the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

A further aspect relates to a nucleic acid cassette or any vector thereof, comprising at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or at least one cellular input-output unit comprising the at least one nucleic acid molecule. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), the synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions. Still further, the controlled expression of at least one of the nucleic acid sequence of interest, results in at least one desired phenotype adapted to the suspension condition. In more specific embodiments, the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif:

A further aspect of the present disclosure relates to a cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. The platform comprising at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), the at least one synthetic conditional nucleic acid sequence of the disclosed platform is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell suspension conditions. In some embodiments, the transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83„ or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

A further aspect of the present disclosure relates to a cell or a population of the cells comprising and/or genetically engineered by, at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or at least one cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cell suspension conditions. In some embodiments, the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83„ or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

These and other aspect of the present disclosure will become apparent by the hand of the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

FIGURE 1A-1B. ACBM production stages

Fig. 1A. Stage 1 : High cell mass production. This stage includes isolation of cells from an animal, culturing the cells, growth and cell expansion in suspension, growth in high cell concentration and upscaling.

Fig. IB. Stage 2: Production of a muscle/meat-like tissue. This stage includes growing cells in adherent conditions, inducing differentiation and specified tissue architecture, and forming a food product.

FIGURE 2. The experimental and computational pipeline for identifying cell state-specific promoters

The figure illustrates the various stages required for identifying cell state-specific promoters Stage A: Library construction. Stage B: Cell infection either under adherent or suspension conditions. Stage C: Next generation sequencing (NGS) is performed for analysis of barcode content. Stage D: Analysis for assessment of promoter suspension/adherence specificity and activity. Stage E: The promoter specificity is further verified by flow cytometry.

FIGURE 3: Identification of suspension (S) promoters using next generation sequencing (NGS)

HEK 293 cells adapted to suspension were infected in suspension with the SPECS library. Fortyeight hours after infection cells were divided to two; half cultured under adherent and half cultured under suspension conditions. Cells were then cultured in the two conditions for additional 5-7 days. The cells were then harvested, RNA was isolated from the cells and library specific cDNA was produced using library specific primers, cDNA was amplified by PCR using library specific primers. PCR products were sent to NGS with a target of 2X10⁶ reads per sample (n=3 biological repeats). The S complete screen was repeated twice. NGS results were analyzed by program developed in-house and S- and A-promoters were identified. Twenty-five S-promoter and of 10 A-promoter were identified (ten results of S-promoters are presented). The results for each promoter in both the screens performed (Screen 1 and 2) are presented. Each graph depicts the distribution of the activity of a single promoter (the number of each promoter is found on the upper left corner of each graph) in suspension (Sus) and adherent (Ad) conditions for 3 biological repeats. The distribution of each promoter activity is seen for the two screens performed, screen 1 (upper graphs) and screen 2 (lower graph). The Fold change of each S-promoter activity (log2) between suspension and adherent conditions is shown as well as the statistical significance of the results as p adjusted (padj).

FIGURE 4A-4B. validated suspension promoters

Identified S-promoters were cloned so that they controlled the expression of GFP. Cloned promoters were infected into HEK293 cells in suspension mode. After 48hrs infected cells were divided to 2 and half were cultured in suspension while half were cultured in adherent conditions. Five days following cell division into suspension and adherent conditions cells were photographed by a fluorescent microscope (Fig. 4A) and analyzed by flow cytometry (Fig. 4B). In the Y axis the fold change represents the following calculation: Median (sus) GFP(+) cells minus median (sus) GFP(-) cells/median (Ad) GFP(+) cells minus (Ad) GFP(-). (n=2 or 3 repeats). Positive control (PC) represents GFP expression under a constitutive promoter.

FIGURE 5A-5C. motif 1 of SEQ ID NO: 73 and 74

Fig. 5A. The motif, as denoted by SEQ ID NOs: 71 and 73.

Fig. SB. the reverse complement as denoted by SEQ ID NOs: 72 and 74. Fig. 5C. scatter plots and statistics for two promoters for examples, results from screen one and two.

FIGURE 6A-6D. Motif 3 of SEQ ID NO: 66 and 77

Fig. 6A. The motif, as denoted by SEQ ID NO: 66.

Fig. 6B. reverse complement, as denoted by SEQ ID NO: 77.

Fig. 6C. motif locations.

Fig. 6D. the sequences of three promoters do not contain motif 3, GCTAACCACAGACTT, as denoted by SEQ ID NO: 67, as well as GACCGAAACC, as denoted by SEQ ID NO: 78, and GGTTTCGGTC as denoted by SEQ ID NO: 79. Motif 1 is underlined in the sequences.

FIGURE 7A-7C. motif 3 in the library

Fig. 7A. motif variation; the nucleic acid sequences of the varied motifs are denoted by SEQ ID NO: 12, 5, 56, 57, 58, 11, 59, 2, 60, 3, 6, 61, 8, 13, 62, 63, 63, 10, 10, 17, 17 and 7, respectively. Fig. 7B. motif to search in the library, as denoted by SEQ ID NO: 64.

Fig. 7C. reverse complement of the motif, as denoted by SEQ ID NO: 65.

FIGURE 8. Venn diagram

Figure shows Venn diagram between the lists of promoters for each motif.

FIGURE 9A-9C. Isolation and characterization of porcine cells

Porcine adipose derived stem cells (AdMCs) (Fig. 9AI) and dermal fibroblasts (Fig. 9AII) were isolated from abdominal skin + fat and adapted to culture. (Fig. 9AIII) UMNSH/DF-1 immortalized chicken fibroblasts were purchased from ATCC and expanded. AdMSCs were induced to differentiate to fat (Fig. 9B) and were expanded to passage (Fig. 9C) 22 when they stopped proliferating and demonstrated a senescence phenotype.

FIGURE 10A-10D. AdMSC immortalization

AdMSCs were infected with combinations of genes known to induce cell immortalization or with GFP as control. On 12 (Fig. 10A) and 19 days (Fig. 10B) post infections cells were imaged. Cells infected with construct encoding the cell immortalization gene combination, are shown in the figure (clones Imm #1, Imm #2, Imm #3) The cells of clone Imm #3 were termed immortalized porcine cells (IPOCs) demonstrated a phenotypic change as early as 12 days post infection (Fig. 10A). Cells were than passaged to passage 20 and their doubling time was examined regularly (Fig. 10C). IPOCs were further expanded to passage 34, demonstrating a stable doubling time (Fig. 10C) and a stable phenotype (Fig. 10D). FIGURE 11A-11B. IPOC adaptation to serum-free- medium

Fig. 11A. IPOCs previously cultured in serum containing medium were adapted to growth in serum-free- -medium and were expanded for additional 12 passages demonstrating a stable phenotype (Fig. 11A), and doubling time (Fig. 11B) from passage 18 to passage 30. The specific “phenotype” of the IPOCs in serum-free medium was due to their inferior adhesion in these conditions, since upon returning IPOCs to serum containing media they regained their original look (Fig 11A).

FIGURE 12A-12B. IPOCs secreting recombinant transferrin expand long-term in serum- free medium (-) transferrin

Transferrin was cloned under the regulation of a constitutive promoter. Successful expression and secretion of transferrin from IPOCs was detected by ELISA following vector introduction by infection.

Fig. 12A. IPOCs secreting recombinant transferrin were able to proliferate for 8 passages in serum- free medium (-) transferrin demonstrating high viability.

Fig. 12B. IPOCs grown in serum-free medium (-) transferrin demonstrated low proliferation capacity and death of all cells after 16 days.

FIGURE 13A-13B. Adaptation of IPOCs to suspension growth

Fig. 13A. a scheme of the protocol of IPOCs to suspension growth.

Fig. 13B. images of suspension IPOCs in different days following their transfer to shaking flasks. FIGURE 14A-14B. S-Promoters in IPOCs

Fig. 14A. Comparison of suspension-specific promoter activity. The data show the normalized fold change in promoter activity in suspension culture relative to adherent culture. Promoter expression was quantified by measuring GFP fluorescence via flow cytometry. The GFP signal for each promoter was normalized to that of the human ubiquitin C (UbC) promoter, which served as the control. The fold change values are displayed above each bar.

Fig. 14B. Suspension-specific promoter's relative activity in suspension and adherent conditions. The data show the normalized expression level of tested promoters in suspension (striped) and adherent culture (white) relative to the human ubiquitin C (UbC) promoter, which served as the control. The data presented in logarithmic scale based on the measured GFP signal quantified via flow cytometry.

FIGURE 15A-15B. A-Promoters in IPOCs

Fig. ISA. Comparison of adherent-specific promoter activity. The data show the normalized fold change in promoter activity in adherent culture relative to suspension culture. Promoter expression was quantified by measuring GFP fluorescence via flow cytometry. The GFP signal for each promoter was normalized to that of the human ubiquitin C (UbC) promoter, which served as the control. The fold change values are displayed above each bar.

Fig. 15B. Adherent-specific promoters' relative activity in suspension and adherent conditions. The data show the normalized expression level of tested promoters in suspension (striped) and adherent culture (white) relative to the human ubiquitin C (UbC) promoter, which served as the control. The data presented in logarithmic scale based on the measured GFP signal quantified via flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above and as also illustrated by Figure 1, animal cell-based meat products (ACBM) production processes can be divided into two main stages. The first major stage, illustrated by Fig. 1A, relates to high cell mass production, and requires the isolation of target cells, growing them in suspension, and inducing their extensive cell proliferation to achieve high cell mass. The second stage involves production of a muscle/meat-like tissue composed of muscle, fat, blood vessels and connective tissue, as illustrated by Fig. IB. More specifically, once a sufficient cell mass is reached at stage 1, cells must stop proliferating and start differentiating into fat cells, muscle cells, and blood vessel cells. The differentiated cells are then mounted to a surface/scaffold to create the muscle tissue (a steak). Several techno-economic assessments have analyzed current status and potential pathways to allow the profitability of the ACBM sector and point out major issues requiring technology development to enable the feasibility and profitability of large-scale ACBM production. Among those the most prominent issues are the following:

Animal-free cell media components, with a focus on the growth factors supplements. In order to reduce the costs significantly, media supplements consumption should be minimized.

Cell biology related factors such as reducing cell metabolism rate, cell maturation time and cell doubling time; increasing of achievable cell concentrations, efficiency of cell differentiation and capability of cells to build a tissue and reducing the overall time of the complete ACBM production process.

Adapting large scale bioreactors to the requirements of growing massive amounts of animal cells.

Reaching controllable, reproducible and cost-effective ACBM production processes present enormous biological challenges to the ACBM field till this very day. These challenges are currently faced with outdated tools such as using spontaneous cell adaptation to the highly versatile conditions of ACBM production. Furthermore, attempts to solve these huge challenges are made in scattered efforts in various companies and in a cell-type specific manner, each company develops its own specific ACBM producing cell-type, and no generic solution is offered. The present disclosure provides a completely different solution by applying a novel transformative synthetic biology platform that enables autonomous control and adaptation of cells phenotype to dynamic environmental conditions through the various ACBM production stages. More specifically, disclosed herein are specific novel synthetic promoters enabling high scale autonomous cell growth, differentiation and tissue maturation upon sensing of dynamic environmental conditions.

Thus, in a first aspect, the present disclosure relates to a method for the production of a cell-based product. The method comprising the step of introducing into at least one cell, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or a cellular platform thereof. The conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition. Optionally, the cellular or environmental conditions sensed during and/or associated with cell culturing and/or production of the cell-based product. More specifically, the controlled expression of at least one nucleic acid sequence of interest, results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site.

It should be noted that in some particular and non-limiting embodiments, the at least one synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure may be also referred to herein as synthetic promoters or as any part of a synthetic promoter. Synthetic, as used herein, relates to an artificially designed and created sequence of nucleotides that make up DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). It should be further understood that in some embodiments, the use of natural, or non- synthetic conditional nucleic acid sequence is also encompassed by the disclosed methods.

As indicated above, the nucleic acid molecule of the present disclosure, and of the methods disclosed herein, comprises at least one synthetic conditional nucleic acid sequence. As used herein, the term "conditional" in the context of the synthetic nucleic acid sequence used in the disclosed methods, refers to a sequence that detects dynamic or changing conditions or is specifically adapted to sense and identify a particular environmental or cellular state or condition. Upon detecting the specified condition, the conditional synthetic sequence (also referred to herein as a promoter, for brevity), became operated and functional. In some embodiments, this operation and function of the synthetic condition regulates the transcription of an operably linked sequence. The term "configured for sensing" refers to a promoter that is designed, modified, or naturally adapted to detect and respond to specific environmental, chemical, or cellular conditions. Upon sensing the relevant condition, the promoter modulates gene expression by initiating or regulating transcription of an operably linked sequence of interest. Sensing, in the context of a promoter (conditional nucleic acid sequence), refers to the ability of a promoter to detect, recognize, and respond to specific environmental, chemical, or biological signals, leading to a change in its activity, such as the initiation or regulation of transcription. Still further, as used herein, "configured for controlling the expression of a sequence upon sensing conditions" refers to a promoter or any synthetic conditional nucleic acid sequence designed to detect a specific environmental, chemical, or biological stimulus and modulate gene expression in response. In some embodiments, this modulation or "controlled expression" can involve activating, repressing, or adjusting transcription, ensuring that gene expression occurs only under predefined conditions. Still further, transcription refers to the process in which genetic information encoded in a DNA sequence is copied into a complementary RNA molecule. This process is carried out by the enzyme RNA polymerase, which synthesizes RNA based on the DNA template. The process involves initiation where the RNA polymerase binds the promoter, along with transcription factors, to start RNA synthesis. In the next step, elongation - RNA polymerase moves along the DNA template strand, synthesizing a complementary RNA strand in the 5' to 3' direction. In termination the transcription ends when RNA polymerase encounters a termination signal, leading to the release of the newly synthesized RNA. In some embodiments, such nucleic acid sequence may be also referred to herein as a synthetic promoter. In some embodiments this synthetic promoter comprises at least two transcription factor binding sites. In some embodiments, the region of the at least two transcription factor binding sites may be referred to herein as a transcription factor binding region of the disclosed synthetic conditional nucleic acid sequence. More specifically, a "promoter" refers to a control region of an operably linked nucleic acid sequence at which initiation and rate of transcription of a nucleic acid sequence are controlled. A promoter regulates (e.g., activates or represses) expression or transcription of the nucleic acid sequence that it is operably linked to, and/or located adjacent to the promoter region. In the context of gene expression, sequences controlled by a promoter, that in accordance with the present disclosure are the nucleic acid sequences of interest, refer to nucleotide sequences whose transcription is regulated [e.g., by level (enhancement or repression), timing, cell/tissue specificity, and physical conditions such as temperature, pH, etc.] by the presence and activity of a promoter located upstream of the coding or regulatory region. In some embodiments, the controlled sequence of interest is located downstream of the promoter in the 5' to 3' direction relative to transcription. In yet some further embodiments, the distance between the promoter (the "conditional sequence") and the controlled sequence of interest can vary depending on the promoter type (e.g., core promoter vs. distal enhancer-regulated promoter). Such distance may range between about 0 to about 1,000 bp. Still further, in some embodiments, the sequence is typically located downstream of a transcription start site (TSS), which marks the beginning of the transcript. Typically -40 to +40 bp relative to the TSS. As indicated herein, the synthetic promoter (synthetic conditional nucleic acid sequence) controls the expression of an operably linked nucleic acid sequence of interest. More specifically, the term "operably linked" refers to the functional connection between two components, specifically, the synthetic promoter and the nucleic acid sequence of interest, specifically, that the at least two sequences are positioned in a manner that allows the effective interaction or influence of the synthetic promoter on the expression of the nucleic acid sequence of interest attached thereto. The term ensures that the components are physically or functionally connected in a way that allows them to fulfill their intended roles in the disclosed methods.

A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific, cell type- specific, cell state-specific, or any combination thereof.

As used herein, the term "synthetic promoter" is a promoter that is not "naturally occurring", specifically, cannot be found in nature as a promoter sequence. However, such synthetic promoter may be composed of naturally occurring elements, e.g., transcription factor binding site/s. The synthetic promoters of the present disclosure may be produced synthetically (e.g., via chemical synthesis), or using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR).

In some embodiments, a synthetic promoter may be 3-350, specifically, 3-100, 4-100, 5-100, 6- 100, 7-100, 8-100, 9-100, 10-350 nucleotides long. For example, the length of a synthetic promoter may be 10-300, 10-290, 10-280, 10-270, 10-260, 10- 250, 10-240, 10-230, 10-220, 10-210, 10- 210, 10-200, 10- 190, 10-180, 10- 170, 10- 160, 10-150, 10- 140, 10-130, 10- 120, 10- 110, 10- 100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10- 20, 20-300, 20-290, 20-280, 20-270, 20- 260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-210, 20-200, 20-190, 20- 180, 20- 170, 20-160, - 150, 20- 140, 20-130, 20- 120, 20- 110, 20-100, 20- 90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-, 30-300, 30-290, 30-280, 30-270, 30-260, 30-250, 30-240, 30-230, 30-220, 30-210, 30-210, 30-0, 30- 190, 30-180, 30- 170, 30- 160, 30-150, 30- 140, 30- 130, 30-120, 30- 110, 30- 100, 30-, 30-80, 30-70, 30-60, 30-50, 30-40, 40-300, 40-290, 40-280, 40-270, 40-260, 40-250, 40-240,-230, 40-220, 40-210, 40-210, 40-200, 40-190, 40- 180, 40- 170, 40-160, 40- 150, 40- 140, 40-0, 40- 120, 40- 110, 40-100, 40-90, 40-80, 40-70, 40- 60, 40-50, 50-300, 50-290, 50-280, 50-0, 50-260, 50-250, 50-240, 50-230, 50-220, 50-210, 50-210, 50-200, 50- 190, 50- 180, 50-170,- 160, 50- 150, 50-140, 50- 130, 50- 120, 50-110, 50- 100, 50-90, 50-80, 50-70, 50-60, 60-300,-290, 60-280, 60-270, 60-260, 60-250, 60-240, 60- 230, 60-220, 60-210, 60-210, 60-200, 60-0, 60- 180, 60- 170, 60-160, 60- 150, 60- 140, 60-130, 60- 120, 60-110, 60- 100, 60-90, 60-80,-70, 70-300, 70-290, 70-280, 70-270, 70-260, 70-250, 70-240, 70-230, 70-220, 70-210, 70-210,-200, 70- 190, 70-180, 70- 170, 70- 160, 70-150, 70- 140, 70- 130, 70-120, 70- 110, 70- 100,-90, 70-80, 80-300, 80-290, 80-280, 80-270, 80-260, 80-250, 80-240, 80-230, 80-220, 80-210,-210, 80-200, 80-190, 80- 180, 80- 170, 80-160, 80- 150, 80- 140, 80-130, 80- 120, 80- 110, 80-0, 80-90, 90-300, 90-290, 90-280, 90-270, 90-260, 90-250, 90-240, 90-230, 90-220, 90-210, 90-0, 90-200, 90-190, 90- 180, 90- 170, 90-160, 90- 150, 90- 140, 90-130, 90- 120, 90- 110, 90-0, 100-300, 100-290, 100-280, 100-270, 100-260, 100-250, 100-240, 100-230, 100-220, 100-0, 100-210, 100-200, 100-190, 100-180, 100- 170, 100- 160, 100-150, 100- 140, 100- 130, 100-0, 100-110, 110-300, 110-290, 110-280, 110-270, 110-260, 110-250, 110-240, 110-230, 110-0, 110-210, 110-210, 110-200, 110-190, 110- 180, 110- 170, 110-160, 110- 150, 110- 140, I I Q-0, 110-120, 120-300, 120-290, 120-280, 120-270, 120-260, 120-250, 120-240, 120-230, 120-0, 120-210, 120-210, 120-200, 120-190, 120- 180, 120- 170, 120-160, 120- 150, 120- 140, 120-0, 130-300, 130-290, 130-280, 130-270, 130-260, 130-250, 130-240, 130-230, 130-220, 130-0, 130-210, 130-200, 130-190, 130-180, 130- 170, 130- 160, 130-150, 130- 140, 140-300, 140-0, 140-280, 140-270, 140-260, 140-250, 140-240, 140-230, 140-220, 140-210, 140-210, 140-0, 140-190, 140- 180, 140-170, 140-160, 140- 150, 150-300, 150-290, 150-280, 150-270, 150-0, 150-250, 150-240, 150-230, 150-220, 150-210, 150-210, 150-200, 150- 190, 150- 180, 150-0, 150-160, 160-300, 160-290, 160-280, 160-270, 160-260, 160-250, 160-240, 160-230, 160-0, 160-210, 160-210, 160-200, 160-190, 160- 180, 160- 170, 170-300, 170-290, 170-280, 170-0, 170-260, 170-250, 170-240, 170-230, 170-220, 170-210, 170-210, 170-200, 170- 190, 170-0, 180-300, 180-290, 180-280, 180-270, 180-260, 180-250, 180-240, 180-230, 180-220, 180-0, 180-210, 180-200, 180-190, 190-300, 190-290, 190-280, 190-270, 190-260, 190-250, 190-0, 190-230, 190-220, 190-210, 190-210, 190-200, 200-300, 200-290, 200-280, 200-270, 200- 260, 200-250, 200-240, 200-230, 200-220, 200-210, 200-210, 210-300, 210-290, 210-280, 210-

270, 210-260, 210-250, 210-240, 210-230, 210-220, 220-300, 220-290, 220-280, 220-270, 220-

260, 220-250, 220-240, 220-230, 230-300, 230-290, 230-280, 230-270, 230-260, 230-250, 230-

240, 240-300, 240-290, 240-280, 240-270, 240-260, 240-250, 250-300, 250-290, 250-280, 250-

270, 250-260, 260-300, 260-290, 260-280, 260-270, 270-300, 270-290, 270-280, 280-300, 280-

290, or 290-300, 300-310, 300-320, 300-330, 300-340, 300-350 nucleotides. Promoters may be longer than 300 nucleotides. In some embodiments, a synthetic promoter may be longer than 300 nucleotide (e.g., 300, 350, 400, 450, or 500 nucleotides long or longer).

In some embodiments, the length of a synthetic promoter is 350 nucleotides or shorter. In some embodiments, a synthetic promoter may be 10, 11, 12, 13, 14, 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, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,

120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,

139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,

158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,

177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,

215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,

234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,

253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,

272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,

291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,

310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,

329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,

348, 349, 350 nucleotides long.

Other aspects of the present disclosure provide synthetic promoters having differential activities in different cell source or different cellular states or environmental conditions. The phrase "promoter activity" as used herein, typically refers to the efficiency or strength of a promoter in driving gene expression, specifically, initiating and supporting transcription of an operably linked nucleic acid sequence of interest. "Having differential activities" means the activity of a synthetic promoter is higher or lower in one type of cell or at a cellular state, or environmental condition, compared to in a different type of cell or at a different cellular state, or different environmental conditions, respectively. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state or environmental condition is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state or another environmental condition, specifically, by at least 10 percent (e.g., at least 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent 100 percent, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold). In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state or an environmental condition, is different from (higher or lower, or "on'7"off") the activity of the synthetic promoter in another cell type or another cellular state, or another environmental condition, by 5 percent -100 percent. For example, the activity of a synthetic promoter in one cell type or a cellular state or environmental condition may be different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state or another environmental condition, by 5% - 100%, 10 % - 100 %, 10 % -90 %, 10 % -80 %, 10 % -70 %, 10 % - 60 %, 10 % -50 %, 10 % -40 %, 10 % -30 %, 10 % -20 %, 20 % - 100 %, 20

% -90 %, 20 % -80 %, 20 % -70 %, 20 % -60 %, 20 % -50 %, 20 % -40 %, 20 % -30 %, 30 % -

100 %, 30 % -90 %, 30 % -80 %, 30 % -70 %, 30 % - 60 %, 30 % -50 %, 30 % -40 %, 40 % - 100

%, 40 % -90 %, 40 % -80 %, 40 % -70 %, 40 % -60 %, 40 % -50 %, 50 % -100 %, 50 % -90 %,

50 % -80 %, 50 % -70 %, 50 % -60 %, 60 % -100 %, 60 % -90 %, 60 % -80 %, 60 % -70 %, 70 % -100 %, 70 % -90 %, 70 % -80 %, 80 % -100 %, 80 % -90 %, or 90 % -100 %. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state or an environmental condition, is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state by 1-1000 fold. For example, the activity of a synthetic promoter in one cell type or a cellular state or an environmental condition, may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state or another environmental condition, by 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1- 300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1- 4, 1-3, 1-2, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 5-90, 5- 80, 5- 70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, 5-6, 10-1000, 10-900, 10-800, 10-700, 10- 600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10- 30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-1000, 30-900, 30-800, 30-700, 30-600, 30- 500, 30- 400, 30-300, 30-200, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-1000, 40- 900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-90, 40-80, 40-70, 40- 60, 40-50, 50- 1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50- 70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60- 90, 60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70- 100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80- 400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90- 100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200- 1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300- 800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400- 800, 400-700, 400-600, 400- 500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700- 1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 fold. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state or an environmental condition, may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state or another environmental condition, by 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 100 percent, 2 fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold.

In some embodiments, a synthetic promoter may be inactive in one cell type and active in another. In some embodiments, a synthetic promoter may be inactive in one cellular state or environmental condition and active in another state or condition. Methods of measuring the activities of a promoter (e.g., a synthetic promoter) are known to those skilled in the art.

As indicated above, the synthetic conditional nucleic acid sequence disclosed herein comprises at least two repeats of a transcription factor binding sites. A transcription factor binding site (TFBS) is a specific sequence of DNA to which a transcription factor binds to regulate the expression of a gene. These binding sites are typically located in the promoter or enhancer regions of a gene and play a critical role in controlling the initiation of transcription. Transcription factors, which are proteins or complexes, recognize and bind to these sites, either activating or repressing the transcription of the associated gene, depending on the specific transcription factor and cellular context. Transcription factors possess several key characteristics that define their function in gene regulation. They contain DNA-binding domains (DBDs), which enable them to recognize and attach to specific DNA sequences, typically within promoter or enhancer regions. Their regulatory function allows them to act as either activators, enhancing transcription, or repressors, inhibiting it. Additionally, transcription factors frequently interact with other proteins, such as RNA polymerase, co-activators, and co-repressors, to modulate transcriptional activity effectively.

The core of a TFBS usually consists of a short sequence of nucleotides (often 6-12 base pairs) that is specifically recognized by the transcription factor. This sequence is often palindromic or symmetrical in nature to accommodate the structure, composition and size of the transcription factor's DNA-binding domain. It should be appreciated that the size (length) of a single TFBS may range between about 5 to 30 nucleotides, specifically, between 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, specifically, between 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 11 to 25, 12 to 25, between 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, between 5 to 23, 6 to 23, 7 to 23, 8 to 23, 9 to 23, 10 to 23, 11 to 23, 12 to 23, between 5 to 22, 6 to 22, 7 to 22, 8 to 22, 9 to 22, 10 to 22, 11 to 22, 12 to 22, between 5 to 21, 6 to 21, 7 to 21, 8 to 21, 9 to 21, 10 to 21, 11 to 21, 12 to 21, between 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20 nucleotides. Still further, in some embodiments, the TFBS may be in the length of any one of: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides. Still further, the disclosed synthetic promoter or "synthetic conditional nucleic acid sequence", comprise at least two repeats of a TFBS. Specifically, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty repeats of a TFBS. In some embodiments, the disclosed synthetic promoter or synthetic conditional nucleic acid sequence, comprise 4, 5, 6, 7 or 8 repeats of a TFBS. It should be understood that when referring to "repeats of a TFBS", the present disclosure encompasses multiple occurrences of the same (identical) and/or similar TFBS within the synthetic promoter. "Similar," in the context of the repeated TFBSs, refers to any sequences that have 75% to 99% identity to the sequence of a specific TFBS, which is the most frequently repeated in the disclosed synthetic promoter. In some embodiments, the at least two TFBSs form within the synthetic promoter of the present disclosure, a TF binding region. In some embodiments, the TF binding region formed by the at least two TF binding sites of the synthetic conditional nucleic acid sequence (referred to herein as the synthetic promoter), comprises at least 50, 60, 70, 75, 80, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, or 175 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the TF binding region comprises at least 50 nucleotides. In some embodiments, the TF binding region comprises at least 100 nucleotides. In some embodiments, the TF binding region comprises at least 170 nucleotides. In some embodiments, the TF binding region comprises at least 150 nucleotides. In some embodiments, the TF binding region comprises at most 180, 190, 200, 210, 220, 225, 230, 240, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2200, 2400, 2500, 2600, 2800, 3000, 3500, 4000, 4500, or 5000 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the TF binding region comprises at most 3000 nucleotides. In some embodiments, the TF binding region comprises at most 190 nucleotides. In some embodiments, the TF binding region comprises at most 200 nucleotides. In some embodiments, the TF binding region comprises at most 250 nucleotides. In some embodiments, the TF binding region consists of between 50 and 3000 nucleotides. In some embodiments, the TF binding region consists of between 100 and 200 nucleotides. In some embodiments, the TF binding region consists of between 150 and 250 nucleotides. In some embodiments, the TF binding region consists of between 170 and 200 nucleotides.

In some embodiments, the transcription factor (TF) binding region within the synthetic conditional nucleic acid sequence (also referred to herein as a synthetic promoter), disclosed herein may further comprise a spacer between the at least two TF binding site repeats. In some embodiments, the TF binding region further comprises a spacer between two TF binding site repeats. In some embodiments, the nucleic acid molecule further comprises a spacer between TF binding site repeats. In some embodiments, there is a spacer between each repeat. In some embodiments, each repeat is separated from an adjacent repeat by a spacer. In some embodiments, the 5’ repeat is proceeded by a spacer. In some embodiments, the 3’ repeat is followed by a spacer. In some embodiments, each spacer between each two TF binding sits (TFBS) is the same. In some embodiments, each spacer between each two TF binding sits (TFBS) is different. In some embodiments, a spacer is between 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 2-20, 2-15, 2-10, 2-7, 2-5, 2-3, 3-20, 3-15, 3-10, 3-7 or 3-5 nucleotides in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the spacer is 1 to 5 nucleotides in length. In some embodiments, the spacer consists of 1-5 nucleotides. In some embodiments, the spacer is 1- 3 nucleotides in length. In some embodiments, the spacer consists of 1-3 nucleotides. In some embodiments, the spacer comprises 3 nucleotides. In some embodiments, the spacer consists of 3 nucleotides. In some embodiments, the spacer is 3 nucleotides in length. It will be understood that the sequence of the spacer is not of any importance, however, it should provide a sufficient distance between each binding site such that a TF can bind at each binding site.

The disclosed method is directed to the production of a cell-based product. According to some embodiments, such product refers to a product that is derived from, or incorporates living cells, or is produced using cells as a fundamental component in its creation. This can include products where cells are cultured, modified, or utilized for various purposes, such as in industrial processes or even in therapeutic applications, diagnostic purposes, or basic research purpose.

As indicated above, the controlled expression of at least one nucleic acid sequence of interest by the methods of the present disclosure, results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition. A desired phenotype, in connection with some embodiments of the present disclosure, refers to a specific set of observable traits or characteristics (phenotype) that have been intentionally modified or selected to suit a particular cellular environment or condition. The "adapted" or "desired" phenotype is one that enables the cell to thrive, function, or express certain traits effectively within the context of the specified cellular environment. In some embodiments of the disclosed methods, the at least one desired phenotype that is a result of the controlled expression of the nucleic acid of interest, comprises at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity. More specifically, the term "cell growth" may refer to an increase in the size or mass of individual cells, often associated with increased protein synthesis and accumulation of cellular components. "Cell expansion", as used herein, refer to the overall increase in the total number of cells in a population or culture, which may result from a combination of cell proliferation and cell survival. As used herein, "cell differentiation" refers to the process by which a less specialized cell becomes a more specialized cell type. This process typically involves changes in gene expression, cellular morphology, and function, resulting in cells with distinct characteristics and roles. The term "cell immortalization" as used herein, may refer to the acquisition of the ability of cells to divide indefinitely, bypassing normal cellular senescence. This may involve changes in gene expression or cellular processes that allow continued cell division beyond typical limits. "Cell maturation" as used herein, may refer to the process by which cells progress to their fully developed and functional state. This may involve changes in cellular structure, metabolism, and gene expression patterns that enable the cell to perform its specialized functions. As used herein, "tissue and/or organ formation" may refer to the process by which cells organize and arrange themselves into complex, three-dimensional structures that form functional tissues or organs. This may involve cell-cell interactions, extracellular matrix deposition, and coordinated cellular activities to create specific tissue architectures and organ structures.

Still further, in some embodiments, the cellular- and/or environmental- state and/or conditions sensed by the synthetic conditional nucleic acid sequence used by the methods disclosed herein, may include any biotic and/or abiotic environmental conditions. Specifically, biotic and/or abiotic environmental conditions, as used herein, refer to the factors that influence the survival, growth, and function of the cells used in the disclosed methods. Biotic conditions encompass living factors, including interactions between the cells, which may involve competition, symbiosis, microbial contamination or co-cultured cells of various origins, in case applied. Abiotic conditions refer to non-living factors, such as temperature, pH, humidity, light (intensity, wavelength and duration), oxygen levels, nutrient availability, salinity, and chemical composition, media composition, osmolarity, and gas exchange. Such cellular- and/or environmental- state and/or conditions may comprise according to some embodiments, at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D- scaffold), levels of lactate, levels of ammonia, pH conditions, salinity conditions, osmolarity conditions, strength of magnetic field, oxidative stress, humidity, temperature conditions, level of nutrients, levels of available amino acids, levels of ions, level of growth factors, level of signaling molecules, cell motility, shear force, levels of cytokines, levels of toxins, levels of oxidants, presence of pathogens, levels of hormones, metabolite accumulation, accumulation of cell waist, and/or cell metabolic state. More specifically, in addition to suspension conditions and adherence conditions that are described in more detail herein after, the disclosed synthetic conditional nucleic acid sequences (referred to herein as synthetic promoters) may sense and active in each of the conditions disclosed herein. Specifically, "adherence to three-dimensional scaffold (3D-scaffold)" refers herein to the attachment and growth of cells on or within a three-dimensional structure designed to support cell culture and tissue formation. In some embodiments, the disclosed synthetic promoters control transcription of a nucleic acid sequence of interest when sensing adherence into 3D-scaffold. "Levels of lactate" as used herein, refers to the concentration of lactic acid or lactate ions present in the cell culture medium or cellular environment. Lactate accumulation in cell culture can inhibit cell growth by lowering the pH and disrupting cellular metabolism, specifically in levels of over 30 rnM. Thus, in some embodiments, the disclosed synthetic promoters may induce transcription of a nucleic acid sequence of interest in high lactate levels. "Levels of ammonia" as used herein, refers herein to the concentration of ammonia or ammonium ions present in the cell culture medium or cellular environment conditions". At low concentrations, ammonia may have minimal effects, but as it accumulates, it can lead to cytotoxicity, altered gene expression, and reduced protein production. High ammonia levels disrupt cellular metabolism by affecting intracellular pH, impairing mitochondrial function, and inhibiting key enzymatic reactions. More specifically, various levels of ammonia are considered toxic and lead to inhibition of cell growth. Specifically, low ammonia levels as used herein refer to a concentration ranging between 2 to 4mM, moderate levels ranging between 4 to 8mM, and high ammonia levels that lead to severe cell toxicity range between 8 to lOrnM. Thus, in some embodiments, the disclosed synthetic promoters may induce transcription of a nucleic acid sequence of interest in ammonia levels that lead to low, moderate and/or severe toxicity as defined herein.

"pH levels", as used herein, refers to the measure of acidity or alkalinity in the cell culture medium or cellular environment. pH levels in cell culture play a critical role in maintaining cell viability, growth, and function. Most mammalian cell cultures thrive in a pH range of 7.0 to 7.4, with an optimal range typically around 7.2 to 7.4. Deviation from this range can negatively impact cell metabolism, protein expression, and overall viability. Thus, in some embodiments, the synthetic promoters used by the disclosed methods may sense changing pH conditions and control transcription of the nucleic acid sequence of interest in pH conditions that are acidic (below pH 7.0), or alternatively, in alkaline pH (above 7.4). Similarly, "Salinity conditions" as used herein, refers to the concentration of dissolved salts in the cell culture medium or cellular environment. "Osmolarity conditions" as used herein, refers to the concentration of osmotically active particles in the cell culture medium or cellular environment. "Strength of magnetic field" as used herein, refers to the intensity of a magnetic field applied to or present in the cell culture environment. "Oxidative stress" refers to an imbalance between the production of reactive oxygen species and the cellular antioxidant defenses. "Humidity" as used herein, refers to the amount of water vapor present in the air surrounding the cell culture. "Temperature conditions" refer to the ambient or controlled temperature of the cell culture environment. Generally, cell cultures are maintained at 37°C, which mimics the physiological temperature. Some cells may tolerate mild hypothermia (32-35°C), or alternatively, high temperature, that may be 39°C or above. "Level of nutrients" may refer to the concentration of essential nutrients, including carbohydrates, proteins, and lipids, available in the cell culture medium. In some embodiments, the synthetic promoters used by the disclosed methods may sense reduction in nutrients level, and for example, induce transcription of a nucleic acid sequence of interest that led to nutrient supplementation. Similarly, "Levels of available amino acids" may refer to the concentration of free amino acids present in the cell culture medium. "Levels of ions" may refer to the concentration of various ionic species, such as sodium, potassium, calcium, and chloride, in the cell culture medium or cellular environment. "Level of growth factors" may refer to the concentration of proteins that stimulate cellular growth, proliferation, and differentiation in the cell culture medium. "Level of signaling molecules" may refer to the concentration of molecules involved in cellular communication and signal transduction pathways. "Cell motility" may refer to the ability of cells to move independently, either through the culture medium or on a substrate. "Shear force" may refer to the mechanical stress applied to cells due to fluid flow in the culture system. "Levels of cytokines" may refer to the concentration of small proteins involved in cell signaling, particularly in immune responses and inflammation. "Levels of toxins" may refer to the concentration of substances that can cause cellular damage or death when present in the cell culture environment. "Levels of oxidants" refer to the concentration of molecules capable of oxidizing other molecules in the cellular environment. "Presence of pathogens" may refer to the detection or existence of microorganisms capable of causing cellular damage or infection in the culture system. "Levels of hormones" may refer to the concentration of signaling molecules that regulate various physiological processes in the cell culture environment. "Metabolite accumulation" may refer to the buildup of byproducts of cellular metabolism in the cell culture medium or cellular environment. "Accumulation of cell waste" may refer to the buildup of cellular debris, dead cells, or other waste products in the cell culture environment. "Cell metabolic state" may refer to the overall condition of cellular metabolism, including the rate and efficiency of various metabolic pathways within the cell.

It should be understood that in some embodiments, for each of the disclosed environmental and cellular conditions, a specific synthetic promoter may be used by the disclosed methods. Such that it specifically senses the condition and act on controlling (e.g., enhancing or repressing the transcription of an operably linked nucleic acid sequence of interest. That is to say that in some embodiments, each synthetic promoter is specific for one of the conditions. However, for certain cellular or environmental conditions, as specified above, several different synthetic promoters may be used (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more). In yet some other alternative embodiments, a specific promoter may be activated in more than one condition, especially, in transition between conditions, for example, in transition between different temperatures, and levels of nutrients or waste components.

The synthetic promoter (synthetic conditional nucleic acid sequence) controls the expression of an operably linked nucleic acid sequence of interest. More specifically, a "nucleic acid sequence of interest", in connection with the preset disclosure, relates to any gene, regulatory element, target, or any other sequence that is being utilized for a specific purpose, specific, for facilitating or enabling the phenotype that is adapted to the specified cellular- or environmental condition. The nucleic acid sequence of interest is typically introduced into a host cell to achieve a desired function, such as protein expression, gene editing, that leads to a desired phenotype as disclosed herein. In some embodiments, the nucleic acid sequence of interest controlled by the at least one synthetic conditional nucleic acid sequence used by the disclosed methods, may comprise at least one coding and/or non-coding inhibitory and/or modulatory nucleic acid molecule.

In some embodiments, at least one inhibitory and/or modulatory non-coding nucleic acid molecule is a ribonucleic acid (RNA) molecule, said RNA molecule is at least one of a double-stranded RNA (dsRNA), an antisense RNA, a single-stranded RNA (ssRNA), and a Ribozyme. In some embodiments, at least one inhibitory and/or modulatory non-coding nucleic acid molecule is at least one of a microRNA (miRNA), MicroRNA-like RNAs (milRNA), artificial miRNAs (amiRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). As indicated herein above, the synthetic conditional nucleic acid sequence of the present disclosure, that was used by the methods disclosed herein, may control the expression of any nucleic acid sequence of interest operably linked thereto. In some embodiments, the nucleic acid sequence of interest may be either a coding or a non-coding nucleic acid sequence that display inhibitory or modulatory action towards at least one target. In yet some further embodiments, such modulatory or inhibitory nucleic acid sequence may affect directly or indirectly the expression, levels, stability and/or activity of at least one target gene or product involved with the desired phenotype. In more specific embodiments, the at least one inhibitory and/or modulatory non-coding nucleic acid molecule provided as the nucleic acid sequence of interest in the present disclosure, may be dsRNA molecules participating in RNA interference. More specifically, the dsRNA encompassed by the present disclosure may be selected from the group consisting of small interfering RNA (siRNA), MicroRNA (miRNA), artificial miRNA (amiRNA), miRNA-like RNAs (miRNA), short hairpin RNA (shRNA), PIWI interacting RNAs (piRNAs). RNA interference (RNAi) is a general conserved eukaryotic pathway which down regulates gene expression in a sequence specific manner. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. Gene silencing is induced and maintained by the formation of partly or perfectly doublestranded RNA (dsRNA) between the target RNA and the siRNA/shRNA derived ‘guide” RNA strand. The expression of the gene is either completely or partially inhibited. As known in the art RNAi is a multistep process. In a first step, there is cleavage of large dsRNAs into 21-23 ribonucleotides-long double-stranded effector molecules called “small interfering RNAs” or “short interfering RNAs” (siRNAs). These siRNAs duplexes then associate with an endonucleasecontaining complex, known as RNA-induced silencing complex (RISC). The RISC specifically recognizes and cleaves the endogenous mRNAs/RNAs containing a sequence complementary to one of the siRNA strands. One of the strands of the double-stranded siRNA molecule (the “guide” strand) comprises a nucleotide sequence that is complementary to a nucleotide sequence of the target gene, or a portion thereof, and the second strand of the double-stranded siRNA molecule (the passenger” strand) comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target gene, or a portion thereof. After binding to RISC, the guide strand is directed to the target mRNA cleaved between bases 10 and 11 relative to the 5' end of the siRNA guide strand by the cleavage enzyme Argonaute-2 (AG02). Thus, the process of mRNA translation can be interrupted by siRNA.

In more particular embodiments, siRNAs comprise a duplex, or double-stranded region, of about 5-50 or more, 10-50 or more, 15-50 or more, 5-45, 10-45, 15-45, 5-40, 10-40, 15-40, 5-35, 10-35, 15-35, 5-30, 10-30 and 15-30 or more nucleotides long. In yet some more particular embodiments, the siRNAs used as the nucleic acid sequence of interest in the present disclosure, comprise a nucleic acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. Often, siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least a portion of one strand of the duplex or doublestranded region of a siRNA is substantially homologous to or substantially complementary to a target sequence within the gene product (i.e., RNA) molecule as herein defined. The strand complementary to a target RNA molecule is the “antisense guide strand”, the strand homologous to the target RNA molecule is the “sense passenger strand” (which is also complementary to the siRNA antisense guide strand). siRNAs may also be contained within structured such as miRNA and shRNA which has additional sequences such as loops, linking sequences as well as stems and other folded structures.

More specifically, the strands of a double-stranded interfering RNA (e.g., siRNA) may be connected to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as mentioned above the at least one inhibitory and/or modulatory non-coding nucleic acid molecule controlled by the nucleic acid molecules of the present disclosure, and used by the methods disclosed herein, may also be short hairpin RNA (shRNA).

According to other embodiments the at least one inhibitory and/or modulatory non-coding nucleic acid molecule provided as the nucleic acid sequence of interest in the present disclosure may be a micro-RNA (miRNA). miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the "pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA." The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA. The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity and usually repress translation without affecting steady-state RNA levels. Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (RISC). More specifically, microRNAs (miRNAs) form a class of endogenous, 20-22nt long regulatory RNA molecules. They exert their function of post-transcriptional gene regulation through mRNA cleavage, RNA degradation, and translation inhibition. Most canonical miRNAs are transcribed by RNA polymerase II (Pol II) to produce pri-miRNA transcripts, which are then cleaved by RNase Ill- type enzymes called Dicer-like proteins into stem-loop structured precursors in the nucleus. Stemloop pre-miRNAs are subsequently cleaved into miRNA/miRNA* duplexes by Dicer or Dicer-like enzymes in the cytoplasm. The mature miRNAs are then incorporated into ARGONAUTE (AGO)- containing RNA-induced silencing complexes (RISC) in the cytoplasm to exert their regulatory effects by guiding the RISC to target transcripts through perfect or partially complementary base pairing. Non-canonical microRNAs have been discovered in various organisms. Non-canonical miRNAs have structural and function similar with canonical miRNAs, but they can skip one or more steps of classic miRNA biogenesis pathway. Small nucleolar RNA-derived miRNAs, endogenous short hairpin RNAs derived miRNAs and tRNA-derived miRNA are three Dicerdependent, Dgcr 8 -Independent miRNAs. Multiple distinct miRNA-like RNAs can arise from a single miRNA precursor and they have been reported in plant species and mammals. The nucleic acid sequence of interest according to the present disclosure may encode miRNA-like RNAs. Still further, in some embodiments, the nucleic acid sequence of interest, may encode artificial miRNA (amiRNA). amiRNAs have been explored as alternative RNAi-triggering molecules and are designed to mimic primary miRNA stem-loops. The mature miRNA duplex in the central stem is replaced by sequences specifically designed for a specific target transcript, but the native flanking recognition sequences for cleavage by Drosha and Dicer are preserved. The artificial miRNAs are transcribed in larger transcripts and can be linked to RNA polymerase II- based expression systems.

More specific embodiments relate to the at least one inhibitory and/or modulatory non-coding nucleic acid molecule provided as the nucleic acid sequence of interest that may be at least one shRNA molecule. The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence. The degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions. The first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.

An “antisense RNA” is a single strand RNA (ssRNA) molecule that is complementary to an mRNA strand of a specific target gene product. Antisense RNA may inhibit the translation of a complementary mRNA by base-pairing to it and physically obstructing the translation machinery. By "complementary" it is meant the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. Still further, in some embodiments, at least one inhibitory and/or modulatory non-coding nucleic acid molecule provided as the nucleic acid sequence of interest in the present disclosure, may comprise an antisense oligonucleotide, or any derivatives thereof. In more specific embodiments such oligonucleotide is an antisense oligonucleotide (ASO). As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

In more specific embodiments, the nucleic acid sequence of interest controlled by the at least one synthetic conditional nucleic acid sequence, used in the disclosed methods, may encode, or control the production and/or activity and/or levels of, at least one product that directly or indirectly leads to, or involved with, the phenotype adapted to the cellular- and/or environmental- state and/or condition. In yet some alternative or additional embodiments, the nucleic acid sequence of interest controlled by the at least one synthetic conditional nucleic acid sequence, may encode, or control the production and/or activity and/or levels of at least one product of interest.

In some embodiments of the disclosed methods, the nucleic acid sequence of interest encodes, or controls at least one of: (a), at least one product that directly or indirectly leads to, or involved with, the phenotype adapted to the cellular- and/or environmental- state and/or condition. Alternatively, the nucleic acid sequence of interest controls the production and/or activity and/or levels of the product. In yet an alternative or additional embodiments (b), the nucleic acid sequence of interest encodes at least one product of interest, or alternatively, controls the production and/or activity, and/or levels of the product.

In some embodiments, the product that directly or indirectly leads to, or involved with, the phenotype adapted to the cellular- and/or environmental- state and/or condition may be at least one of: at least one growth factor, at least one survival factor, at least one differentiation factor, at least one immortalization factor, at least one cell metabolic factor, at least one adhesion molecule, at least one protease, and/or at least one cell migration factor.

In some embodiments, the product of interest may be a therapeutic agent, a cosmetic agent, a food product, an in vitro multicellular system (e.g., engineered tissue and/or artificial organs). Still further, in some embodiments, the product of interest may be any product produced by precision fermentation, e.g., enzymes for the food, laundry, and any industrial products, or products used in any industry.

In some embodiments, the cell applicable in the disclosed methods may be a eukaryotic cell or a prokaryotic cell.

In yet some further embodiments, the eukaryotic cell applicable in the disclosed methods may be cell/s of at least one unicellular or multicellular organism of the biological kingdom Animalia or of the biological kingdom Plantae.

In yet some further embodiments, the cell/s applicable in the methods of the present disclosure may be any cells of any organism of the biological kingdom Animalia. In yet some more specific embodiments, the cells applicable in the disclosed methods may be of any organism, specifically, any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

In some embodiments, the cellular- and/or environmental- state and/or condition sensed by the synthetic conditional nucleic acid sequence (synthetic promoters) of the nucleic acid molecule of the present disclosure, that are used in the disclosed methods, may comprise suspension conditions. Suspension conditions as used herein, refer to specific environmental factors and culture conditions necessary for the growth and proliferation of cells that are suspended in a liquid medium rather than adhering to a surface. In accordance with some embodiments of the present disclosure, suspension conditions may include particular liquid medium in which the cells are suspended, optimal temperature range for the growth of the cells in suspension, adequate oxygen supply to support cellular respiration and metabolism. In some embodiments, these conditions may further involve agitation of the culture to enhance gas exchange or the use of specialized culture vessels. More specifically, in some embodiments, mechanical agitation or stirring of the culture may be applicable for the provision of suspension conditions.

Accordingly, in some embodiments, the resulting at least one desired phenotype comprises at least one of: cell proliferation, cell growth and/or cell expansion in suspension conditions.

In some specific embodiments, the at least one synthetic conditional nucleic acid sequence of the disclosed nucleic acid molecule, used in the methods of the present disclosure, is configured for controlling the expression of at least one nucleic acid sequence of interest.

In some embodiments, such conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in suspension conditions, may comprise at least one transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76, and/or any functional fragments thereof. It should be noted that A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

In yet some further embodiments, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in suspension conditions, used in the disclosed methods, may comprise at least one transcription factor binding site comprising the nucleic acid motif: GTTTCNNT , as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70; and/or any functional fragments thereof. Still further, A is adenine, G is Guanin, C is cytosine, T is thymine and N is any nucleic acid residue.

In more specific embodiments, the motif of at least one transcription factor binding site of the disclosed synthetic conditional nucleic acid sequence used in the disclosed methods, may comprise the nucleic acid sequence of :GTTTCRRT , as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof. It should be understood that Y is a pyrimidine, specifically, C or T, and wherein R is G or A.

Still further, in some embodiments, the synthetic conditional nucleic acid sequence of the present disclosure used in the disclosed methods for suspension conditions, may comprise a motif comprising the nucleic acid sequence of: GTTTCGGT, as denoted by SEQ ID NO: 73, and/or the reverse complement ACCGAAAC, as denoted by SEQ ID NO: 74, and/or any functional fragments thereof. In yet some alternative or additional embodiments, the motif of at least one transcription factor binding site of the synthetic conditional nucleic acid sequence of the present disclosure used in the disclosed methods, may comprise the nucleic acid sequence of: RSTTTCRNWWY, as denoted by SEQ ID NO: 64; and/or any functional fragments thereof. As indicated above, R is A or G, S is G or C, W is A or T, and Y is C or T, specifically, [AG][GC]TTTC[GA]N[TA][TA][TC], and/or the reverse complement RWWNYGAAASY, as denoted by SEQ ID NO: 65. The R is A or G, S is G or C, W is A or T, and Y is C or T, specifically, [AG][AT][AT]N[CT]GAAA[CG][TC]. In some embodiments, the motif of at least one transcription factor binding site of the synthetic conditional nucleic acid sequence of the present disclosure, comprises the nucleic acid sequence of: AGTTTCGNTTT, as denoted by SEQ ID NO: 66, and/or the reverse complement AAANCGAAACT, as denoted by SEQ ID NO: 77; and/or any functional fragments thereof.

In some embodiments, the at least one transcription factor binding site (TFBS) of the synthetic conditional nucleic acid sequence of the present disclosure used in the disclosed methods, for suspension conditions, is in the length of 8 to 20 nucleotides, specifically, 10 to 17 nucleotides.

In some particular embodiments, for suspension conditions, the at least one transcription factor binding site of the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, used in the disclosed methods, may comprise the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86 and/or the reverse complement thereof, or any combinations thereof, and/or any functional fragments thereof. In some embodiments, the at least one synthetic conditional nucleic acid sequence comprises 3 to 100 repeats of the transcription factor binding site.

In some embodiments, the at least one synthetic conditional nucleic acid sequence is in the length of 250 to 500 nucleotides, specifically, 270 to 300 nucleotides.

In some embodiments, the at least one synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, used in the disclosed methods in suspension conditions, may comprise the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47 and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, where the desired phenotype is production and/or secretion of at least one growth factor in suspension conditions for providing cell proliferation, cell growth and/or cell expansion, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, encodes, or controls the production and/or activity and/or levels of, at least one growth factor. In some specific embodiments, the at least one growth factor comprises at least one of: transferrin, fibroblast growth factor-2 (FGF-2), insulin and Platelet-derived growth factor BB (PDGF-BB).

In some embodiments, the desired phenotype is cell proliferation in suspension conditions. Accordingly, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, encodes or controls the production and/or activity, and/or levels of at least one of: transforming growth factor beta induced (TGFBI), A disintegrin and metalloprotease 12 (ADAM12), Plakophilin-3 (PKP3), yes-associated protein 1 (YAP1) or TAFAZZIN (TAZ) genes. Still further, in some alternative or additional embodiments, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequences used by the disclosed methods, may comprise at least one protease. In some specific embodiments, such proteases may facilitate growth in suspension conditions, preventing formation of cell aggregates. Non-limiting embodiment for proteases useful in the present methods may include Matrix metalloproteinases (MMPs), natural proteases (Dispase), and the like.

In some embodiments, the desired phenotype is cell proliferation at high cell density. According to these embodiments, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, encodes or controls the production and/or activity, and/or levels of at least one of: gene/s related to metabolic shifts, enhanced glucose uptake, reduced lactate production, enhanced lactate degradation and/or enhanced glutamine production and ammonia degradation.

In some embodiments, the conditional nucleic acid sequence used by the disclosed methods is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. It should be understood that "adherence conditions" refer to the specific environmental or culture conditions that enable cells to attach to a surface, such as a culture dish, scaffold, or substrate. These conditions typically include factors such as the type of surface or coating (e.g., extracellular matrix proteins like collagen or fibronectin), the composition of the culture medium (e.g., nutrient levels, presence of adhesion molecules), temperature, pH, and the mechanical properties of the surface. Accordingly, the cellular- and/or environmental- state and/or condition sensed by such synthetic promoter comprises adherence conditions, in such case, the desired phenotype may comprise at least one of: non-proliferative state of the cells and/or cell differentiation and/or tissue maturation in adherence conditions. In some embodiments, the desired phenotype in adherence conditions may be differentiation. Cell differentiation is the process by which a less specialized cell becomes a more specialized cell type with distinct structural and functional characteristics. During differentiation, stem cells or progenitor cells undergo a series of changes in gene expression, morphology, and function, ultimately acquiring the specialized attributes needed to become part of the cell-based product. This process is tightly regulated by intrinsic genetic programs and external signals such as growth factors, cytokines, and environmental cues. Differentiation allows for the development of various cell types required for the disclosed cell-based product, such as muscle cells, adipose cells, endothelial cells, or epithelial cells, from a common precursor cell. In some embodiments, the desired phenotype in adherence conditions may be non-proliferative state of the cells. More specifically, non-proliferative state refers to a condition in which a cell is no longer actively dividing or replicating. In this state, the cell remains in a quiescent or dormant phase, temporarily or permanently halting progression through the cell cycle. Cells in a non-proliferative state may remain viable and functional, but they do not undergo mitosis or generate daughter cells. In some cases, cells may exit the cycle at key checkpoints, such as the Gl/S or G2/M transition, and enter phases like GO, where they are temporarily withdrawn from active division. In some embodiments, the desired phenotype in adherence conditions may be tissue maturation. Tissue maturation refers to the process by which cultured or engineered tissue undergoes structural and functional development over time, ultimately reaching a state that closely mimics natural, fully developed tissue. In the context of cell-based products, tissue maturation involves the organization, differentiation, and functionalization of cells into a mature, functional tissue. This process can include the formation of extracellular matrices, the development of appropriate cellular architecture, and the establishment of necessary biochemical and mechanical properties.

In some embodiments, the at least one transcription factor binding site of the synthetic conditional nucleic acid sequence of the present disclosure used in the disclosed methods, is in the length of 8 to 25 nucleotides, specifically, 15 to 22 nucleotides.

In more specific embodiment, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions used by the disclosed methods, comprises the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T. In some embodiments of the disclosed methods, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in the adherence conditions, may comprise at least one transcription factor binding site, specifically, at least one NF-KB binding site. More specifically, an NF-KB binding site refers to a specific DNA sequence that is recognized and bound by the nuclear factor kappa-light-chain- enhancer of activated B cells (NF-KB) transcription factor. NF-KB is a protein complex that plays a crucial role in regulating immune response, inflammation, cell survival, and other biological processes. When NF-KB binds to its binding site, it can either activate or repress the transcription of nearby genes, thereby influencing various cellular processes.

In some embodiments, the disclosed methods may use synthetic conditional nucleic acid sequence, specifically synthetic promoters specific for adherence conditions comprising the at least one transcription factor binding site having the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof.

In some embodiments, the at least one synthetic conditional nucleic acid sequence comprises 3 to 100 repeats of the transcription factor binding site. In some embodiments, the at least one synthetic conditional nucleic acid sequence is in the length of 250 to 500 nucleotides, specifically, 270 to 300 nucleotides.

In yet some further embodiments, for adherent conditions, the at least one synthetic conditional nucleic acid sequence specific for adherence conditions used in the disclosed methods may comprise the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

It should be understood that the disclosed TF binding motifs are present in the TFBS of the synthetic promoters identified and used by the present disclosure, may be presented as a complete motif, or as a partial motif, that is also indicated herein as "functional fragment", for example, in the TFBS of the suspension promoters, some of the promoters contain part of the motif, for example, about a half of the motif. Thus, in some embodiments a fragment" of the motif, refers to herein to any fragment that enables binding of the transcription factor and transcription control. For example, the promoter D6M_483 (SEQ ID NO: 4), contains part of the motif as denoted by SEQ ID NO: 77, namely, GAAACT,

It should be understood that the disclosed transcription factor (TF) binding motifs are present in the TF binding sites (TFBS) of the synthetic promoters identified and used in the present disclosure. These motifs may be presented either as a complete motif or as a partial motif, which is referred to herein as a "functional fragment." For instance, in the TFBS of the suspension promoters, some promoters contain only a portion of the motif, such as approximately half of the full motif. Alternatively, a quarter, third, fifth, or other fractional portions of the motif may also function as a "functional fragment" as long as they retain the necessary structural elements to enable binding of the transcription factor and facilitate transcriptional regulation. Therefore, in some embodiments, a "fragment" of the motif refers to any segment that retains the ability to bind the transcription factor and regulate transcription. For example, the promoter may contain a functional fragment that is sufficient for transcriptional control. In some embodiments, the disclosed fragment of the TFBS in promoter D6M_483 (SEQ ID NO: 4), contains part of the motif as denoted by SEQ ID NO: 77, namely, GAAACT.

In some embodiments, the use of these adherent specific synthetic conditional nucleic acid sequences (synthetic promoters) by the disclosed methods, allows achieving the desired phenotype in adherence conditions. In some embodiments, the desired phenotype comprises differentiation of the cell/s under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell. According to such embodiments, the at least one nucleic acid sequence of interest, controlled by the adherence-specific synthetic promoter, encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

In some embodiments, the desired phenotype is differentiation and/or tissue maturation. Therefore, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, the adherence-specific synthetic promoter, encodes, or controls the production and/or activity and/or levels, of at least one gene related to adipogenesis or myogenesis.

In some embodiments, at least gene related to adipogenesis comprises at least one of ZFP423, API AP-1 (Activator Protein-1), C/EBPa, P and 5 and PPARy. More specifically, ZFP423 (Zinc Finger Protein 423) is a transcription factor that belongs to the C2H2 zinc finger protein family. It plays a crucial role in regulating gene expression, particularly in neural development, adipogenesis, and stem cell differentiation. CCAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors that regulate gene expression in differentiation, metabolism, and immune responses. The three key members — C/EBPa, C/EBP , and C/EBPy — share a basic leucine zipper (bZIP) domain, which enables them to bind DNA and form homo- or heterodimers, modulating transcriptional activity. These factors play a crucial role in regulating cell differentiation, metabolism, and proliferation, particularly in adipogenesis, myelopoiesis, and liver function. PPARy (Peroxisome Proliferator- Activated Receptor Gamma) is a nuclear receptor and transcription factor that plays a central role in adipogenesis, glucose metabolism, and inflammation regulation.

In some embodiments, at least gene related to myogenesis comprises at least one of Sixl/4, Pax3, Pax7, Myf5, MyoD and MyoG. More specifically, Pax3 (Paired Box 3) is a transcription factor belonging to the PAX family, specifically, Pax 3, and 7, which plays a crucial role in embryonic development, neural crest formation, and muscle precursor cell differentiation. Myf5 (Myogenic Factor 5) is a transcription factor that plays a critical role in muscle development. MyoD (Myogenic Differentiation 1) and Myogenin (MyoG) are both transcription factors that play essential roles in muscle development and differentiation.

It should be understood that nucleic acid sequences encoding each of these specified factors may be used in some embodiments as the nucleic acid sequence of interest controlled by the synthetic promoters of the present disclosure. Still further, in some embodiments, the desired phenotype is cell immortalization. According to such embodiments, the at least one nucleic acid sequence of interest controlled by the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure, encodes or controls the production and/or activity, and/or levels of at least one of: gene/s related to cell immortalization comprise at least one of TERT and CDK4.

In yet some further embodiments, the disclosed methods involve the use of at least one cellular input-output (or sensor-output) unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition. More specifically, the unit/s used in the disclosed methods may comprise: (i), at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and (ii), at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence of (i). More specifically, the at least one synthetic conditional nucleic acid sequence of

(i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of the at least one operably linked nucleic acid sequence of interest of

(ii), upon sensing the cellular- and/or environmental- state and/or conditions.

In yet some further alternative or additional embodiments, the methods of the present disclosure may use a nucleic acid cassette or any vector or vehicle thereof, comprising the at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence (specifically, a synthetic promoter), or at least one cellular input-output unit comprising the at least one nucleic acid molecule. Accordingly, the disclosed method may comprise the step of introducing into at least one cell, at least one nucleic acid cassette comprising the synthetic conditional nucleic acid sequence of the present disclosure, or any a cellular input-output unit thereof. In some embodiments, the nucleic acid cassette used in the disclosed methods optionally further comprises at least one genetic element. It should be understood that genetic elements included in the cassette used by the disclosed methods are described in more detail in connection with other aspects of the present disclosure and are thus encompassed by the present aspect as well.

In yet some further alternative or additional embodiments, the disclosed methods may use a cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. Accordingly, the disclosed method may comprise the step of introducing into at least one cell, at least one cellular platform. More specifically, the disclosed platform, that according to some embodiments is used by the methods of the present disclosure, may comprise at least one synthetic conditional nucleic acid sequence (e.g. synthetic promoter/s), or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cellular- and/or environmental- state and/or conditions.

In some embodiments, the cellular platform of the present disclosure used in the disclosed methods, may comprise at least one cellular input-output unit or any nucleic acid cassette or vector comprising the same. Such unit comprising: (i), at least one nucleic acid molecule comprising the at least one synthetic conditional nucleic acid sequence (e.g., synthetic promoter); and (ii), at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of the at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions.

In some particular and non-limiting embodiments, the cellular platform used by the methods of the present disclosure may comprise at least two of the following components:

(a), at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in cell suspension conditions; (b), at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in adherence conditions; (c), at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in high cell density conditions; and/or

(d), at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in three-dimensional scaffold (3D-scaffold) (or aggregate) conditions. As indicated above, the present disclosure provides methods directed at the production of a cellbased product. In some embodiments, a cell-based product is at least one of: a food product, an additive, a medicament, a cosmetic product or an in vitro multicellular system.

In some embodiments, the disclosed methods are particularly applicable for producing a food product. In more specific embodiments, such food product is an animal cell-based meat (ACBM) product.

In some embodiments, the cell of the present disclosure, that may be also applicable in the disclosed cell-based products and methods, may be a eukaryotic cell or a prokaryotic cell.

Prokaryotic cells are any cells that lack a membrane-bound nucleus and other membrane-bound organelles. The cells of the present disclosure may be therefore in some embodiments any bacterial or archaeal cell. Still further, the cell of the present disclosure may be a unicellular or multicellular eukaryotic cell. More specifically, the eukaryotic cell of the present disclosure may be any unicellular or multicellular eukaryotic cell of the biological kingdom Fungi (e.g. yeast cells), Protists (algae, protozoa), the biological kingdom Animalia or the biological kingdom Plantae.

It should be further understood that the term "Cell", is defined here as to comprise any type of cell, a prokaryotic cell or a eukaryotic cell, isolated or not, cultured or not, differentiated or not, and comprising also higher levels organizations of cells such as tissues, organs, calli, organisms or parts thereof.

In more specific embodiments, the cell of the disclosed product may be a eukaryotic cell. Such eukaryotic cell may be of at least one unicellular or multicellular organism of the biological kingdom Animalia or of the biological kingdom Plantae. In some embodiments, the cells of the preset disclosure, that may be also applicable in the disclosed methods and cell-based products, may be derived from any eukaryotic organism of the biological kingdom Animalia. In other embodiments, such organism may be any one of a vertebrate or an invertebrate.

Invertebrates are animals that neither possess nor develop a vertebral column (commonly known as a backbone or spine), derived from the notochord. This includes all animals apart from the subphylum Vertebrata. Familiar examples of invertebrates include insects; crabs, lobsters and their kin; snails, clams, octopuses and their kin; starfish, sea-urchins and their kin; jellyfish and worms. Vertebrates comprise all species of animals within the subphylum Vertebrata (chordates with backbones). Vertebrates represent the overwhelming majority of the phylum Chordata, with currently about 66,000 species described. Vertebrates include the jawless fish and the jawed vertebrates, which include the cartilaginous fish (sharks, rays, and ratfish) and the bony fish.

Still further, in some embodiments, the cells of the preset disclosure may be derived from anyone of a non-human mammal, an avian, an insect, a fish, an amphibian, a reptile, a crustacean, a crab, a lobster, a snail, a clam, an octopus, a starfish, a sea-urchin, jellyfish, and worms.

In more specific embodiments, the cells of the preset disclosure may be derived from any mammal, specifically, a non-human mammal. In yet some further embodiments, such mammalian organisms may include any member of the mammalian nineteen orders, specifically, Order Artiodactyla (even-toed hoofed animals), Order Carnivora (meat-eaters), Order Cetacea (whales and purpoises), Order Chiroptera (bats), Order Dermoptera (colugos or flying lemurs), Order Edentata (toothless mammals), Order Hyracoidae (hyraxes, dassies), Order Insectivora (insect-eaters), Order Lagomorpha (pikas, hares, and rabbits), Order Marsupialia (pouched animals), Order Monotremata (egg-laying mammals), Order Perissodactyla (odd- toed hoofed animals), Order Pholidata, Order Pinnipedia (seals and walruses), Order Primates (primates), Order Proboscidea (elephants), Order Rodentia (gnawing mammals), Order Sirenia (dugongs and manatees), Order Tubulidentata (aardvarks).

More specifically, the present nucleic acid molecules, product, methods and units or platforms thereof as disclosed by the present disclosure offer great economic advantage for any industrial or agricultural use of animals, specifically, livestock. Thus, in some specific embodiments, the cells of the preset disclosure may be derived from any mammalian livestock. Livestock are domesticated animals raised in an agricultural setting to produce labor and commodities such as meat . The term includes but is not limited to Cattle, sheep, domestic pig (swine, hog), horse, goat, alpaca, lama and Camels. Of particular interest are cattle applicable in the meat industry. More specifically, in certain embodiments, the cells of the preset disclosure may be derived from a Cattle, colloquially cows, that are the most common type of large, domesticated ungulates, that belong to the In some embodiments, the cells of the preset disclosure may be derived from any mammalian organisms of the Order Artiodactyla, including members of the family Suidae, subfamily Suinae and Genus Sus, and members of the family Bovidae, subfamily Bovinae including ungulates. Still further, the Bovidae are the biological family of cloven-hoofed, ruminant mammals that includes bison, African buffalo, water buffalo, antelopes, wildebeest, impala, gazelles, sheep, goats, muskoxen. The biological subfamily Bovinae includes a diverse group of ten genera of medium to large-sized ungulates, including domestic cattle, bison, African buffalo, the water buffalo, the yak, and the four-horned and spiral-horned antelopes. Of particular interest in the present invention may be the domestic cattle are the most widespread species of the genus Bos and are most commonly classified collectively as Bos taurus. More specifically, Bos is the genus of wild and domestic cattle. Bos can be divided into four subgenera: Bos, Bibos, Novibos, and Poephagus. Subgenus Bos includes Bos primigenius (cattle, including aurochs), Bos primigenius primigenius (aurochs), Bos primigenius taurus (taurine cattle, domesticated) and Bos primigenius indicus (zebu, domesticated).

Still further, in some embodiments, the present disclosure (specifically, the methods, products, nucleic acid molecules, cells, units and platforms), may be applicable for cells derived from pigs. The pig Sus domesticusf is of the mammalian family Equidae, and the order Artiodactyla. Pigs may be also referred to herein as swine, hog, or domestic pig when distinguishing from other members of the genus Sus, is an omnivorous, domesticated, even-toed, hoofed mammal. It is variously considered a subspecies of Sus scrofa (the wild boar or Eurasian boar) or a distinct species.

When used as livestock, pigs are farmed primarily for the production of meat.

Still further, in some embodiments, the present disclosure (specifically, the methods, products, nucleic acid molecules, cells, units and platforms), may be applicable for the mammalian family Equidae, more specifically a horse. The "horse" (Equus ferus caballus) is a domesticated, one-toed, hoofed mammal. It belongs to the taxonomic family Equidae and is one of two extant subspecies of Equus ferus.

In yet some further embodiments, the cells of the present disclosure may be derived from any avian organisms. In yet some further specific embodiments, birds are provided as a source for the cells of the present disclosure. More specifically, domesticated and undomesticated birds are also suitable organisms for the cells of the present disclosure. In more specific embodiment, the avian organism may be any one of a poultry or a game bird. The term "avian" relates to any species derived from birds characterized by feathers, toothless beaked jaws, the laying of hard-shelled eggs, a high metabolic rate, a four-chambered heart, and a lightweight but strong skeleton. In some specific embodiments, the avian organism may be of the order Galliformes which comprise without limitation, chicken, quail, turkey, duck, Gallinacea sp, goose, pheasant and other fowl. In yet some specific embodiments, the cells of the present disclosure may be derived from a chicken.

In yet some further embodiments, the cells of the present disclosure may be derived from any organism of the aquaculture industry.

Fish, as used herein refer to gill-bearing aquatic craniate animals that lack limbs with digits. They form a sister group to the tunicates, together forming the olfactores. Included in this definition are the living hagfish, lampreys, and cartilaginous and bony fish as well as various related groups. It should be noted that the present invention relates to any group, class, subclass or any family of fish. Specifically, any fish of the following families, specifically, Cyprinidae, Gobiidae, Cichlidae, Characidae, Loricariidae, Balitoridae, Serranidae, Labridae, and Scorpaenidae.

In some specific embodiments, the cells of the preset disclosure may be derived from any organism of the genus tilapia. Tilapia, as used herein is the common name for nearly a hundred species of cichlid fish from the tilapiine cichlid tribe.

In more specific embodiments, the tilapia fish may be of the Oreochromis niloticus species. In yet other embodiment, the tilapia fish may be of any one of the species Oreochromis aureus, Oreochromis karongae or Pelmatolapia mariae.

Still further, the invention may be useful for cells derived from crustaceans organisms. Crustaceans, as used herein, form a large, diverse arthropod taxon which includes crabs, lobsters, crayfish, shrimp, krill, woodlice, and barnacles, that are all encompassed by the present invention. The crustacean group is usually considered as a paraphyletic group and comprises all animals in the Pancrustacea clade other than hexapods. In some embodiments, such crustaceans may be shrimp. The term shrimp is used to refer to decapod crustaceans and covers any of the groups with elongated bodies and a primarily swimming mode of locomotion i.e. Caridea and Dendrobranchiata.

As mentioned above, in some embodiments thereof, the present disclosure concerns cells derived from members of the biological kingdom Plantae. Thus, in some further embodiments, the eukaryotic organisms may be a dioecious plant. In yet some further embodiments, the cell of the present disclosure, that may be also applicable in the disclosed methods and cell-based products is of an organism of the biological kingdom Animalia. More specifically, such organism is any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

In some embodiments, the cell-based product of the present disclosure is at least one of: a food product, an additive, a medicament, a cosmetic product or an in vitro multicellular system. More specifically, in some embodiments, the cell-based product may be a food product. A food product, as used herein, refers to any substance that is processed, prepared, or manufactured for human or animal consumption, either as a whole food, ingredient, or formulated composition. It may further include in addition to the disclosed cells, natural, semi-processed, or fully processed items derived from plant, animal, microbial, or synthetic sources. Food products as provided herein, can be solid, liquid, or semi-solid and may undergo preservation, fortification, or modification to enhance shelf life, nutritional value, taste, texture, or safety. This term as used herein, encompasses a wide range of consumables, including packaged goods, beverages, dietary supplements, and functional foods designed for specific health benefits.

In some embodiments, the cell-based product may be an additive. Specifically, an additive refers to a substance that is intentionally introduced into a product to modify its properties, such as stability, appearance, texture, taste, or functionality. Additives can be used in food, pharmaceuticals, cosmetics, and industrial applications and may include preservatives, colorants, emulsifiers, stabilizers, or bioactive compounds.

In yet some further embodiments, the cell-based product provided herein, may be, or may be used as a medicament. A medicament is a substance or composition formulated for therapeutic use in the prevention, diagnosis, treatment, or alleviation of disease or medical conditions in humans or animals. Medicaments can include pharmaceuticals, biologies, and natural or synthetic compounds administered in various forms, such as tablets, injections, or topical applications.

Still further, the disclosed cell-based product may be in some embodiments, a cosmetic product. A cosmetic product is a formulation designed for external application to the human body to cleanse, protect, enhance appearance, or maintain skin, hair, nails, or oral hygiene. Cosmetics include skincare products, makeup, shampoos, deodorants, and sunscreens, which primarily exert their effects on the body’s surface without therapeutic action.

In yet some further embodiments, the cell-based product provided herein, may be, or may be used as an in vitro multicellular system. An in vitro multicellular system refers to a culture or model composed of multiple interacting cell types grown outside a living organism under controlled laboratory conditions. These systems are used to study cell-cell interactions, tissue function, drug responses, or disease mechanisms and may include organoids, co-cultures, or 3D tissue models, as well as for screening for therapeutic compounds.

In yet some further embodiments, the cell-based product is a food product, specifically, an animal cell-based meat (ACBM) product.

In some embodiments, any of the cell-based products of the present disclosure, and specifically, the ACBM product may be prepared by the methods according to the present disclosure, as defined herein above.

A cell-based product, as provided by the present disclosure and prepared by the disclosed methods, refers to a product that contains living cells, specially, the cells of the present disclosure, which can be derived from any source as defined herein. It should be understood that the cell-based products provided by the present disclosure and prepared by the disclosed methods, encompassed products that contain the cells disclosed herein, while the percentage of cells in such a product can vary significantly, depending on the formulation and purpose. In a product, the concentration of cells may range from 5% to 100. For example, a product with 5% cells means that only a small proportion of the product consists of living cells, with the rest comprising other materials or substances. A 10% concentration of cells indicates a slightly higher proportion of living cells, while 15% and 20% show gradual increases in cell content. As the percentage increases, such as 25%, 30%, and 35%, the product contains more cells relative to other components. At 50%, the product consists of an equal proportion of cells and other substances. A 60% cell concentration and higher, such as 70% or 75%, indicate that cells make up a significant majority of the product. For products with 80%, 90%, or 100% cell content, the majority or entirety of the product is composed of living cells, with minimal to no other components. In addition to living cells, a cellbased product may contain various other components and ingredients to support function, and stability. These can include amino acids, vitamins, sugars, and salts. Buffering agents help maintain the proper pH in the product. Preservatives might be incorporated to extend the shelf life of the product, particularly in the case of cryopreserved cells. Additionally, scaffolds or extracellular matrix components may be included in tissue engineering applications to support cell attachment and tissue formation. Other ingredients can include cryoprotectants for cells stored at low temperatures, and biological buffers for controlling osmolarity or maintaining ionic balance within the product.

In some specific and non-limiting embodiments, a cell-based product may be a product composed of a cell mass composed of a single cell type, or two or three cell types. For example, in some embodiments, a cell-based product may be a mass of adipose cells. Specifically, a product composed of fat cells, and/or fat tissue. In some embodiments, such product may be composed of 5% to 100% of adipose cells, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of adipose cells in the cell-based product. Adipose cells, also known as adipocytes, are specialized cells primarily responsible for storing energy in the form of lipids. Adipocytes can be classified into white adipose cells, which store large lipid droplets for long-term energy storage, and brown adipose cells, which contain multiple smaller lipid droplets and a high number of mitochondria, enabling thermogenesis through heat production. A third type, beige adipocytes, exhibits characteristics of both white and brown adipose cells and can be induced under certain physiological conditions. It should be understood that the disclosed product may be composed of each of the specified adipose cells, or any combinations thereof, or any combination or tissue-like product composed of combination of these adipose cells with other cells, or any product produced by these cells (e.g. lipids). Still further, in some specific and non-limiting embodiments, a cell-based product may be a mass of muscle cells. Specifically, a product composed of muscle cells, and/or muscle tissue. In some embodiments, such product may be composed of 5% to 100% of muscle cells, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of muscle cells in the cell-based product. More specifically, muscle cells, also known as myocytes, are specialized cells responsible for generating force and movement through contraction. They are the fundamental units of muscle tissue and can be categorized into three main types: skeletal muscle cells, cardiac muscle cells, and smooth muscle cells. It should be appreciated that the products of the preset disclosure may be composed of muscle cell mass of any of the specified types or any combinations thereof, or any product produced by these cells. In yet some further embodiments, the disclosed product may be composed of fibroblast cells. In some embodiments, such product may be composed of 5% to 100% of fibroblast cells, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of fibroblast cells in the cell-based product. More specifically, fibroblast cells are specialized mesenchymal cells responsible for producing and maintaining the extracellular matrix (ECM) and connective tissue.

Still further, the present disclosure further provides methods for producing cell-based products, that may be any product produced by the cells of the present disclosure (e.g., cell that comprise the synthetic promoters of the present disclosure). Still further, the present disclosure provides a cellbased product that may be any product produced by the disclosed cells. Products produced by the cels of the present disclosure may include proteins, Carbohydrates, Lipids (polyunsaturated fatty acids (PUFAs) like omega-3 and omega-6), Vitamins, Metabolites and bioactive compounds, and cellular components. More specifically, cell-based product as used herein may be categorized based on their function and application. Therapeutic proteins and biologies include monoclonal antibodies used in immunotherapy, cytokines like interleukins and interferons for immune modulation, and hormones such as insulin, erythropoietin, and growth hormones for metabolic and hematopoietic disorders. Coagulation factors, including Factor VIII and Factor IX, (e.g., for the treatment of hemophilia). Enzymes for therapeutic use include tissue plasminogen activator (tPA) for dissolving blood clots, glucocerebrosidase for lysosomal storage disorders, and asparaginase for leukemia treatment. Nutritional and metabolic products include vitamins such as vitamin B12, essential amino acids like lysine and tryptophan, and polyunsaturated fatty acids such as omega-3 and omega-6, produced using microbial or mammalian cell cultures. These compounds are used in dietary supplements, functional foods, and pharmaceutical formulations, and in some embodiments, may be provided as the cell-based product by the preset disclosure. Industrial and specialty biomolecules in some embodiments, may be provided as the cell-based product by the preset disclosure, include enzymes used in textiles, such as proteases for bio-polishing and amylases for desizing fabrics. Lipases and oxidoreductases are applied in laundry detergents for stain removal and fabric care. In cosmetics, recombinant collagen and hyaluronic acid are produced for anti-aging and skincare applications, while bioengineered keratin and elastin are used in hair and nail treatments. Additionally, biodegradable bioplastics, biofuels, and silk proteins for medical sutures and textiles are derived from cell-based systems, may be provided in some embodiments, as the cell-based product by the preset disclosure. A further aspect of the present disclosure relates to a method of preparing an animal cell-based meat (ACBM) product. The method comprising: (a), culturing under suitable conditions at least one source cell or at least one population of the cells. It should be understood that the source cell/s comprise and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence (specifically, a synthetic promoter), or an input-output (or sensor-output) unit, a nucleic acid cassette and/or a platform comprising the nucleic acid molecule. The next or additional step (b) involves processing the cells and/or at least one product produced by, or produced from, the cell to prepare the food product. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition. Still further, the controlled expression of at least one of the nucleic acid sequences of interest results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

In some embodiments, any of the nucleic acid molecules of the present disclosure may be used for the disclosed methods. Still further, the disclosed methods may use any of the input-output (or sensor-output) unit as defined by the present disclosure, and/or any of the cassettes disclosed herein, and/or any of the cellular platform/s as define by the present disclosure.

In some embodiments, the source cells are programed for at least one of: autonomous growth, proliferation, expansion, differentiation, immortalization, tissue and/or organ formation, maturation, production of at least one product of interest and/or modulated cell activity, in dynamic cellular- and/or environmental- state and/or conditions. In some embodiments, the product of the present disclosure is prepared by a method comprising the methods for producing a cell-based product, as described by the present disclosure, and indicated herein above. Thus, the disclosed methods for preparing the ACBM product, comprise in step (a) thereof, at least part of, and preferably, all steps used for the preparation of a cell-based product, as defined by the present disclosure. In more particular embodiments, step (a) of the disclosed methods for preparing the ACBM product, that involves the culturing of the source cell/s that comprise and/or genetically engineered by the synthetic promotes of the present disclosure, comprises all steps as define herein above in connection with other aspects of the present disclosure, specifically, the steps of the methods for producing the cell-based product as defined by the present disclosure.

As indicated herein, the disclosed methods for preparing the cell-based products and in particular the methods for preparation of ACBM product, provides cells that are programed for autonomous actions such as at least one of growth, proliferation, expansion, differentiation, immortalization, tissue and/or organ formation, maturation, production of at least one product of interest and/or modulated cell activity, in dynamic cellular- and/or environmental- state and/or conditions. More specifically, in some embodiments, the cells used in the disclosed methods, that are genetically modified by, and/or comprise the synthetic conditional nucleic acid sequence (e.g., synthetic promoters), or any unit, cassette or platform thereof, are cells thar independently acquire or express a specific, desired characteristic or function (phenotype), such as growth, proliferation etc., without requiring continuous external intervention. This occurs through the introduction of the synthetic promoters of the present disclosure and the input/outputs units thereof, which are integrated into the own molecular machinery of the cell. These elements enable the cell to autonomously sense changing culture conditions (dynamic) and to produce or regulate the desired phenotype, such as altered metabolism, enhanced resistance to stress, or specific protein production, depending on the intended modification. The term "adapted" in this context implies that the phenotype is specifically tailored to respond to or function within particular environmental or cellular conditions, allowing the cell to adapt its behavior in a controlled and predictable manner. In some embodiments, the cellular- and/or environmental- state and/or conditions comprise at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D-scaffold), levels of lactate, levels of ammonia, pH conditions, salinity conditions, osmolarity conditions, strength of magnetic field, oxidative stress, humidity, temperature conditions, level of nutrients, levels of available amino acids, levels of ions, level of growth factors, level of signaling molecules, cell motility, shear force, levels of cytokines, levels of toxins, levels of oxidants, presence of pathogens, levels of hormones, metabolite accumulation, accumulation of cell waist, and/or cell metabolic state.

Still further, in some embodiments of the disclosed methods, the dynamic cellular- and/or environmental- state/s and/or condition/s comprise at least one change in the cellular- and/or environmental- state/s and/or condition/s over time. More specifically, dynamic cellular and/or environmental state(s) and/or condition(s)" refers to the continuously changing biological or external factors that influence the cells in the culture over time (hours, days). A dynamic cellular state or condition encompasses variations in physiological, biochemical, or genetic characteristics, such as fluctuations in gene expression, metabolic activity, differentiation status, or signaling pathway activation in response to internal or external stimuli received from the changing culture conditions, and cell-cell interactions. Similarly, a dynamic environmental state or condition involves external factors that change over time, including variations in temperature, pH, nutrient availability, osmotic pressure, mechanical forces, or the presence of signaling molecules.

In some embodiments the cell used by the disclosed methods is a eukaryotic cell or a prokaryotic cell.

In Some embodiments the eukaryotic cell used by the disclosed methods is of at least one unicellular or multicellular organism of the biological kingdom Animalia or of the biological kingdom Plantae.

Still further, the source cell used by the methods of the present disclosure is of an organism of the biological kingdom Animalia. Such organism is any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

In some embodiments the source cells applicable in the present disclosure may be any pluripotent cells, IPSCs, specifically, any embryonic stem cells derived from any germ layers (mesoderm, ectoderm, endoderm). In some embodiments, the source cells may be mesenchymal cells that can differentiate into adipocytes, chondrocytes, myocytes, osteoblasts, and neurocytes, in addition to other cell types. Still further, it should be appreciated that any cell disclosed by the present disclosure is also applicable for the present aspect as well.

In some embodiments, at least one source cell used in the disclosed methods is a mesenchymal multipotent cell. Specifically, a mesenchymal multipotent cell refers to a type of progenitor cell derived from mesenchymal tissues that retains the ability to differentiate into multiple cell lineages. These cells, often referred to as mesenchymal stem cells (MSCs), exhibit multipotency, meaning they can give rise to various mesodermal-derived cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).

In yet some further embodiments, the at least one source cell is at least one of an adipose stem cell, a satellite cell, and/or a dermal fibroblast. More specifically, an adipose stem cell (ASC) is a type of mesenchymal stem cell found in adipose (fat) tissue, capable of differentiating into adipocytes (fat cells), osteoblasts (bone cells), chondrocytes (cartilage cells), and other mesodermal-derived cell types. A satellite cell is a type of muscle stem cell located between the basal lamina and sarcolemma of muscle fibers. A dermal fibroblast is a connective tissue cell found in the dermis of the skin, responsible for producing extracellular matrix components such as collagen and elastin. In some embodiments, the source mesenchymal stem cells are of a non-human mammal. More specifically, the non-human mammal is at least one of Cattle, domestic pig (swine, hog), sheep, horse, goat, buffalo, alpaca, lama and Camels.

A further aspect of the present disclosure relates to an animal cell-based meat (ACBM) product comprising at least one cell or at least one population of the cells, and/or any product of interest produced by, or produced from said cells. The cell/s of the ACBM disclosed herein may comprise and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising the at least one nucleic acid molecule. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, thereby the cell of the disclosed ACBM, autonomously exhibits at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition. In some embodiments, the ACBM product of the present disclosure is prepared by the method as defined by the present disclosure herein above.

In some embodiments, the animal cell-based meat (ACBM) product may be prepared from, or comprise cells of non-human mammal, an avian, a fish, a crustacean, a crab and/or a lobster. In some embodiments, the ACBM product may be prepared from cells of a non-human mammal. More specifically, cells of at least one of Cattle, domestic pig (swine, hog), sheep, horse, goat, buffalo, alpaca, lama and Camels. In specific some embodiments, the ACBM may be prepared from cells of a domestic pig (swine, hog).

An "animal cell-based meat product", as used herein, refers to a food product that is derived from or contains meat produced through the in vitro cultivation of animal cells, as defend defined by the methods of the present disclosure, as opposed to conventional livestock farming. These products encompass compositions where animal cells are the primary ingredient, as well as hybrid products that combine cultured animal cells with plant-based or synthetic components. Such hybrid formulations aim to enhance the taste, texture, and nutritional profiles of the final product. Still further, animal cell-based meat products can be categorized into several types, depending on their structure and method of production. It should be understood that all the disclosed categories, for example, unstructured products, structured products and hybrid products, are encompassed by the ACBM product of the present disclosure. More specifically, unstructured products, such as ground meat, meatballs, sausages, or nuggets, are composed of cultured animal cells in a dispersed or aggregated form. These products rely on the cultivation of animal cells to provide meat-like characteristics, but the final structure may not resemble that of conventional cuts of meat. On the other hand, structured products, including steaks, fillets, or whole cuts, are created using scaffolding, bioprinting, or tissue engineering techniques. These methods aim to replicate the texture and structure of traditionally farmed meat, providing a more authentic eating experience. Additionally, hybrid products, is a category of products that may incorporate plant-based proteins, fats, or other food-grade ingredients along with cultured animal cells to enhance sensory properties or improve cost efficiency. Still further, it should be understood that the percentage of animal cells in a product can vary, which directly influences its texture, nutritional profile, and similarity to conventional meat. The ACBM products of the present disclosure comprise any rate of cell content. In some embodiments, products with low cell content (5%-30%) typically consist primarily of plant-based, fungal, or synthetic components, with a small proportion of cultured animal cells used to enhance flavor, aroma, or texture. Examples of such products are hybrid plant- cultured meat formulations. Moderate cell content products (31%-60%) strike a balance between cultured animal cells and other ingredients, offering a meat-like experience while maintaining cost efficiency. These products may incorporate plant-based scaffolds or binders to help achieve the desired consistency. High cell content products (61%-90%) contain a dominant proportion of cultured animal cells, with only minimal additional ingredients for structural support or enhancement. These products more closely mimic the texture and taste of conventional meat. Near-pure cell content products (91 %-99%) are almost entirely made from cultured animal cells, with only minor additives such as food-grade stabilizers or nutrients. Finally, pure cell-based products (100%) are made exclusively from cultured animal cells, without any non-animal-derived components. This category represents the closest replication of conventional meat, offering a fully cultured product. Still further, in some embodiments of the animal cell-based meat products encompassed by the present disclosure, various non-cellular components are incorporated to support the structure, texture, taste, and nutritional value of the final product. These components play a crucial role in ensuring that the cultured meat closely resembles conventional meat in terms of appearance, mouthfeel, and overall sensory experience. One of the primary categories of non- cellular components includes structural elements, such as plant-based scaffolds, biodegradable polymers, and extracellular matrix (ECM) proteins. Scaffolds derived from soy protein, pea protein, or alginate provide a framework for cell attachment and tissue formation, mimicking the structural integrity of muscle fibers. Biodegradable polymers, such as collagen, gelatin, and chitosan, further enhance texture and enable proper cellular organization. Additionally, ECM proteins like laminin and fibronectin play a critical role in promoting cell adhesion and supporting tissue maturation. Hydrogels, including agarose, hyaluronic acid, and methylcellulose, help retain moisture and contribute to the meat’s juiciness, while gelling agents such as pectin, carrageenan, and konjac gum improve stability and mouthfeel. Still further, cultured meat products, derived from animal cells of the present disclosure, can be categorized based on their processing and preparation methods. Ground and minced cultured meats include products like cultured ground beef, pork, and chicken, which can be used to create patties, meatballs, and meatloaf. Processed cultured meats include alternatives to traditional deli meats such as cultured ham, turkey slices, and roast beef, as well as luncheon meats like bologna and mortadella. Sausages made from cultured cells, including fresh, smoked, and cured varieties like cultured salami and pepperoni, replicate conventional sausage textures and flavors. Ready-to-eat or convenience cultured meat products include nuggets, hot dogs, and canned meats, offering easy-to-prepare alternatives. Dry and preserved cultured meats, such as lab-grown jerky and meat sticks, provide extended shelf life. Specialty cultured meat products include pate, terrines, and stuffed meats like cultured cordon bleu. Additionally, structured cultured meat cuts, such as steaks, ribs, and fillets, aim to replicate wholemuscle textures. In some embodiments, the ACBM products of the present disclosure may be cultured meat and fat products derived from muscle and adipose cells, that include ground meats, structured cuts like steaks, sausages, nuggets, and other processed meat alternatives. In yet some further embodiments, the ACBM products of the present disclosure may be cultured fat, that is rich in bioengineered lipids and may be used to enhance the taste and texture of various meat products, making them more comparable to conventional meat.

Still further, in some embodiments, ACBM may also encompass any product produced by fish cells. Fish meat products include ground fish, structured fish fillets, fish steaks, and other seafood alternatives such as fish nuggets and fish sausages, produced from cultured muscle cells.

It should be appreciated that the cell-based product, as well as the ACBM as used herein may contain in addition to the cells of the present disclosure or any combination of these cells, or any combinations of these cells with any other cells, also any combinations of the cells of the present disclosure with any product produced by the cells.

A further aspect of the present disclosure relates to a nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions. The controlled expression of at least one nucleic acid sequence of interest results in at least one desired phenotype adapted to the suspension condition. It should be understood that the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. More specifically, the transcription factor binding site comprising the nucleic acid motif:

STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

In some embodiments of the disclosed nucleic acid molecule, the at least one desired phenotype comprises at least one of: cell proliferation, cell growth and/or cell expansion in suspension conditions.

In yet some further embodiments, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, comprises at least one transcription factor binding site comprising the nucleic acid motif: GTTTCNNT, as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70; and/or any functional fragments thereof; wherein A is adenine, G is Guanin, C is cytosine, T is thymine and N is any nucleic acid residue.

In some embodiments, the motif of at least one transcription factor binding site of the disclosed nucleic acid molecules, may comprise the nucleic acid sequence of: GTTTCRRT, as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof; wherein Y is a pyrimidine, specifically, C or T, and wherein R is G or A.

In yet some further embodiments, the motif of at least one transcription factor binding site of the disclosed nucleic acid molecule may comprise the nucleic acid sequence of: GTTTCGGT, as denoted by SEQ ID NO: 73, and/or the reverse complement ACCGAAAC, as denoted by SEQ ID NO: 74: and/or any functional fragments thereof.

In yet some alternative or additional embodiments, the motif of at least one transcription factor binding site comprises the nucleic acid sequence of: RSTTTCRNWWY, as denoted by SEQ ID NO: 64, wherein R is A or G, S is G or C, W is A or T, and Y is C or T, specifically, [AG][GC]TTTC[GA]N[TA][TA][TC], and/or the reverse complement RWWNYGAAASY, as denoted by SEQ ID NO: 65, wherein R is A or G, S is G or C, W is A or T, and Y is C or T, specifically, [AG][AT][AT]N[CT]GAAA[CG][TC]. In some embodiments, motif of at least one transcription factor binding site comprises the nucleic acid sequence of: AGTTTCGNTTT, as denoted by SEQ ID NO: 66, and/or the reverse complement AAANCGAAACT, as denoted by SEQ ID NO: 77; and/or any functional fragments thereof.

In yet some further embodiments, the at least one transcription factor binding site is in the length of 8 to 20 nucleotides, specifically, 10 to 17 nucleotides.

In some particular embodiments, the at least one transcription factor binding site of the nucleic acid molecules of the present disclosure may comprise the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86 and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the at least one synthetic conditional nucleic acid sequence comprises 3 to 100 repeats of said transcription factor binding site.

In some embodiments, the at least one synthetic conditional nucleic acid sequence of the nucleic acid molecules of the preset disclosure, comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47 and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the desired phenotype is production and/or secretion of at least one growth factor in suspension conditions, thereby providing cell proliferation, cell growth and/or cell expansion, said at least one nucleic acid sequence of interest encodes, or controls the production and/or activity and/or levels of, at least one growth factor, said at least one growth factor comprises at least one of: transferrin, fibroblast growth factor-2 (FGF-2), insulin and Platelet-derived growth factor BB (PDGF-BB).

In some embodiments, the desired phenotype is cell proliferation in suspension conditions, and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity, and/or levels of at least one of at least one of: transforming growth factor beta induced (TGFBI), A disintegrin and metalloprotease 12 (ADAM12), Plakophilin-3 (PKP3), yes-associated protein 1 (YAP1) or TAF AZZIN (TAZ) genes.

In some embodiments, the desired phenotype is cell proliferation at high cell density and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity, and/or levels of at least one of: gene/s related to metabolic shifts, enhanced glucose uptake, reduced lactate production, enhanced lactate degradation and/or enhanced glutamine production and ammonia degradation.

Still further, the present disclosure provides in another aspect thereof, a nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing adherence conditions. The controlled expression of at least one of the nucleic acid sequences of interest, results in at least one desired phenotype adapted to the adherence condition. More specifically, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. Specifically, the transcription factor binding site comprising the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments of the disclosed nucleic acid sequence, the cellular- and/or environmental- state and/or condition comprises adherence conditions. Accordingly, the at least one desired phenotype comprises at least one of: a non-proliferative state and/or cell differentiation in adherence conditions.

In some embodiments, the desired phenotype is differentiation and/or tissue maturation and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels, of at least one gene related to adipogenesis or myogenesis.

In some embodiments, at least gene related to adipogenesis comprises at least one of ZFP423, API , C/EBPa, P and 5 and PPARy.

In some embodiments, at least gene related to myogenesis comprises at least one of Sixl/4, Pax3, Pax7, Myf5, MyoD and MyoG.

In some embodiments, the desired phenotype is cell immortalization and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity, and/or levels of at least one of: gene/s related to cell immortalization comprise at least one of TERT and CDK4. Accordingly, in some embodiments (I), the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in the adherence conditions, comprises at least one transcription factor binding site, specifically, NFKB binding site, as defined herein before. More specifically, such binding site comprising the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof. Still further, in some alternative or additional embodiments (II), at least one synthetic conditional nucleic acid sequence for adherence conditions, comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

In more specific embodiments, the desired phenotype comprises differentiation of the cell under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell, and wherein the at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

In some embodiments, the disclosed nucleic acid molecules of the present disclosure, and specifically the synthetic conditional nucleic acid sequence (promoters), disclosed herein, may be useful for over-expression of at least one nucleic acid sequence operably linked thereto.

The present disclosure provides nucleic acid sequences and/or molecules. The term “nucleic acid”, “nucleic acid sequence”, or "polynucleotide" and “nucleic acid molecule” refers to polymers of nucleotides, and includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA/RNA hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. Preparation of nucleic acids is well known in the art.

It should be noted that the nucleic acid molecules (or polynucleotides) according to the invention can be produced synthetically, or by recombinant DNA technology. Methods for producing nucleic acid molecules are well known in the art.

The nucleic acid molecule according to the present disclosure may be of a variable nucleotide length. For example, in some embodiments, the nucleic acid molecule according to the invention comprises 1-100 nucleotides, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In other embodiments the nucleic acid molecule according to the invention comprises 100-1,000 nucleotides, e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides. In further embodiments the nucleic acid molecule according to the invention comprises 1,000-10,000 nucleotides, e.g., about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 nucleotides. In yet further embodiments the nucleic acid molecule according to the present disclosure comprises more than 10,000 nucleotides, for example, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000 nucleotides.

A further aspect of the present disclosure relates to a cellular input-output (or sensor-output) unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition. More specifically, the unit comprising: (i), at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and (ii), at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence of (i). More specifically, the at least one synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of the at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions. In some embodiments, the at least one nucleic acid molecule that comprises the at least one synthetic conditional nucleic acid sequence, is any of the nucleic acid molecules of the present disclosure, as defined herein above.

In some embodiments, the disclosed cellular input-output unit/s of the present disclosure autonomously provides to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition. Such desired phenotype may be at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity.

Still further, in some embodiments, the cellular input-output unit of the present disclosure may autonomously provides to a cell any of the desired phenotypes disclosed above, that are adapted to a cellular- and/or environmental- state and/or conditions that may comprise at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D- scaffold), levels of lactate, levels of ammonia, pH conditions, salinity conditions, osmolarity conditions, strength of magnetic field, oxidative stress, humidity, temperature conditions, level of nutrients, levels of available amino acids, levels of ions, level of growth factors, level of signaling molecules, cell motility, shear force, levels of cytokines, levels of toxins, levels of oxidants, presence of pathogens, levels of hormones, metabolite accumulation, accumulation of cell waist, and/or cell metabolic state.

As indicated above, the cellular input-output unit of the present disclosure comprises (i), at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and (ii), at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence of (i). In some embodiments, the at least one nucleic acid sequence of interest may comprise at least one coding and/or non-coding inhibitory and/or modulatory nucleic acid molecule.

In some embodiments, at least one inhibitory and/or modulatory non-coding nucleic acid molecule is a ribonucleic acid (RNA) molecule, said RNA molecule is at least one of a double-stranded RNA (dsRNA), an antisense RNA, a single-stranded RNA (ssRNA), and a Ribozyme.

In some embodiments, at least one inhibitory and/or modulatory non-coding nucleic acid molecule is at least one of a microRNA (miRNA), MicroRNA-like RNAs (milRNA), artificial miRNAs (amiRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).

In some embodiments, the at least one of the nucleic acid sequence of interest of the disclosed cellular input-output unit, encodes, or controls the production and/or activity and/or levels of, at least one product that directly or indirectly leads to, or involved with, the desired phenotype adapted to the cellular- and/or environmental- state and/or condition. In yet some alternative, or additional embodiments, the at least one of the nucleic acid sequence of interest of the disclosed cellular input-output unit, encodes, or controls the production and/or activity and/or levels of at least one product of interest (produced by, or produced from the cells).

As indicated above, in some embodiments, the nucleic acid sequence of interest of the disclosed cellular input-output unit, encodes, or controls the production and/or activity and/or levels of, at least one product that directly or indirectly leads to, or involved with, the desired phenotype adapted to the cellular- and/or environmental- state and/or condition. According to such embodiments, the product may be at least one of: at least one growth factor, at least one survival factor, at least one differentiation factor, at least one cell metabolic factor, at least one adhesion molecule, at least one immortalization factor, and/or at least one cell migration factor.

In more specific embodiments, the desired phenotype may be the production and/or secretion of at least one growth factor in suspension conditions, thereby providing cell proliferation, cell growth and/or cell expansion, in suspension. According to these embodiments, the at least one nucleic acid sequence of interest of the disclosed cellular input-output unit encodes or controls the production and/or activity and/or levels of at least one growth factor, specifically, growth factor comprising at least one of: transferrin, fibroblast growth factor-2 (FGF-2), insulin, Platelet-derived growth factor BB (PDGF-BB).

A further aspect disclosed herein, relates to a cellular input-output unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cell and/or environmental state and/or condition. The unit comprising: (i) at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The at least one synthetic conditional nucleic acid sequence of (i), is configured for controlling the expression of at least one nucleic acid sequence of interest of (ii), upon sensing one of the following conditions. In some embodiments (I), suspension conditions, accordingly, the controlled expression of at least one of the nucleic acid sequence of interest results in at least one desired phenotype adapted to the suspension condition. More specifically, the synthetic conditional nucleic acid sequence of the disclosed unit comprises at least two repeats of a transcription factor binding site, and the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. Alternatively, or additionally, (II), adherence conditions, in such case the controlled expression of at least one of the nucleic acid sequence of interest results in at least one desired phenotype adapted to the adherence condition. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. More specifically, the transcription factor binding site comprising the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83„ or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest (e.g., at least one growth factor), specifically, the synthetic promoter of the disclosed cellular input-output unit, may comprise at least one transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof. It should be noted that A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

In some embodiments, at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest (e.g., at least one growth factor), also referred to herein as synthetic promoter/s, of the disclosed cellular input-output unit, comprises at least one transcription factor binding site comprising the nucleic acid motif: GTTTCNNT, as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70. Specifically, A is adenine, G is Guanin, C is cytosine, T is thymine and N is any nucleic acid residue.

In some embodiments, the motif of at least one transcription factor binding site of the synthetic promoters of the disclosed cellular input-output unit, comprises the nucleic acid sequence of: GTTTCRRT, as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof. More specifically, Y is a pyrimidine, specifically, C or T, and R is G or A. Still further, the motif of at least one transcription factor binding site of the synthetic promoter/s of the disclosed cellular input-output unit, comprises the nucleic acid sequence of: GTTTCGGT, as denoted by SEQ ID NO: 73, and/or the reverse complement ACCGAAAC, as denoted by SEQ ID NO: 74; and/or any functional fragments thereof.

In yet some further embodiments, at least one transcription factor binding site of the synthetic promoter/s of the units of the present disclosure, may be in the length of 8 to 20 nucleotides, specifically, 10 to 17 nucleotides (the negatives are longer 21nt).

In more specific embodiments, the at least one transcription factor binding site of the synthetic promoters of the units of the present disclosure may be any of the promoters disclosed by the present disclosure, for example, any promoter that may comprise the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86, and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the at least one synthetic conditional nucleic acid sequence (specifically, synthetic promoter), may comprise 3 to 100 repeats of the transcription factor binding site.

In some embodiments, the at least one synthetic conditional nucleic acid sequence (specifically, the synthetic promoter of the disclosed units) comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47, and/or the reverse complement thereof, or any combinations thereof.

In more specific embodiment, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions used by the disclosed methods, comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83„ or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, V is G, C, or A and K is G or T.

In some embodiments of the disclosed cellular input-output unit, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in the adherence conditions, may comprise at least one transcription factor binding site, specifically, at least one NFKB binding site. In some embodiments, the disclosed cellular input-output unit may use synthetic conditional nucleic acid sequence for adherence conditions, specifically synthetic promoters comprising the at least one transcription factor binding site having the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof.

In yet some further embodiments, for adherent conditions, the at least one synthetic conditional nucleic acid sequence used in the disclosed methods for adherence conditions, may comprise the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

A further aspect relates to a cellular input-output unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition. The unit comprising: (i) at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The at least one synthetic conditional nucleic acid sequence of (i), is configured for controlling the expression of at least one nucleic acid sequence of interest of (ii), upon sensing suspension conditions. The controlled expression of at least one of the nucleic acid sequences of interest results in at least one desired phenotype adapted to the suspension condition. More specifically, the synthetic conditional nucleic acid sequence of the disclosed unit comprises at least two repeats of a transcription factor binding site, and the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. Still further, the present disclosure provides a cellular input-output unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition. The unit comprising: (i) at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence; and at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The at least one synthetic conditional nucleic acid sequence of (i), is configured for controlling the expression of at least one nucleic acid sequence of interest of (ii), upon sensing adherence conditions. The controlled expression of at least one of the nucleic acid sequence of interest results in at least one desired phenotype adapted to the adherence condition. More specifically, the synthetic conditional nucleic acid sequence of the disclosed unit comprises at least two repeats of a transcription factor binding site, and the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments, the at least one nucleic acid molecule of the disclosed cellular input-output units, is as defined by the present disclosure.

In some embodiments of the disclosed of the disclosed cellular input-output units, at least one desired phenotype comprises at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity.

In yet some further embodiments, the at least one nucleic acid sequence of interest controlled by the conditional nucleic acid sequence of the of the disclosed cellular input-output units, comprises at least one coding and/or non-coding inhibitory and/or modulatory nucleic acid molecule.

In some embodiments of the disclosed cellular input-output units, the nucleic acid sequence of interest encodes, or controls at least one of:

(a) the production and/or activity and/or levels of, at least one product that directly or indirectly leads to, or involved with, said phenotype adapted to the suspension condition/s; and (b) the production and/or activity, and/or levels of at least one product of interest.

In some embodiments of the disclosed cellular input-output units, the product is at least one of: at least one growth factor, at least one survival factor, at least one differentiation factor, at least one cell metabolic factor, at least one adhesion molecule, at least one immortalization factor, and/or at least one cell migration factor.

In yet some further embodiments of the disclosed cellular input-output units, the desired phenotype is production and/or secretion of at least one growth factor in suspension conditions, thereby providing cell proliferation, cell growth and/or cell expansion. Accordingly, the at least one nucleic acid sequence of interest encodes, or controls the production and/or activity and/or levels of at least one growth factor, the growth factor comprising at least one of: transferrin, fibroblast growth factor-2 (FGF-2), insulin, Platelet-derived growth factor BB (PDGF-BB), transforming growth factor p.

In yet some further embodiments of the cellular input-output unit according to the present disclosure, at least one of the nucleic acid sequence of interest encodes, or controls at least one of: (a), the production and/or activity and/or levels of, at least one product that directly or indirectly leads to, or involved with, the phenotype adapted to the adherent condition/s; and (b), the production and/or activity, and/or levels of at least one product of interest. In some embodiments, the desired phenotype comprises differentiation of the cell under adherence conditions into at least one of: a fat cell, a muscle cell, fibroblasts (producing ECM components) and a blood vessel cell. The at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

Still further, the cellular input-output unit according to the present disclosure, wherein the desired phenotype comprises differentiation of the cell under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell, and wherein the at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

A further aspect of the present disclosure relates to a nucleic acid cassette or any vector thereof, comprising at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence (specifically, a synthetic promoter), or at least one cellular input-output unit comprising the at least one nucleic acid molecule. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or conditions.

In some embodiments, the nucleic acid cassette or any vector or vehicle thereof in accordance with the present disclosure may comprise any of the nucleic acid molecules as disclosed by the present disclosure, and/or any of the cellular input-output unit disclosed by the present disclosure.

In yet some further embodiments, the nucleic acid cassette of the present disclosure may further comprise at least one genetic element. In some embodiments, the genetic element is at least one of: IRES, a 2A peptide coding sequence, a promoter or any functional fragments thereof (a minimal promoter as used in the results), a polyadenylation site, a signal peptide a stop codon, and a transcription enhancer.

A further aspect relates to a nucleic acid cassette or any vector or vehicle thereof, comprising at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or at least one cellular input-output unit comprising the at least one nucleic acid molecule. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site.

In some embodiments (I), the synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions. Still further, the controlled expression of at least one of the nucleic acid sequence of interest results in at least one desired phenotype adapted to said suspension condition. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In more specific embodiments, the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments, the nucleic acid molecule of the disclosed cassette is as defined by the present disclosure.

In yet some further embodiments, the disclosed nucleic acid cassette, further comprising at least one genetic element. The present disclosure further provides a nucleic acid cassette or construct, that comprise the nucleic acid molecule disclosed herein and specifically, the at least one synthetic conditional nucleic acid sequence (also referred to herein as a synthetic promoter). The term "nucleic acid cassette" refers to a polynucleotide sequence comprising at least one regulatory sequence, that is the synthetic conditional nucleic acid sequence of the present disclosure, that may be optionally, operably linked to a sequence encoding a nucleic acid sequence encoding or forming the nucleic acid sequence of interest disclosed herein. All elements comprised within the cassette of the invention are operably linked together. The term "operably linked", as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the nucleic acid sequences are linked in a manner that enables regulated expression of the linked structural nucleotide sequence. Specifically, that the at least two sequences are at a correct functional location and orientation in relation to one another. It should be understood that in some embodiments, in addition to the synthetic conditional nucleic acid sequence (synthetic prompter), the cassette of the present disclosure may comprise one or more additional genetic elements. In some embodiments, the cassette may further comprise in addition to the synthetic conditional nucleic acid sequence, also a minimal promoter.

As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.

In some embodiments, promoters applicable in the present disclosure may be either inducible or constitutive. In yet some further embodiments, a functional fragment of a promoter applicable in the nucleic acid molecules of the present disclosure, or any cassettes of the invention may be a minimal promoter. The term "minimal promoter" includes partial promoter sequences that define the start site of transcription for the linked sequence to be transcribed which by itself is not capable of initiating transcription. Thus, the activity of such a minimal promoter is dependent upon the binding of a transcriptional activator to an operably linked regulatory sequence, e.g., enhancer. In certain embodiments a minimal promoter may be included in the nucleic acid molecules or cassettes of the invention. A "constitutive promoter" refers to a promoter that allows for continual transcription of the coding sequence or gene under its control. In yet some further embodiments, a promoter suitable in the nucleic acid molecules, vectors and/or cassette of the invention may be an inducible promoter. An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of the regulatory region. An "inducible promoter" refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. Still further, in some embodiments, the nucleic acid cassette of the present disclosure may further comprise a 2A sequence. By an "2A peptide sequence", it is meant a nucleotide sequence that allows for the initiation of protein translation in the middle of a messenger RNA (mRNA) sequence. More specifically, a 2A peptide sequence or a CHYSEL site causes a eukaryotic ribosome to release the growing polypeptide chain, but continue translating, thereby giving rise to two separate polypeptides from a single translating ribosome.

In yet some further embodiments, the nucleic acid cassette provided by the disclosure may comprise at least one signal peptide leader. "Signal peptide leader", as used herein, shall mean a peptide chain (of about 3-60 amino acids long) that directs the post-translational transport of a protein to the endoplasmic reticulum and may be cleaved off.

In some embodiments, the nucleic acid molecule of the present disclosure may comprise a promoter. In some embodiments, the promoter is a minimal promoter. In some embodiments, the promoter is a constitutively active promoter. In yet some further embodiments, the operation of the promoter necessities binding of at least one transcription factor to at least one of the TF binding site. In some embodiments, binding of a TF to the TF binding site or TF binding region activates the promoter. In some embodiments, the TF binding site repeats are operably linked to the minimal promoter, thereby forming a synthetic promoter, also referred to herein as the synthetic conditional nucleic acid sequence. In some embodiments, the TF binding region is operably linked to the promoter. In some embodiments, the TF binding site repeats are placed 5’ to the promoter. In some embodiments, the TF binding region is placed 5’ to the promoter. In some embodiments, the TF binding site repeats are placed 3’ to the promoter. In some embodiments, the TF binding region is placed 3’ to the promoter. In some embodiments, the TF binding site repeats are placed within the promoter. In some embodiments, the TF binding region is placed within the promoter. In some embodiments, the TF binding region or the at least two TF binding sites act as a regulatory element controlling the promoter. In some embodiments, the TF binding sites activate the minimal promoter, thereby controlling the expression of the nucleic acid sequence of interest. Still further, the nucleic acid molecules of the present disclosure or any cassettes thereof, or any of the disclosed units or platforms, may be comprised within vector/s. Vector/s, as used herein, are nucleic acid molecules of particular sequence that can be introduced into a host cell, thereby producing a transformed host cell or be transiently expressed in the cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression. Many vectors, e.g. plasmids, cosmids, minicircles, phage, viruses, (as detailed below) useful for transferring nucleic acids into target cells may be applicable in the present invention. The vectors comprising the nucleic acid(s) may be maintained episomally, e.g. as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus-derived vectors such as AAV, MMLV, HIV-1, ALV, etc.

As indicated above, in some embodiments, viral vectors may be applicable in the present disclosure. The term "viral vector" refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host.

In some embodiments such viral vectors may be used for transient or stable expression of the nucleic acid molecule, cassette, unit or platform of the present disclosure in the cell.

The term "virus" refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane. Examples of viruses useful in the practice of the present invention include baculoviridiae, parvoviridiae, picornoviridiae, herepesviridiae, poxviridiae, adenoviridiae, picotmaviridiae. The term recombinant virus includes chimeric (or even multimeric) viruses, i.e., vectors constructed using complementary coding sequences from more than one viral subtype. In yet some particular embodiments, such viral vector may be any one of retroviral vector and lentiviral vectors, recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self-complementary rAAV (scAAV), Simian vacuolating virus 40 (SV40) vector, Adenovirus vector and/or helper-dependent Adenoviral vector.

More specifically, in some embodiments, the nucleic acid molecules of the present disclosure may be comprised within retroviral vector/s. A retroviral vector consists of pro viral sequences that can accommodate the gene of interest, to allow incorporation of both into the target cells. The vector may also contain viral and cellular gene promoters, to enhance expression of the gene of interest in the target cells. Retroviral vectors stably integrate into the dividing target cell genome so that the introduced gene is passed on and expressed in all daughter cells. They contain a reverse transcriptase that allows integration into the host genome.

In some specific embodiments, lentiviral vectors may be used in the present invention. Lentiviral vectors are derived from lentiviruses which are a subclass of Retroviruses. Commonly used retroviral vectors are "defective", i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising the nucleic acids sequence of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing the retroviral vectors comprising the nucleic acid molecules of the invention that contains the nucleic acids sequence of interest into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.

In yet some further specific embodiments, an appropriate vector that may be used by the invention may be an Adeno-associated virus (AAV). The term "adenovirus" is synonymous with the term "adenoviral vector". AAV is a single-stranded DNA virus with a small (~20nm) protein capsule that belongs to the family of parvoviridae and specifically refers to viruses of the genus adenoviridiae. The term adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (AdllA and Ad IIP), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.

Due to its inability to replicate in the absence of helpervirus coinfections (typically Adenovirus or Herpesvirus infections) AAV is often referred to as dependovirus. AAV infections produce only mild immune responses and are considered to be nonpathogenic, a fact that is also reflected by lowered biosafety level requirements for the work with recombinant AAVs (rAAV) compared to other popular viral vector systems. Due to its low immunogenicity and the absence of cytotoxic responses AAV-based expression systems offer the possibility to express genes of interest for months in quiescent cells.

Production systems for rAAV vectors typically consist of a DNA-based vector containing a transgene expression cassette, which is flanked by inverted terminal repeats (payload). Construct sizes are limited to approximately 4.7-5.0 kb, which corresponds to the length of the wild-type AAV genome.

It should be appreciated that the usage of rAAV constructs with a self-complementing structure (sc AAV) in which the two halves of the single-stranded AAV genome can form an intra-molecular double-strand, may be also applicable in the present disclosure. This approach reduces the effective genome size usable for gene delivery to about 2.3kB but leads to significantly shortened onsets of expression in comparison with conventional single-stranded AAV expression constructs (ssAAV). Thus, in some embodiments, ssAAV may be applicable as a viral vector by the invention.

In yet some further embodiments, HDAd vectors may be suitable for the present disclosure. The Helper-Dependent Adenoviral (HDAd) vectors HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity.

Still further, in some embodiments, SV40 may be used as a suitable vector. SV40 vectors (SV40) are vectors originating from modifications brought to Simian virus-40 an icosahedral papovavirus. Recombinant SV40 vectors are good candidates for gene transfer, as they are well-known viruses, non-replicative vectors that are easy-to-make, and also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types.

In some alternative embodiments, the vector may be a non-viral vector. More specifically, such vector may be in some embodiments any one of plasmid, minicircle and linear DNA, ssDNA or RNA (useful to avoid long term expression and or integration) or a modified polynucleotide (mainly chemically protective modifications to protect RNA or DNA-RNA chimeras to enhance specificity and or stability).

Nonviral vectors, in accordance with the invention, refer to all the physical and chemical systems except viral systems and generally include either chemical methods, such as cationic liposomes and polymers, or physical methods, such as gene gun, electroporation, particle bombardment, ultrasound utilization, and magnetofection. Efficiency of this system is sometimes less than viral systems in gene transduction, but their cost-effectiveness, availability, and more importantly reduced induction of immune system and no limitation in size of transgenic DNA compared with viral system have made them attractive also for gene delivery. For example, physical methods applied for in vitro and in vivo gene delivery are based on making transient penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA, RNA or RNP entrance into the targeted cells is facilitated.

In more specific embodiments, the vector may be a naked DNA vector. More specifically, such vector may be for example, a plasmid, minicircle or linear DNA.

Naked DNA alone may facilitate transfer of a nucleic acid sequence (2-200Kb or more) into skin, thymus, cardiac muscle, and especially skeletal muscle and liver cells when directly injected. It enables also long-term expression. Although naked DNA injection is a safe and simple method, its efficiency for gene delivery is quite low.

Minicircles are modified plasmid in which a bacterial origin of replication (ori) was removed, and therefore they cannot replicate in bacteria.

Linear DNA or Doggybone™ are double-stranded, linear DNA construct that solely encodes a payload expression cassette, comprising antigen, promoter, polyA tail and telomeric ends.

A further aspect of the present disclosure relates to a cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. More specifically, the disclosed platform comprising at least one synthetic conditional nucleic acid sequence (e.g. synthetic promoter/s), or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cellular- and/or environmental- state and/or conditions.

In some embodiments, the cellular platform of the present disclosure may comprise at least one cellular input-output unit or any nucleic acid cassette or vector comprising the same. Such unit comprising: (i), at least one nucleic acid molecule comprising the at least one synthetic conditional nucleic acid sequence (e.g., synthetic promoter); and (ii), at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of the at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions.

In some embodiments, the disclosed cellular platform may comprise one or more synthetic promoter/s, specifically, any of the promoters disclosed by the present disclosure. In yet some further embodiments, the platform of the present disclosure may comprise at least one input-output (or sensor-output) unit, as define by the present disclosure. In yet some further embodiments, the platform of the present disclosure may comprise any of the nucleic acid cassette/s as disclosed by the present disclosure.

In some embodiments, the at least one desired phenotype autonomously provided by the cellular platform of the present disclosure, upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s, comprises at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity under said dynamic conditions.

In yet some further embodiments, the cellular platform of the present disclosure autonomously provides the desired phenotype as discussed above, following cellular- and/or environmental- state and/or conditions comprise at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D- scaffold), levels of lactate, levels of ammonia, pH conditions, salinity conditions, osmolarity conditions, strength of magnetic field, oxidative stress, humidity, temperature conditions, level of nutrients, levels of available amino acids, levels of ions, level of growth factors, level of signaling molecules, cell motility, shear force, levels of cytokines, levels of toxins, levels of oxidants, presence of pathogens, levels of hormones, metabolite accumulation, accumulation of cell waist, and/or cell metabolic state.

In some embodiments, dynamic cellular- and/or environmental- state/s and/or condition/s comprise at least one change in said cellular- and/or environmental- state/s and/or condition/s over time.

Thus, according to this aspect of the present disclosure, a cellular platform is provided, the platform is configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. The platform comprising at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), the at least one synthetic conditional nucleic acid sequence of the disclosed platform is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell suspension conditions. In some embodiments, the transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments, the cellular platform of the present disclosure comprises at least two of the following components:

(d), at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in three-dimensional scaffold (3D-scaffold) (or aggregate) conditions.

In yet some further embodiments of the disclosed cellular platform, the at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in cell suspension conditions comprises at least one synthetic conditional nucleic acid sequence (also referred to herein as the synthetic promoter) comprising at least one transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof. It should be noted that A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

In some embodiments, at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest (e.g., at least one growth factor) of the disclosed cellular platform, comprises at least one transcription factor binding site comprising the nucleic acid motif: GTTTCNNT, as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70; and/or any functional fragments thereof. Specifically, A is adenine, G is Guanin, C is cytosine, T is thymine, and N is any nucleic acid residue.

In some embodiments, the motif of at least one transcription factor binding site of the synthetic promoters of the disclosed cellular input-output unit of the disclosed cellular platform, comprises the nucleic acid sequence of: GTTTCRRT, as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof. More specifically, Y is a pyrimidine, specifically, C or T, and R is G or A.

Still further, the motif of at least one transcription factor binding site of the synthetic promoter/s of the disclosed cellular input-output unit, comprises the nucleic acid sequence of: GTTTCGGT, as denoted by SEQ ID NO: 73, and/or the reverse complement ACCGAAAC, as denoted by SEQ ID NO: 74; and/or any functional fragments thereof.

In more specific embodiments, the at least one transcription factor binding site of the synthetic promoters of the platforms of the present disclosure may be any of the promoters disclosed by the present disclosure, for example, any promoter that may comprise the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86, and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the at least one synthetic conditional nucleic acid sequence (specifically, synthetic promoter), may comprise 3 to 100 repeats of said transcription factor binding site.

In some embodiments, the at least one synthetic conditional nucleic acid sequence (specifically, the synthetic promoter of the disclosed units) for suspension conditions comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47, and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the synthetic conditional nucleic acid sequence of the disclosed cellular platform, is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest. Such at least one nucleic acid sequence of interest, encodes or controls the production and/or activity and/or levels, of at least one growth factor. In some embodiments, the growth factor comprising at least one of: transferrin, fibroblast growth factor-2 (FGF-2), insulin, Platelet-derived growth factor BB (PDGF-BB).

In more specific embodiment, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions used by the disclosed cellular platforms, comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

In some embodiments of the disclosed platform, the at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in the adherence conditions, may comprise at least one transcription factor binding site, specifically, at least one NFKB binding site. In some embodiments, the disclosed platform may use synthetic conditional nucleic acid sequence, specifically synthetic promoters for adherence conditions, comprising the at least one transcription factor binding site having the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof.

In yet some further embodiments, for adherent conditions, the at least one synthetic conditional nucleic acid sequence used in the disclosed platforms may comprise the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

In some embodiments, the use of these adherent specific promoters by the disclosed platforms, allows achieving the desired phenotype in adherence conditions. In some embodiments, the desired phenotype comprises differentiation of the cell/s under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell. According to such embodiments, the at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor. A further aspect of the present disclosure relates to a cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. The platform comprising at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), The at least one synthetic conditional nucleic acid sequence of the disclosed platform is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell suspension conditions. In some embodiments, the transcription factor binding site comprising the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, V is G, C, or A and K is G or T.

In some embodiments, the cellular platform of the present disclosure comprises at least one cellular input-output unit or any nucleic acid cassette or vector comprising the same. Specifically, the platform of the present disclosure may comprise at least one, at least two, at least three, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more, for example, at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least one hundred or more different units. More specifically, such unit may comprise: (i) at least one nucleic acid molecule comprising said at least one synthetic conditional nucleic acid sequence; and (ii) at least one nucleic acid sequence of interest, operably linked to the synthetic conditional nucleic acid sequence. The at least one synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of the at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions.

In some embodiments, the nucleic acid molecule/s, the input-output unit/s and/or the cassette/s of the of the disclosed cellular platform are as defined by the present disclosure.

In some embodiments, the cellular platform of the present disclosure may comprise at least two or more, specifically, at least three, at least four of: a. at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in cell suspension conditions; b. at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in adherence conditions; c. at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in high cell density conditions; and d. at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in three-dimensional scaffold (3D-scaffold) (or aggregate) conditions.

A further aspect of the present disclosure relates to a cell or a population of the cells comprising and/or genetically engineered by, at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or at least one cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s. The synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site. In some embodiments (I), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cell suspension conditions. In some embodiments, the transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue. In yet some additional or alternative embodiments (II), the at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions. More specifically, the transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, or the motif DDKGRAHDYHMY, as denoted by SEQ ID NO: 68, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T. A further aspect of the present disclosure relates to a cell or a population of these cells comprising and/or genetically engineered by, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising the at least one nucleic acid molecule. More specifically, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, thereby the cell autonomously exhibits at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

The present disclosure provides cells that comprise the nucleic acid molecule of the present disclosure and/or are genetically engineered and/or genetically modified by these nucleic acid molecules. A "genetically engineered" generally refers to a cell that comprise and encodes a heterologous nucleic acid sequence, and/or exogenously added nucleic acid sequence, and/or non- naturally occurring nucleic acid sequence, or one or more additional nucleic acid sequences that are not normally endogenous to the cell (collectively referred to herein as "transgenes"). These exogeneous elements and sequences may be in some embodiments chromosomally integrated into the cells, or expressed as non-integrated nucleic acid sequences. As a result of such transfer and integration, the transferred sequence may be transmitted through any cell progeny. It is understood that such terms refer not only to the particular subject cells but to the progeny or potential progeny of such a cell. Because certain modification may occur in succeeding generation due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

In some embodiments, nucleic acid molecule of the cell of the present disclosure is any of the nucleic acid molecules of the present disclosure, specifically, any of those defined herein above. Still further, the input-output (or sensor-output) unit of the disclosed cell, is as defined by the present disclosure, the cassette of the cell is any of the cassettes disclosed herein, and/or the cellular platform used for the cells of the present disclosure is any of the platforms disclosed by the present disclosure.

In some embodiments, the cells of the present disclosure are primary cells. In some embodiments, there are several types of cells, specifically, eukaryotic cells, that may be used at the cells of the present disclosure. As indicated above, these cells may be derived from any unicellular or multicellular prokaryotic or eukaryotic organisms. In yet some further embodiments, in case the cells are of eukaryotic organisms, specifically, any of the multicellular organisms disclosed above, they may be derived from any tissue or organ of any differentiation stage of the organism and of any embryonic germ layer (e.g., ectoderm, mesoderm, and endoderm). By way of example, eukaryotic cells may be, but are not limited to, stem cells, embryonic stem cells, totipotent stem cells, pluripotent stem cells or induced pluripotent stem cells and multipotent progenitor cells.

The term "stem cells," as used herein, refers to undifferentiated cells possessing the unique capacity for self-renewal and the potential to differentiate into various specialized cell types. These cells are characterized by their ability to undergo asymmetric cell division, generating one daughter cell that retains stem cell properties and another committed to differentiation into specific lineages. Stem cells encompass pluripotent, multipotent, and unipotent cell populations, each exhibiting distinct differentiation potentials and lineage commitments.

Pluripotent stem cells have the capability to differentiate into cell types representing all three embryonic germ layers, including ectoderm, mesoderm, and endoderm. Multipotent stem cells possess a more limited differentiation potential, typically differentiating into cell types within a specific germ layer. Unipotent stem cells, on the other hand, are committed to differentiating into a single specialized cell type.

Stem cells may be isolated from various sources, such as embryonic tissues, fetal tissues, adult tissues, or induced pluripotent stem cells (iPSCs) derived from reprogrammed somatic cells. Additionally, the term "stem cells" as used herein, encompasses both naturally occurring and genetically modified or engineered stem cells, wherein the latter may be altered to exhibit enhanced or targeted differentiation characteristics. Still further, stem cells are generally known for their unique characteristics, specifically, the unique ability to renew themselves continuously; the ability to differentiate into somatic cell types; and the ability to limit their own population into a small number. In mammals, there are two broad types of stem cells, namely embryonic stem cells (ESCs), and adult stem cells. In some embodiments, the cells according to the present disclosure may be embryonic stem cells, or human embryonic stem cells (hESCs), that were obtained from self-umbilical cord blood just after birth, of any of the eukaryotic organisms disclosed herein, specifically of any of the non-human mammals disclosed by the present disclosure. Embryonic stem cells are pluripotent stem cells derived from the early embryo that are characterized by the ability to proliferate over prolonged periods of culture while remaining undifferentiated and maintaining a stable karyotype, with the potential to differentiate into derivatives of all three germ layers. hESCs may be also derived from the inner cell mass (ICM) of the blastocyst stage (100- 200 cells) of embryos generated by in vitro fertilization. However, methods have been developed to derive hESCs from the late morula stage (30^-0 cells) and, recently, from arrested embryos (16-24 cells incapable of further development) and single blastomeres isolated from 8 -cell embryos. It should be understood that the cells of any of the embryonic stages discussed herein, are encompassed by the present disclosure.

In further embodiments, the cells according to the disclosure are totipotent stem cells. Totipotent stem cells are versatile stem cells, and have the potential to give rise to any and all mammalian cells, such as brain, liver, blood or heart cells or to an entire functional organism (e.g. the cell resulting from a fertilized egg). The first few cell divisions in embryonic development produce more totipotent cells. After four days of embryonic cell division, the cells begin to specialize into pluripotent stem cells. Embryonic stem cells may also be referred to as totipotent stem cells.

In further embodiments, the cells according to the disclosure are pluripotent stem cells. Similar to totipotent stem cells, a pluripotent stem cell refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type. However, unlike totipotent stem cells, they cannot give rise to an entire organism. On the fourth day of development, the embryo forms into two layers, an outer layer which will become the placenta, and an inner mass which will form the tissues of the developing human body. These inner cells are referred to as pluripotent cells. In still further embodiments, the cells that may be applicable for present disclosure, are multipotent progenitor cells. Multipotent progenitor cells have the potential to give rise to a limited number of lineages. As a non-limiting example, but of a particular interest in the present disclosure, a multipotent progenitor stem cell may be a mesenchymal stem cell, which can differentiate into osteoblasts, chondrocytes, and adipocytes. Another example hematopoietic cell, which is a blood stem cell that can develop into several types of blood cells but cannot into other types of cells. Multipotent progenitor cells may be obtained by any method known to a person skilled in the art.

In yet further embodiments, the cells according to the disclosure are induced pluripotent stem cells. Induced pluripotent stem cells, commonly abbreviated as iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, even a patient’s own. Such cells can be induced to become pluripotent stem cells with apparently all the properties of hESCs. Induction requires only the delivery of transcription factors found in embryos to reverse years of life as an adult cell back to an embryo-like cell.

As indicated above, in some embodiments, the cells of the present disclosure may be Mesenchymal Stem Cells (MSCs), that are distinct population of multipotent, self-renewing cells characterized by their origin in the mesodermal germ layer and their capacity to differentiate into various mesenchymal cell lineages. MSCs are commonly isolated from various tissues, including but not limited to bone marrow, adipose tissue, umbilical cord tissue, and dental pulp. These cells demonstrate multilineage differentiation potential, giving rise to cells of mesodermal lineages such as osteoblasts, adipocytes, and chondrocytes.

In some embodiments, the cells of the present disclosure may be Adipose-Derived Stem Cells (ADSCs). These cells form a specific subpopulation of mesenchymal stem cells (MSCs) that are isolated from adipose tissue.

Still further, in some embodiments, the cells of the present disclosure may be myocytes. More specifically, "Myocytes," as employed herein, denote specialized muscle cells responsible for contractile function within skeletal, cardiac, or smooth muscle tissues. These cells exhibit distinctive morphological features and are characterized by the presence of contractile proteins, including actin and myosin, organized into sarcomeres. Still further, "Satellite cells," as referenced in present disclosure, refer to a population of quiescent, mononucleated cells situated in close proximity to myocytes, predominantly within skeletal muscle tissue. Satellite cells possess the unique ability to serve as a reservoir for myogenic regeneration. Upon activation, satellite cells undergo proliferation and subsequently differentiate into myocytes.

In yet some further embodiments, the cells of the present disclosure may be cardiomyocytes. Cardiomyocytes are specialized muscle cells that constitute the myocardium, the muscular tissue of the heart. These cells are responsible for the contraction and pumping action of the heart, facilitating the circulation of blood throughout the body. Cardiomyocytes exhibit unique structural and functional characteristics that are tailored to their role in cardiac function.

In yet some further embodiments, the cells of the present disclosure may be fibroblasts. Fibroblasts are a type of connective tissue cell, that can be derived from mesenchymal cells, and plays a crucial role in the synthesis of extracellular matrix components, such as collagen, elastin, and various glycoproteins. These cells are essential for providing structural support to tissues and organs and are involved in wound healing, tissue repair, and maintaining the integrity of the extracellular matrix. Fibroblasts are found in various tissues throughout the body and contribute to the production and maintenance of the connective tissue framework.

Still further, in some embodiments, the cells of the present disclosure may be endothelial cells. Endothelial Cells, as utilized in the present disclosure, pertain to a specialized cell type forming the innermost layer of blood vessels, lymphatic vessels, and the heart.

In yet some further embodiments, the cells of the present disclosure may be keratinocytes. Keratinocytes are the primary cell type found in the epidermis. These cells constitute the majority of the epidermal cells, produce keratin and play a crucial role in maintaining the integrity and protective functions of the skin.

In some embodiments, the cell of the present disclosure may be engineered by the nucleic acid molecule of the present disclosure that comprise the synthetic promoters disclosed herein, or any cellular input-output unit, nucleic acid cassette or platform comprising the synthetic promoters. It should be understood that the cells of the present disclosure may be engineered by any means known in the art. The synthetic nucleic acid sequence of the present disclosure may be inserted or introduced to the cells at any appropriate site, either randomly, or in a specific target site. In some embodiments, the cell may comprise only the synthetic promoters of the present disclosure (also referred to herein as the synthetic conditional nucleic acid sequence of the nucleic acid molecule of the present disclosure). According to such embodiments, the synthetic promoter/s of the present disclosure may be inserted in a specific target site, such that it is placed in an operable linkage to a target nucleic acid of interest. Non-limiting examples for appropriate target nucleic sequence of interest may include endogenous sequences controlling and/or encoding growth factors. In some embodiments, when specific targeting of the nucleic acid molecule of the present disclosure is required, any appropriate gene editing system may be used. More specifically, in some embodiments, the CRISPR-Cas system may be used. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a bacterial immune system that has been modified for genome engineering. CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. More specifically, Class 1 may be divided into types I, III, and IV and class 2 may be divided into types II, V, and VI. It should be understood that the present disclosure contemplates the use of any of the known CRISPR systems. In some embodiments, a CRISPR type II system may be used for engineering the cells of the present disclosure.

The type II CRISPR-Cas systems include the 'HNH'-type system (Streptococcus-like; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9, a single, very large protein, seems to be sufficient for generating crRNA and cleaving the target DNA, in addition to the ubiquitous Casl and Cas2. Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein. It should be appreciated that any type II CRISPR- Cas systems may be applicable in the present invention, specifically, any one of type II- A or B, that comprise the following nucleases. More specifically, the endonuclease may be a Cas9, CasX, Casl2, Casl3, Casl4, Cas6, Cpfl, CMS1 protein, or any variant thereof that is derived or expressed from Methanococcus maripaludis C7, Corynebacterium diphtheria, Corynebacterium efficiens YS-314, Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum R, Corynebacterium kroppenstedtii (DSM 44385), Mycobacterium abscessus (ATCC 19977), Nocardia farcinica IFM10152, Rhodococcus erythropolis PR4, Rhodococcus jostii RFIA1 , Rhodococcus opacus B4 (uid36573), Acidothermus cellulolyticus 11 B, Arthrobacter chlorophenolicus A6, Kribbella flavida (DSM 17836), Thermomonospora curvata (DSM43183), Bifidobacterium dentium Bdl, Bifidobacterium longum DJO10A, Slackia heliotrinireducens (DSM 20476), Persephonella marina EX H 1, Bacteroides fragilis NCTC 9434, Capnocytophaga ochracea (DSM 7271), Flavobacterium psychrophilum JIP02 86, Akkermansia muciniphila (ATCC BAA 835), Roseiflexus castenholzii (DSM 13941), Roseiflexus RSI, Synechocystis PCC6803, Elusimicrobium minutum Peil91, uncultured Termite group 1 bacterium phylotype Rs D17, Fibrobacter succinogenes S85, Bacillus cereus (ATCC 10987), Listeria innocua, Lactobacillus casei, Lactobacillus rhamnosus GG, Lactobacillus salivarius UCC118, Streptococcus agalactiae-5-A909, Streptococcus agalactiae NEM316, Streptococcus agalactiae 2603, Streptococcus dysgalactiae equisimilis GGS 124, Streptococcus equi zooepidemicus MGCS10565, Streptococcus gallolyticus UCN34 (uid46061), Streptococcus gordonii Challis subst CHI, Streptococcus mutans NN2025 (uid46353), Streptococcus mutans, Streptococcus pyogenes Ml GAS, Streptococcus pyogenes MGAS5005, Streptococcus pyogenes MGAS2096, Streptococcus pyogenes MGAS9429, Streptococcus pyogenes MGAS 10270, Streptococcus pyogenes MGAS6180, Streptococcus pyogenes MGAS315, Streptococcus pyogenes SSI-1, Streptococcus pyogenes MGAS10750, Streptococcus pyogenes NZ131, Streptococcus thermophiles CNRZ1066, Streptococcus thermophiles LMD-9, Streptococcus thermophiles LMG 18311, Clostridium botulinum A3 Loch Maree, Clostridium botulinum B Eklund 17B, Clostridium botulinum Ba4 657, Clostridium botulinum F Langeland, Clostridium cellulolyticum H10, Finegoldia magna (ATCC 29328), Eubacterium rectale (ATCC 33656), Mycoplasma gallisepticum, Mycoplasma mobile 163K, Mycoplasma penetrans, Mycoplasma synoviae 53, Streptobacillus, moniliformis (DSM 12112), Bradyrhizobium BTAil, Nitrobacter hamburgensis X14, Rhodopseudomonas palustris BisB18, Rhodopseudomonas palustris BisB5, Parvibaculum lavamentivorans DS-1, Dinoroseobacter shibae. DFL 12, Gluconacetobacter diazotrophicus Pal 5 FAPERJ, Gluconacetobacter diazotrophicus Pal 5 JGI, Azospirillum B510 (uid46085), Rhodospirillum rubrum (ATCC 11170), Diaphorobacter TPSY (uid29975), Verminephrobacter eiseniae EF01 -2, Neisseria meningitides 053442, Neisseria meningitides alphal4, Neisseria meningitides Z2491, Desulfovibrio salexigens DSM 2638, Campylobacter jejuni doylei 269 97, Campylobacter jejuni 81116, Campylobacter jejuni, Campylobacter lari RM2100, Helicobacter hepaticus, Wolinella succinogenes, Tolumonas auensis DSM 9187, Pseudoalteromonas atlantica T6c, Shewanella pealeana (ATCC 700345), Legionella pneumophila Paris, Actinobacillus succinogenes 130Z, Pasteurella multocida, Francisella tularensis novicida U 112, Francisella tularensis holarctica, Francisella tularensis FSC 198, Francisella tularensis, Francisella tularensis WY96- 3418, or Treponema denticola (ATCC 35405).

The present disclosure provides at least one cell or any populations comprising the cell, that comprises or is modified by the nucleic acid molecules disclosed herein. Such cell may be also referred to herein as a host cell. The term "host cell" includes a cell into which a heterologous (e.g., exogenous) nucleic acid and/or protein (e.g., nucleic acid molecule or any unit or platform thereof,), has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, developmental maturation, or due to the intended action of the present disclosure, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell". In some embodiments, the host cells provided by the invention are transduced or transfected by the nucleic acid sequences provided by the invention This may refer in some embodiments, to cells that underwent a transfection procedure, meaning the introduction of a nucleic acid, e.g., an expression vector, or a replicating vector, into recipient cells by nucleic acid-mediated gene transfer.

For example, transfection of eukaryotic cells may be chemical, e.g., via a cationic polymer (such as DEAE-dextran, polyethyleneimine, dendrimer, polybrene, calcium), calcium phosphate (e.g., phosphate, lipofectin, DOTAP, lipofectamine, CTAB/DOPE, DOTMA) or via a cationic lipid. Transfection of eukaryotic cells may also be physical, e.g. via a direct injection (for example, by Micro-needle, AFM tip, Gene Gun,), via biolistic particle delivery (for example, phototransfection, Magnetofection), or via electroporation (i.e., Lonza Nucleofector), laser-irradiation, sonoporation or a magnetic nanoparticle. Transfection of eukaryotic cells may also be biological (i.e., use of Agrobacterium in plants).

A further aspect of the present disclosure relates to a method for programing a cell or a population of cells for at least one of: autonomous growth, proliferation, expansion, differentiation, immortalization, maturation, production of at least one product of interest and/or modulated cell activity, in dynamic cellular- and/or environmental- state and/or conditions. More specifically, the method comprising introducing into the cell, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or an input-output (or sensor-output) unit, a nucleic acid cassette and/or a platform comprising the at least one nucleic acid molecule. In some embodiments, the synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition. The controlled expression of at least one of the nucleic acid sequence of interest, results in at least one desired phenotype adapted to the cellular- and/or environmental- state and/or condition.

It should be understood that all definitions disclosed herein for each term appearing in the claims are applicable to every aspect of the present disclosure. Specifically, unless otherwise indicated, the definition of a term in connection with one particular aspect applies equally to all other aspects of the present disclosure.

Furthermore, as used herein in connection with all embodiments and aspects of the present disclosure, the phrase "at least one" refers to any number that is 1 or greater. More specifically, this includes, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more, such as several hundred, several thousand, or even higher numbers.

Still further, when referring to the terms "increase" or alternatively, "decrease", for example, in connection with specific conditions of the specific cell culture and other cellular and/or environmental conditions as specified herein, relates to "inhibition", "moderation", “reduction” or "attenuation" of the specified condition or expression of a nucleic aid sequence of interest, that results in a desired phenotype, as referred to herein, relate to the retardation, restraining or reduction of the specific parameter in the specified range. Alternatively, by any one of "increase," "elevation ", "rise" , "elevation" "growth," "boost”, "expansion”, "escalation” , of the specified condition or expression of a nucleic acid sequence of interest, that results in a desired phenotype, as referred to herein, relate to the elevation, or enhancement of the specific parameter in the specified range. More specifically, for "increase" or alternatively, "decrease" the following rates are applicable: about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. It should be appreciated that 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. Therefore, the term inhibit or decrease or alternatively, increase or enhance refers to a change of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more, as compared with the previous level of the discussed parameter.

It should be understood that the terms specified in the claims and defined by the following definitions are applicable for each and every aspect of the invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Throughout this specification and the Examples and claims which follow, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

Experimental procedures

Cells: HEK-293 (ATCC CRL-1573) cells were cultured in adherent conditions in Dulbecco's Modified Eagle Medium (DMEM; high glucose, containing glutamine; Biological Industries) containing 10% fetal bovine serum (FBS), 1% sodium pyruvate, 100 pg/mL Pen-Strep, and 10 mM non-essential amino acids or adapted to suspension (Sartorius) or in suspension conditions in CD293 (Thermo Fisher Scientific) in 8% CO2 in a shaking 6 well plate in 130 RPM. Lentivirus production: The production of lentiviral-based particles was carried out using HEK- 293T cells, a set of three plasmids, Opti-MEM (Thermo Fisher, Cat. Number: 31985062), and FuGENE® HD Transfection Reagent (Promega, Cat. Number: E2312) according to an existing protocol. In brief, for each sample, pVSVG (0.5 pg), pPAX (0.5 pg), and an expression plasmid of choice (1 pg) were used. The plasmids mixture was added to a mix of 100 pL Opti-MEM and 8 pL FuGENE-HD (per sample) and vortexed for ~5 min. This was added to the HEK-293T cell (2.5 X 10⁶/l mL per sample) and seeded on a 6-well plate with an additional 1 mL medium. After ~12 h, the medium was replaced with 2.5 mL of standard DMEM.

Flow cytometry: The fluorescent signal was measured by cytoFLEX S flow cytometer (Beckman Coulter, California, USA) after preparing the sample according to the manufacturer’s protocol. Briefly, the culture medium was removed from the cells by a centrifuge, and the pellet was resuspended with PBS and measured at the desired laser setting. A minimum of 5000 live cells were measured in each sample.

RNA extraction and next-generation sequencing: RNA extraction and next-generation sequencing: Cells were collected by centrifuging twice at a speed of 3000 rpm for 3 min. RNA extraction was performed using RNeasy Plus Mini Kit (Qiagen, Cat. Number: 74134) according to the kit's protocol. The whole-cell RNA of the cells was aliquoted to 5 pg/12 pL, and RT-PCR was performed in triplicates using primers for the mKate2 gene using Superscript III Rev Transcript protocol (Thermo Fisher, Cat. Number 18080093). The attachment of the NGS adaptors was done by a PCR reaction with relevant primers. The NGS analysis was carried out at the Core Research Facility (CRF) of the Faculty of Medicine, The Hebrew University of Jerusalem, Israel. Cell images: Both bright field and fluorescent images were taken by OLYMPUS 1X70 MICROSCOPE. Fluorescent images of cells in suspension and in adherent conditions were taken with the same exposure time to allow comparing the intensity of fluorescence between conditions in qualitative manner.

SPECS screen: The screen is based on a library of synthetic promoters with enhanced cell-state specificity (SPECS), which correspond to 6,000 known eukaryotic transcription factor binding sites (TF-BSs), extracted from several databases (Kheradpour, P., Kellis, M. et. (2014) Nucleic acids research 2014;42:2976-2987, 5; Weirauch, M. T., et al.(2014) Cell 2014;158:1431-1443). Each of the SPECS is approximately 250 base pairs long and contains tandem identical repeats of a single TF-BS encoded upstream of a minimal promoter. Each of the SPECS regulates the coding sequence of the mKate2 fluorescent protein. In addition, the mKate2 sequence regulated by each of the SPECS includes a unique 20bp barcode (Fig. 2). In the current screen, forty-eight hours after library infection into cells, cells were divided to two; half were cultured under adherent and half were cultured under suspension conditions. After a few days mRNA produced from each subpopulation is used as a template for cDNA synthesis with primers that are complementary to the mKate2 coding sequence, and which therefore reverse-transcribe only the library mRNA. Finally, the cDNA is sequenced via Next Generation Sequencing (NGS) (Fig. 2). The data is analyzed to identify which SPECS are activated in the cell-states of interest. The barcode-based screen is not limited by plasmid copy number and does not require FACS sorting, since each unique SPECS produces a unique reporter (i.e., its corresponding mRNA barcode) rather than an identical fluorescent output. Therefore, it can be accomplished in a few days and requires only a small number of cells (about 60,000).

Construction of plasmids

These identified synthetic promoters were cloned into lentiviral vectors with conventional restriction enzyme cloning, upstream of an adenovirus minimal promoter to control the expression of the green fluorescent protein gene. As an expression control the green fluorescent protein gene was cloned down stream to the ubiquitin C (UbC) constitutive promoter.

Motif Analysis

The identification of common motifs within a list of promoters was conducted using the MEME Suite, motif-based sequence analysis tools (https://meme-suite.org/meme/tools/meme). This process includes motif discovery, creation of motif logos for visualization, precise localization within the promoters, and analysis of variations present within these motifs.

EXAMPLE 1

Designing the "SmartCell platform"

The "SmartCell platform", is a biological software system based on optimization of a next generation synthetic biology technology described in Nissim et al. [1-2]. It is composed of biological switches that provide cells with the ability to switch on/off or tune up/down expression of gene sets as a function of a specific cell state, in a reversible manner. No external intervention is required, as the platform is autonomous. The first version of the system now developed by the inventors, comprises one or more switches providing cells (primary adherent cells in origin) with the following capabilities (also referred to herein as cell phenotypes):

Proliferate optimally in suspension - by turning off adherence growth and turning on suspension growth. Proliferate in an animal and growth-factor (GF) free medium - by turning on expression of a selected set of GFs.

Optimal differentiation to relevant cell types - once cells reach high concentrations - turn off proliferation, turn back to adherent proliferation and turn on differentiation to relevant cell types (fat, muscle and blood vessel cells), thus enhancing differentiation efficiency.

The system is incorporated into cells of choice (e.g., animal cells, as indicted in the preset disclosure) to provide them with enhanced capabilities to efficiently produce ACBM products by providing solutions for two of the major bottlenecks currently faced by this industry.

The first, relates to reduction of production costs. More specifically, reaching cell masses sufficient for ACBM industrial-scale production requires massive cell proliferation that can be achieved only by GF addition to the culture medium. GFs are currently by far, the most significant cost contributors, driving medium costs to account for about 70% of the product’s marginal costs. The SmartCell technology incorporates into cells a switch capable of turning on the expression of a specific GF set in a timely manner, avoiding the need to add GF to the medium and reducing medium cost significantly. Moreover, the technology disclosed herein, enables each cell to sense the dynamic and changing environmental and/or cellular conditions and secrete GFs, or any other desired product, in an autologous manner, only according to its specific needs, significantly enhancing process efficiency. Importantly, when cells reach stage 2 of the process as illustrated in Fig. IB (production of meat), they autonomously turn off the GF switch, enabling cells to perform optimal differentiation and preventing GF contamination of the final food product.

The second bottleneck is increasing differentiation efficiency. Specifically, to date, cell differentiation during ACBM production is induced by external manipulations once cells reach a required quantities and concentration. These manipulations suffer from low conversion rates and a long differentiation period and require careful and intensive cell treatment. This is partly due to cells being in a multi-proliferative state (Stage 1 of the process as illustrated in Fig. 1A) which interferes and contradicts cell differentiation, making this transition highly difficult and inefficient. The SmartCell technology disclosed herein, incorporates into cells also a differentiation switch, activated (turned on) autonomously only when cell reach a sufficient cell concentration, inducing the expression of a specific set of proteins required for cell differentiation to either fat or muscle and significantly enhancing cell differentiation efficiency.

The biological software comprises smart biological switches, each containing two types of biological sequences: The first nucleic acid sequence acting as a "Sensor", is a synthetic promoter sequences activated by specific culture condition. Once cells harboring this promoter sense a specific culture condition (e.g., suspension, adherent versus non-adherent conditions), the promoter is turned on/off according to its specific programming.

The second element is the "Output", that may be one or more genetic elements, specifically a gene or other coding or non-coding nucleic acid sequences, e.g., a set of genes, which, once expressed, provide the cell with a certain ability, e.g. proliferating under non-adherent conditions.

The following switches are developed:

A cellular input-output (also referred to herein, as a sensor-output) unit (previously referred to as Switch) #1- A unit enabling cell immortalization and rapid cell division of the cells. In order to efficiently produce significant biomass quantities, cells need to proliferate rapidly and perform more than thirty population doublings. The switch “turns on” the expression of a set of genes that reduce cell-cycle regulation and allow their “infinite” rapid proliferation. Non-limiting examples for such genes are telomerase reverse transcriptase (TERT), cyclin-dependent kinase 4 (CDK4), and more.

A cellular input-output unit #2 - A unit enabling cells to secrete specific GFs in an autologous manner - once the cell is required to grow in animal and GF-free low-cost medium, the unit “turns on” the expression of a set of GFs that support cell proliferation under these conditions. This enables to replace the need in exogenous supplementation of GFs (e.g. transferrin, fibroblast growth factor-2 (FGF-2), insulin and Platelet-d erived growth factor BB (PDGF-BB)) to the medium.

A cellular input-output unit #3 - A unit enabling cell proliferation in high cell densities (stage 3, see Fig. 1) - once cell expansion in the bioreactor reaches high cell densities (~lX10⁷/ml), the sensor senses the change in culture conditions (e.g. reduced oxygen, reduced glucose, increased lactate, increased ammonia etc.) and activates a set of genes related to metabolic shifts in the cell, e.g. enhanced glucose uptake, enhanced glutamine production to reduce ammonia concentration. This switch enables reaching high cell mass with a significant reduction in medium volume used per batch.

A cellular input-output unit #4 - A unit enabling cell adaptation to suspension expansion (stages 2 and 3, see Fig. 1) - once cells are required to grow in suspension, the circuit senses cell transfer to suspension conditions and modulates (either activate or inhibit) the expression of a suspension- growth-enabling set of proteins, e.g. transforming growth factor beta induced (TGFBI), A disintegrin and metalloprotease 12 (ADAM12), Plakophilin-3 (PKP3), yes-associated protein 1 (YAP1) or TAFAZZIN (TAZ), Components of secreted extra cellular matrix (ECM), various proteases that disrupt the ability to aggregate, etc.

A cellular input-output unit #5 - A unit enabling efficient cell differentiation once cells reach the target cell density (stage 4, see Fig. 1) - Once cell reach their target cell density, a density sufficient to ensure cost-efficient cell-mass production (5X1O⁷-1X1O⁸) the sensor senses the change in culture conditions and all previously activated immortalization/proliferation, GF and suspension adaptation genes are turned off while specific differentiation genes, activation of adipogenesis (e.g. ZFP423, API, C/EBPa, P and 5 and PPARy) or myogenesis inducing genes (e.g. Sixl/4, Pax3, Pax7, Myf5, and MyoD) are turned on enabling efficient cell differentiation and tissue maturation. The SmartCell platform provided herein is incorporated into multiple animal cells of choice, specifically, any cell of any tissue origin of any domestic animal used in agriculture, e.g., pig, or any other domestic mammal used in meat industry, chicken, fish etc. to enable a cost efficient and controllable production process of cells for the ACBM industry.

EXAMPLE 2

Identification of suspension and adherent-specific synthetic promoters (S and A-promoters respectively)

Synthetic promoters that are specifically activated in non-adherent (suspension) conditions and promoters that are specifically activated in adherent conditions were identified by the present disclosure, using an improved SPECS screen.

The stages of the screen are schematically illustrated by Figure 2, stages A to E.

More specifically, cells are cultured under either adherent or suspension conditions and at each condition are infected separately with the improved pooled library of -6,000 synthetic promoters, in which each promoter encodes a unique barcode and an mKate2 coding region.

Five to seven days post library delivery to each subpopulation, cells are harvested, mRNA is produced, and barcode content is analyzed using NGS.

Next, barcodes which are overrepresented under adherent conditions, but not found under suspension, are indicative of adherent specific promoters and vice versa.

The cellular system used for this screen is a cell system that contains two cell types that are highly similar, however, one can proliferate only under adherent conditions while the second is adapted to suspension conditions. Preferably, such a system comprises adherent cells that were adapted to suspension.

More specifically, in order to identify synthetic promoters that are specifically activated in non- adherent (suspension-S) conditions and promoters that are specifically activated in adherent conditions (adherent-A), a cell-line, which is able to proliferate in both adherent and suspension conditions, is needed. To this end, the HEK-293 cell line was acquired from ATCC (ATCC CRL- 1573) and expanded under adherent conditions according to ATCC instructions (DMEM +FCS 10%). HEK-293 cells were then adapted to suspension conditions, using shaking flasks and a commercially available growth medium developed for cell culture in suspension (CD293, Thermo Fisher scientific). Once cells were able to expand in both culture conditions (S and A) they were infected with a modified version of the SPECS library (Synthetic Promoters with Enhanced Cell- State Specificity) first published by Wu et al (PMID: 31253799), while in suspension conditions. Forty-eight hours after infection, cells were divided into two; cells of one half were cultured under adherent and cells of the other half were cultured under suspension conditions. Five to seven days later, cells were harvested, RNA was isolated, and library specific cDNA was produced using library specific primers, and cDNA was amplified by PCR using library specific primers. PCR products were sent to NGS with a target of 2X10⁶ reads per sample (n=3 biological repeats). The above-described screen was performed twice providing strong reproducibility of identified promotors for both adherent and suspension conditions (Fig. 3).

Following NGS, the data was analyzed to identify promoters that have demonstrated significantly enhanced transcription under suspension conditions (S-promoter candidates) and promoters that demonstrates significantly enhanced transcription under adherent conditions (A-promoter candidate). Twenty-five S -promoters and 10 A-promoters demonstrating a significantly enhanced promoter activity in suspension versus adherent or adherent versus suspension respectively, were identified (examples of 10 of the S-promoters are shown in Figure 3).

To validate the identified S-promoters, each of the promoters was cloned upstream to green fluorescent protein (GFP) gene. HEK 293 cells were infected with the cloned S-promoters and GFP expression was compared between cells cultured in adherent versus suspension conditions using fluorescent microscopy and FACS analysis. As can be seen in Figure 4, enhanced expression of GFP under suspension conditions was demonstrated in 10 of the 25 candidate promoters, both by fluorescent microscopy (Fig. 4A) and by flow cytometry (Fig. 4B). Sequences of 10-positive and 12-negtive promoters is disclosed in Table 1. The quantitative results demonstrated a fold change of >10 folds between suspension and adherent in 10 of the validated promoters. To further ensure the validity of the results obtained on the protein level (GFP), comparison of the level of GFP transcription was also validated by real-time PCR demonstrating very similar results. Furthermore Table 1 below provides further information about the sequences of the examined promoters.

Table 1: details and sequences of the examined promoters

More specifically, Table 1 further discloses the size of the transcription factor (TF) binding site, and sequences thereof as denoted by SEQ ID NOs: 1 to 22, and 84, 85 and 86, the full sequence of all identified promoters as denoted by SEQ ID NOs: 23 to 47, the size of the entire promoter and the number of TF repeats in each promoter.

As shown herein, an experimental system enabling the expansion of cells from same origin in both adherent and suspension conditions was established. Moreover, S- promoters were identified by a SPECS library screen. Validation of the bioinformatic analysis code used to analyze screen results was performed with an external expert that confirmed the statistical methods employed and the adequately and robustness of the analysis. The SPECS library screen was repeated twice yielding very similar results further demonstrating the robustness of the technology and experimental system. S-promoters identified in the screen were validated by comparison of GFP expression under the regulation of various S-promoters between suspension and adherent conditions. Promoter activity 14-fold higher under suspension conditions was demonstrated for S-promoters. The results obtained establish for the first time, the validity and power of the SPECS screen of the present disclosure, to identify cultured meat relevant condition-specific promoters. Validated S- promoters are used herein as sensors in the main switch of the first prototype - the suspension unit. EXAMPLE 3

Identification of TF motif for S-promoters

The inventors next analysed the promoter sequences for the identification of a common motif. Three motifs having a core common sequence were identified.

Motif 1

The initial motif common to promoters activated in suspension (S-promoters) was identified within the sequences of seven suspension-specific promoters, which were detected by NGS and subsequently validated in FACS, and is presented in Figures 5A, and SB (motif 1, also denoted by SEQ ID NOs: 71 , 73 and 72, 79). Subsequently, this motif was screened across the entire library, and 26 promoters containing it, were identified. For each of these promoters, the distribution of RNA expression concerning suspension/adherent conditions, was examined. This analysis revealed that most of these promoters exhibited an increase in expression in suspension (Fig. 5C).

Motif 2

Twenty-four out of twenty-six promoters described herein, are associated with IRF. The inventors therefore examined IRF motifs within databases and reveled that the motif identified by the present disclosure, shared similarity with majority IRF motifs. Notably, positions 2 and 3 exhibit heightened dynamism. Furthermore, three additional promoters validated by FACS analysis were found to contain the motif ANNGAAAC (SEQ ID NO: 70), when N represents each nucleotide. Therefore, a search for this motif in the library was conducted and the promoters containing this motif were examined.

Motif 3

Twenty-five promoters exhibited statistical significance in screen 1 of NGS, a significance that was either validated statistically or through an identical trend in the second screening. As exploration was next conducted to identify any shared motifs among these promoters. Figure 6A, illustrates the motif, while its reverse complement is depicted in Figure 6B. The motifs positional distribution in each promoter is depicted in Figure 6C, noting that three promoters lack this motif. Upon reviewing the sequences of these three promoters (Fig. 6D) it becomes evident that two out of three contain the initial motif.

Upon reviewing the sequences of 22 promoters containing the motif (Fig. 7A), it is evident that there is variability in the eighth position of the motif across these sequences. Consequently, in the motif search against the library, the inventors account for each nucleotide in position 8 within the motif, marking this option as 'N' in the sequences (Figure 7B, 7C, these motifs are denoted by SEQ ID NO: 64, 65). It was found that about 50/64 promoters showed an increase in suspension. A Venn diagram (Fig. 8) was created to demonstrate the intersection between the promoters’ groups which obtained as described above.

EXAMPLE 4

Porcine cell adaptation to culture conditions

Abdominal porcine skin tissue, including subcutaneous fat, was purchased from LAHAV CRO and a protocol of the isolation of adipose derived stem cells (AdMSCs) was established. As can be seen in Figure 9AI, AdMSCs adapted to culture from 4 separate isolations were obtained, and a cell frozen stock of each isolation in early passages was prepared. From these isolations one isolation #2 was chosen and its ability to differentiate into fat was next demonstrated, thereby ensuring that the cells used were indeed AdMSCs with a fat differentiation potential. Once fat differentiation was demonstrated (Fig. 9B), using a StemPro® adipocyte differentiation basal medium (StemPro ADM, gibco). A large frozen stock #2 AdMSCs was prepared, and their expansion potential was examined. As can be seen, the AdMCs of the present disclosure were able to expand to -passage 21 when enter senescence and stop to proliferate (Fig. 9CI). The change of phenotype of the cells can be seen in the images of the cells (Fig. 9CII) in which the cells became bigger with passage leading finally to the termination of proliferation.

In parallel to porcine AdMSCs production, dermal fibroblasts were also isolated from abdominal skin samples. As can be seen in Figure 9AII, the inventors were able to isolate and adapt to culture fibroblasts from abdominal cells and from porcine ear. In addition, UMNSAH/DF-1 cells (fibroblast cells o chicken embryo, CRL-3586), were purchased, and their ability to expand in 2D cultures was next evaluated. As can be seen in Figure 9AIII, these cells were immediately expanded in 2D demonstrating very efficient expansion and rapid proliferation (-14 hours population doubling). In addition, UMNSAH/DF-1 cells were adapted to suspension reaching a concentration of -3X10⁶ cells/ml after 45 days in culture suspension (data not shown).

Next, the inventors immortalized the AdMSCs. As can be seen in Figure 10, combinations of genes (e.g., TERT, CDK4), previously suggested to induce immortalization of primary cells (the resulting clones indicated herein as Imm#l, Imm#2, Imm#3), or GFP as negative control, were infected to AdMSCs. Twelve days after infection, a phenotypic change was observed in AdMSCs that were infected with the Imm #3 (Fig. 10A). The observed phenotype persisted a week later and was accompanied also by enhanced proliferation capacity as was demonstrated by the enhanced density of cells (Fig. 10B). All infected cells were then expanded and their population doubling time was compared as a measure of their proliferation rate. In contrast to AdMSCs infected with GFP (negative control), in which doubling time increased significantly with time all cells infected with the different gene combination demonstrated a significant reduction in doubling times until a constant double time was reached. This low doubling time remained constant for several passages until passage 20 indicating all three cells underwent immortalization that allowed their long-term proliferation without going into senescence (Fig. 10C). From these three cells, clone Imm #3, demonstrated the most rapid immortalization as it already reached its low and constant doubling at passage 5. In addition, Imm #3 cells demonstrated the lowest doubling time of all cells ~35 hours. These immortalized porcine cells (IPOCs) were used in the following experiments, and are designated herein, as the IPOCs of the present disclosure. To ensure that the IPOCs retain the proliferation in a sufficient manner to participate in cultured meat production processes, their proliferation was continued to passage 34 which equals to ~70 population doublings. Importantly, the IPOCs retained a stable doubling time ~35 hours throughout the expansion period and a stable cellular phenotype (Fig. 10C and 10D). This stability is highly important once such cells reach industrial production and is known to indicate to the genetic stability of the immortalized cells.

In summary, porcine AdMSCs and dermal fibroblasts were isolated and adapted to culture conditions. Moreover, the AdMSCs demonstrated differentiation to fat and were able to propagate until about passage 20 when proliferation stopped gradually due to senescence.

The AdMSCs were rapidly immortalized after they were introduced with various combinations of genes known to induce immortalization.

EXAMPLE 5

Serum-free medium is able to support IPOC expansion

The inventors next evaluated the expansion ability of the IPOCs, in a serum free medium.

In a first step, the inventors performed experiments aimed at evaluating the proliferation of the IPOCs in 2D using a serum free medium (Based on DMEM-F12 (Dulbecco's Modified Eagle Medium and Ham's F-12 Nutrient Mixture)). As can be seen in Figure 12, a serum-free medium supported IPOC long-term expansion (12 passages) retaining high cell viability >90% and a doubling time of ~36 hours, which is practically identical to the doubling time of these cells when expanded in serum containing medium (Figs. 11A and 11B).

Next, transferrin was cloned into an expression plasmid, expressing it under the regulation of a constitutive promoter, the ubiquitin c (UbC) promoter. Transferrin was introduced with a leader signal that assisted its secretion from the cells. The expression of transferrin was demonstrated by ELISA. As can be seen in Figurel2A, uninfected IPOCs were unable to grow in serum-free medium with no (-) transferrin and died after 16 days in this medium. In contrast, as can be seen in Figure 12B, IPOCs expressing transferrin was able to support IPOC long-term expansion in serum-free medium (-) transferrin. These results provide a first and very important POC to the ability of the first prototype to grow in serum-free medium without essential costly GFs once the cells were program to secrete these GFs.

EXAMPLE 6

Adaptation of IPOCs to suspension conditions in medium +1 % serum

Once the IPOCs of the present disclosure were adapted to growth in serum-free- -medium in 2D, the inventors noticed that in the absence of serum IPOCs demonstrated reduced adherence to plates’ surface. Therefore, adaptation of IPOCs grown in defined medium directly into suspension growth in defined medium, was next performed. Since suspension growth increases the shear force applied on cells, 1% serum and 5ng/ml PDGF-b, were added to the medium (DMEM-F12). As can be seen in Figure 13, adaptation to suspension conditions in shaking flasks was successful with IPOCs demonstrating continued proliferation of ~72 hours doubling time and viability >90% after 20 days in culture.

EXAMPLE 7

Screening and validation of suspension promoters in immortalized porcine cells (IPOCs)

Once IPOCs were developed and adapted to suspension conditions as described in Examples 5 and 6, the activity of S-promoters identified in human 239 cells (Example 2), was next evaluated in porcine cells. More specifically, the previously identified promoters D6M_4838 and D6M_1854 (also referred to in Table 1, as clones #34 and #17, respectively), were cloned upstream to the green fluorescent protein (GFP) gene. IPOCs were then infected with the cloned S-promoters, and GFP expression was compared between cells cultured in adherent versus suspension conditions using fluorescent microscopy and FACS analysis.

Figure 14A and 14B, demonstrate successful enhanced expression of the GFP reporter in suspension conditions, for both, D6M_4838 and D6M_1854 (also referred to in Table 1, as clones #34 and #17, respectively) promoters, demonstrating that the promoters are conserved between species.

Still further, an additional screen for suspension promoters (S-promoters) was next performed in IPOCs More specifically, IPOCs adapted to suspension that were cultured in defined medium with 1 % serum, were infected in suspension lentiviral-based particles containing the SPECS library described in Example 2. One hour after the initiation of infection, cells were divided into two portions, one portion was cultured under adherent in gelatine coated culture plates (see Example 8), and the other portion was cultured under suspension conditions in flasks. Infection was terminated forty-eight hours later and viral particle containing medium was replaced with fresh medium. Cells were cultured in suspension or adherent conditions for additional 5 days. The cells were then harvested, RNA was isolated from the cells and library specific cDNA was produced using library specific primers, cDNA was amplified by PCR using library specific primers. PCR products were sent to NGS with a target of 2X10⁶ reads per sample (n=3 biological repeats).

Following NGS, the data was analysed to identify promoters that demonstrated significantly enhanced transcription under suspension conditions (Porcine S-promoter candidates).

Three of the promoters identified in this screen are disclosed in Table 2, specifically, clones D6M_4839, DM_1745 and DM_4726.

To validate the identified S- promoter candidates, IPOCs were infected with each of the S-promoter candidates cloned upstream to GFP gene. GFP expression was compared between cells cultured in adherent versus suspension conditions using fluorescent microscopy and FACS analysis.

As can be seen in Figure 14A, the five S-promoters demonstrated >10 fold increased expression under suspension, as compared with adherent conditions in porcine cells (IPOCs). These results validate and further approves the successful results demonstrated in 293 human cells (see Figure 4), also in cultured meat relevant cells of a porcine origin. Interestingly, the enhanced expression of S-promoters under suspension conditions is far more robust that in human 293 cells and is as high as -60,000 fold higher in suspension versus adherent conditions. Importantly, as indicated above, two of these S-promoters, specifically, D6M_4838 and D6M_1854, that demonstrated 168- and 38.9-fold expression increase under suspension conditions in porcine cells (Fig. 14A), were previously identified as S-promoters in human 293 cells. The fact that the same synthetic promoters exhibit a specific elevated activity under suspension conditions in two different species demonstrate for the first time the interspecies properties of S-promoters. In addition to their condition-specific expression, the level of expression of the promoters in suspension and their level of residual activity in adherent conditions is also important to their functional use in bioprocess control. In order to evaluate these traits, the level of expression of all five S-promoters was normalized to the expression of the human ubiquitin C (UbC) promoter, which was used as control and is a known ubiquities promoter able to promote medium to high protein expression (Fig. 14B). As can be seen in this figure, the two promoters demonstrating the highest fold increase under suspension conditions are D6M_4838 and D6M_4839, which demonstrate 168 and -60,000 fold increase in suspension versus adherent conditions respectively. Importantly, while the D6M_4838 and D6M_1854 promoters that identified in the 293 cells screen, contain the sequence of motif 3 (see Figures 6B (SEQ ID NO: 77) and 6A (SEQ ID NO: 66, respectively), D6M_4839, which was identified in the IPOC screen also contains the motif although in its shorter form, specifically, TCGAAACT (SEQ ID NO: 87). This further verifies the validity of the identified motif to S- promoter specificity.

Interestingly, however, as is demonstrated by DM_1745 and DM_4726, which do not contain the motif, the large synthetic promoter library allows the identification of more than one motif in specific species (porcine).

These results demonstrate that the motifs controlling the specificity of SPECS are conserved between species.

Table 2: S-promoters identified in IPOCs

EXAMPLE 8

Screening and validation of adhesion promoters in immortalized porcine cells (IPOCs)

As indicated in Example 7, a screen in IPOCs was performed for identifying A-promoters. IPOCs adapted to suspension that were cultured in defined medium with 1% serum were infected in suspension lentiviral-based particles containing the SPECS library. One hour after the initiation of infection cells were cultured under adherent conditions in gelatine coated culture plates. Infection was terminated forty-eight hours later and viral particle containing medium was replaced with fresh medium. Cells were then cultured in the two conditions for additional five days. The cells were then harvested, RNA was isolated from the cells and library specific cDNA was produced using library specific primers, cDNA was amplified by PCR using library specific primers. PCR products were sent to NGS with a target of 2X10⁶ reads per sample (n=3 biological repeats).

Following NGS, the data was analysed to identify promoters that demonstrated significantly enhanced transcription under adherent conditions (porcine A-promoter candidates).

To validate the identified A-promoter candidates, each of the promoters was cloned upstream to green fluorescent protein (GFP) gene. IPOCs were then infected with the cloned A-promoter candidate and GFP expression was compared between cells cultured in adherent versus suspension conditions using fluorescent microscopy and FACS analysis. The A-promotes identified in the screen are disclosed in Table 3.

The promoter screen performed in IPOCs identified also promoters that were specifically expressed under adherent conditions as compared to suspension. The expression of the promoter identified was ~10 fold higher under adherent versus suspension conditions. Figure ISA and 15B, shows representative four promoters, specifically, D6M_3792, D6M_3732, D6M_3787 and D6M_3830, that contain at least one NFKB binding site, specifically, a transcription factor binding site having the motif of GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83, or the motif DDKGRAHDYHMY, SEQ ID NO: 68. It should be understood that R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T. Table 3: A-promoters identified in IPOCs

Altogether, the results of the IPOC promoter screen further substantiate the suggested use of SPECS to improve industrial cell-based bioprocesses. More specifically, demonstrating the feasibility of the disclosed SPECS screen for identifying promoters that are activated by various conditions relevant to industrial process (e.g., suspension and adherent conditions).

EXAMPLE 9

Production of low-cost high quality fat cells, hybrid products and low-cost high-quality muscle tissue

As indicated above, the SmartCell platform is incorporated into multiple animal cells of choice, for example, porcine cells. More specifically, the SmartCell platform is incorporated into porcine cells enabling them to produce low-cost high-quality fat cells. Low cost SmartCell based fat are first manufactured and distributed as an additive to existing plant-based alternative-meat producers. Alternatively, both SmartCell based fat and undifferentiated cell biomass are used as a basis for hybrid products. In addition, the SmartCells are programed and induced to create low- cost high-quality muscle tissue that are used to create various ACBM products.

Claims

CLAIMS:

1. A method for the production of a cell-based product, the method comprising the step of introducing into at least one cell, at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or a cellular platform thereof, wherein said conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said cellular- and/or environmental- state and/or condition, and wherein said conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site.

2. The method according to claim 1 , wherein said at least one desired phenotype comprises at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity.

3. The method according to any one of claims 1 and 2, wherein said cellular- and/or environmental- state and/or conditions comprise at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D- scaffold), levels of lactate and levels of ammonia.

4. The method according to any one of claims 1 to 3, wherein said nucleic acid sequence of interest comprises at least one coding sequence and/or non-coding sequence and/or inhibitory and/or modulatory nucleic acid molecule.

5. The method according to any one of claims 1 to 4, wherein said nucleic acid sequence of interest encodes, or controls at least one of:

(a) at least one product that directly or indirectly leads to, or involved with, said phenotype adapted to the cellular- and/or environmental- state and/or condition, or controls the production and/or activity and/or levels of said product; and

(b) at least one product of interest or controls the production and/or activity, and/or levels of said product.

6. The method according to any one of claims 1 to 5, wherein said product is at least one of: at least one growth factor, at least one survival factor, at least one differentiation factor, at least one immortalization factor, at least one cell metabolic factor, at least one adhesion molecule, at least one protease, and/or at least one cell migration factor.

7. The method according to any one of claims 1 to 6, wherein said cell is a eukaryotic cell or a prokaryotic cell.

8. The method to any one of claims 1 to 7, wherein said eukaryotic cell is of at least one unicellular or multicellular organism of the biological kingdom Animalia or of the biological kingdom Plantae.

9. The method according to claim 8, wherein said organism is of the biological kingdom Animalia, said organism is any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

10. The method according to any one of claims 1 to 9, wherein said cellular- and/or environmental- state and/or condition comprises suspension conditions and wherein said at least one desired phenotype comprises at least one of: cell proliferation, cell growth and/or cell expansion in suspension conditions.

11. The method according to claim 10, wherein said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in suspension conditions, comprises the nucleic acid motif:

STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76, and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue.

12. The method according to any one of claims 10 and 11, wherein said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in suspension conditions, comprises at least one transcription factor binding site comprising the nucleic acid motif:

GTTTCNNT, as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70; and/or any functional fragments thereof; wherein A is adenine, G is Guanin, C is cytosine, T is thymine and N is any nucleic acid residue.

13. The method according to claim 12, wherein said motif of at least one transcription factor binding site comprises the nucleic acid sequence of:

GTTTCRRT, as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof; wherein Y is a pyrimidine, specifically, C or T, and wherein R is G or A.

14. The method according to claim 13, wherein said motif of at least one transcription factor binding site, comprises the nucleic acid sequence of:

GTTTCGGT, as denoted by SEQ ID NO: 73, and/or the reverse complement ACCGAAAC, as denoted by SEQ ID NO: 74; and/or any functional fragments thereof.

15. The method according to any one of claims 10 to 13, wherein said at least one transcription factor binding site comprises the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86and/or the reverse complement thereof, or any combinations thereof.

16. The method according to any one of claims 10 to 15, wherein said at least one synthetic conditional nucleic acid sequence comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47, and/or the reverse complement thereof, or any combinations thereof.

17. The method according to any one of claims 10 to 16, wherein said desired phenotype is production and/or secretion of at least one growth factor in suspension conditions, thereby providing cell proliferation, cell growth and/or cell expansion, said at least one nucleic acid sequence of interest encodes, or controls the production and/or activity and/or levels of, at least one growth factor.

18. The method according to any one of claims 1 to 9, wherein said cellular- and/or environmental- state and/or condition comprises adherence conditions and wherein said at least one desired phenotype comprises at least one of: non-proliferative state and/or cell differentiation in adherence conditions.

19. The method according to claim 18, wherein said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions, comprises the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

20. The method according to any one of claims 18 and 19, wherein said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in said adherence conditions, comprises at least one transcription factor binding site comprising the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof.

21. The method according to any one of claims 18 to 20, wherein said at least one synthetic conditional nucleic acid sequence comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

22. The method according to any one of claims 18 to 21, wherein said desired phenotype comprises differentiation of said cell under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell, and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

23. The method according to any one of claims 1 to 22, wherein said cellular input-output unit, is configured for autonomously providing to a cell at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition, the unit comprising:

(i) at least one nucleic acid molecule comprising said at least one synthetic conditional nucleic acid sequence; and

(ii) at least one nucleic acid sequence of interest, operably linked to said synthetic conditional nucleic acid sequence; wherein said at least one synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of said at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions.

24. The method according to any one of claims 1 to 23, wherein said nucleic acid cassette or any vector or vehicle comprises said at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or at least one cellular input-output unit comprising said at least one nucleic acid molecule, said cassette optionally further comprises at least one genetic element.

25. The method according to any one of claims 1 to 24, wherein said cellular platform is configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s, the platform comprising at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, wherein said at least one synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cellular- and/or environmental- state and/or conditions.

26. The method according to claim 25, wherein said platform comprises at least one cellular input-output unit or any nucleic acid cassette or vector comprising the same, said unit comprising:

(i) at least one nucleic acid molecule comprising said at least one synthetic conditional nucleic acid sequence; and (ii) at least one nucleic acid sequence of interest, operably linked to said synthetic conditional nucleic acid sequence; wherein said at least one synthetic conditional nucleic acid sequence of (i), comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of said at least one operably linked nucleic acid sequence of interest of (ii), upon sensing the cellular- and/or environmental- state and/or conditions.

27. The method according to any one of claims 1 to 26, wherein said platform comprises at least two of:

(a) at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in cell suspension conditions;

(b) at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in adherence conditions;

(c) at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in high cell density conditions; and

(d) at least one input-output unit configured for autonomously providing to a cell at least one desired phenotype in three-dimensional scaffold (3D-scaffold) (or aggregate) conditions.

28. The method according to any one of claims 1 to 27, wherein said cell-based product is at least one of: a food product, an additive, a medicament, a cosmetic product or an in vitro multicellular system.

29. The method according to claim 28, wherein said food product is an animal cell-based meat (ACBM) product.

30. A cell-based product comprising at least one cell or at least one population of said cells, or any product of interest produced by, or produced from said cells, said cell/s comprising at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising said at least one nucleic acid molecule, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, thereby said cell autonomously exhibits at least one desired phenotype.

31. The cell-based product according to claim 30, wherein said cell is a eukaryotic cell or a prokaryotic cell.

32. The cell-based product according to any one of claims 30 to 31 , wherein said eukaryotic cell is of at least one unicellular or multicellular organism of the biological kingdom Animalia or of the biological kingdom Plantae.

33. The cell-based product according to claim 32, wherein said organism is of the biological kingdom Animalia, said organism is any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

34. The cell-based product according to any one of claims 30 to 33, wherein said cell-based product is at least one of: a food product, an additive, a medicament, a cosmetic product or an in vitro multicellular system.

35. The cell-based product according to claim 34, wherein said food product is an animal cellbased meat (ACBM) product.

36. The cell-based product according to any one of claims 30 to 35, wherein said product is prepared by the method according to any one of claims 1 to 28.

37. A method of preparing an animal cell-based meat (ACBM) product, the method comprising: a. culturing under suitable conditions at least one source cell or at least one population of said cells, wherein the source cell/s comprise at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or an input-output unit, a nucleic acid cassette and/or a platform comprising said nucleic acid molecule; and b. processing said cells and/or at least one product produced by, or produced from said cell to prepare said food product; wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the dynamic cellular- and/or environmental- state and/or condition, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype.

38. The method according to claim 37, wherein said source cells are programed for at least one of: autonomous growth, proliferation, expansion, differentiation, immortalization, tissue and/or organ formation, maturation, production of at least one product of interest and/or modulated cell activity, in dynamic cellular- and/or environmental- state and/or conditions, and wherein step (a) of the preparation of said ACBM product comprise the steps of the method as defined by any one of claims 1 to 28.

39. The method according to any one of claims 37 to 38, wherein said cellular- and/or environmental- state and/or conditions comprise at least one of: suspension conditions, adherence conditions, high cell density conditions, cell aggregate formation, adherence to three-dimensional scaffold (3D-scaffold), levels of lactate and levels of ammonia.

40. The method according to any one of claims 37 to 39, wherein dynamic cellular- and/or environmental- state/s and/or condition/s comprise at least one change in said cellular- and/or environmental- state/s and/or condition/s over time.

41. The method according to any one of claims 37 to 40, wherein said source cell is of an organism of the biological kingdom Animalia, said organism is any one of a non-human mammal, an avian, a fish, a crustacean, a crab or a lobster.

42. The method according to any one of claims 37 to 41, wherein said at least one source cell is a mesenchymal multipotent cell.

43. The method according to any one of claims 37 to 42, wherein said at least one source cell is at least one of an adipose stem cell, a satellite cell, and/or a dermal fibroblast.

44. The method according to any one of claims 37 to 43, wherein said source mesenchymal stem cells are of a non-human mammal being at least one of Cattle, domestic pig, sheep, horse, goat, buffalo, alpaca, lama and Camels.

45. An animal cell-based meat (ACBM) product comprising at least one cell or at least one population of said cells, and/or any product of interest produced by, or produced from said cells, wherein said cell/s comprise at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or any cellular input-output unit, nucleic acid cassette or platform comprising said at least one nucleic acid molecule, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing the cellular- and/or environmental- state and/or condition, thereby said cell autonomously exhibits at least one desired phenotype.

46. The ACBM product according to claim 44, wherein said product is prepared by the method as defined in any one of claims 37 to 44.

47. A nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said suspension condition, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, said transcription factor binding site comprising the nucleic acid motif:

48. The nucleic acid molecule according to claim 47, wherein at least one desired phenotype comprises at least one of: cell proliferation, cell growth and/or cell expansion in suspension conditions.

49. The nucleic acid molecule according to any one of claims 47 and 48, wherein said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in suspension conditions, and wherein at least one of: (I) said conditional nucleic acid sequence comprises at least one transcription factor binding site comprising the nucleic acid motif:

GTTTCNNT, as denoted by SEQ ID NO: 69, and/or the reverse complement ANNGAAAC, as denoted by SEQ ID NO: 70; and/or any functional fragments thereof; wherein A is adenine, G is Guanin, C is cytosine, T is thymine and N is any nucleic acid residue;

(II) said motif of at least one transcription factor binding site comprises the nucleic acid sequence of:

GTTTCRRT, as denoted by SEQ ID NO: 71, and/or the reverse complement AYYGAAAC, as denoted by SEQ ID NO: 72; and/or any functional fragments thereof; wherein Y is a pyrimidine, specifically, C or T, and wherein R is G or A; and/or

(III) said motif of at least one transcription factor binding site, comprises the nucleic acid sequence of:

50. The nucleic acid molecule according to claim 49, wherein at least one of:

(I) said at least one transcription factor binding site comprises the nucleic acid sequence as denoted by at least one of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 84, 85 and 86 and/or the reverse complement thereof, or any combinations thereof; and/or

(II) said at least one synthetic conditional nucleic acid sequence comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 45, 46 and 47, and/or the reverse complement thereof, or any combinations thereof.

51. The nucleic acid molecule according to any one of claims 47 to 50, wherein said desired phenotype is production and/or secretion of at least one growth factor in suspension conditions, thereby providing cell proliferation, cell growth and/or cell expansion, said at least one nucleic acid sequence of interest encodes, or controls the production and/or activity and/or levels of at least one growth factor.

52. A nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing adherence conditions, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said adherence condition, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, said transcription factor binding site comprising the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83,; or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

53. The nucleic acid sequence according to claim 52, wherein said cellular- and/or environmental- state and/or condition comprises adherence conditions, and wherein said at least one desired phenotype comprises at least one of: non-proliferative state and/or cell differentiation in adherence conditions.

54. The nucleic acid sequence according to any one of claims 52 and 53, wherein at least one of:

(I) said at least one synthetic conditional nucleic acid sequence configured for controlling the expression of at least one nucleic acid sequence of interest in said adherence conditions, comprises at least one transcription factor binding site comprising the nucleic acid sequence as denoted by any one of SEQ ID NO: 48, 49, 50 and 51, and/or the reverse complement thereof, or any combinations thereof; and/or any functional fragments thereof; and/or

(II) said at least one synthetic conditional nucleic acid sequence comprises the nucleic acid sequence as denoted by any one of SEQ ID NOs: 52, 53, 54 and 55, and/or the reverse complement thereof, or any combinations thereof.

55. The nucleic acid sequence according to any one of claims 52 to 54, wherein said desired phenotype comprises differentiation of said cell under adherence conditions into at least one of: a fat cell, a muscle cell, and a blood vessel cell, and wherein said at least one nucleic acid sequence of interest encodes or controls the production and/or activity and/or levels of, at least one differentiation factor.

56. The nucleic acid molecule according to any one of claims 47 to 55, for use in the expression of at least one nucleic acid sequence of interest operably linked thereto.

57. A cellular input-output unit configured for autonomously providing to a cell, at least one desired phenotype adapted to at least one cellular- and/or environmental- state and/or condition, the unit comprising:

(i) at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site; and

(ii) at least one nucleic acid sequence of interest, operably linked to said synthetic conditional nucleic acid sequence; wherein said at least one synthetic conditional nucleic acid sequence of (i), is configured for controlling the expression of at least one nucleic acid sequence of interest of (ii), upon sensing one of:

(I) suspension conditions, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said suspension condition, and wherein said transcription factor binding site comprises the nucleic acid motif:

STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue; or

(II) adherence conditions, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said adherence condition, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, said transcription factor binding site comprising the nucleic acid motif:

GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO:81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83,, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

58. The cellular input-output unit according to claim 57, wherein:

(a) said at least one nucleic acid molecule for sensing suspension conditions according to (I), is as defined by any one of claims 47 to 51 and 56; and/or

(b) said at least one nucleic acid molecule for sensing adherence conditions according to (II), is as defined by any one of claims 52 to 56.

59. The cellular input-output unit according to any one of claims 57 to 58, wherein said at least one desired phenotype comprises at least one of: cell proliferation, cell growth, cell expansion, cell differentiation, cell immortalization, cell maturation, tissue and/or organ formation, production of at least one product of interest and/or modulated cell activity.

60. The cellular input-output unit according to any one of claims 57 to 59, wherein said at least one nucleic acid sequence of interest comprises at least one coding and/or non-coding inhibitory and/or modulatory nucleic acid molecule.

61. A nucleic acid cassette or any vector thereof, comprising at least one nucleic acid molecule comprising at least one synthetic conditional nucleic acid sequence, or at least one cellular inputoutput unit comprising said at least one nucleic acid molecule, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and wherein at least one of :

(I) said synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest, upon sensing suspension conditions, wherein the controlled expression of at least one of said nucleic acid sequence of interest results in at least one desired phenotype adapted to said suspension condition, and wherein said transcription factor binding site comprises the nucleic acid motif:

STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue; and/or

(II) wherein said at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one nucleic acid sequence of interest in adherence conditions, said transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83, and/or the reverse complement thereof; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

62. The nucleic acid cassette or any vector thereof according to claim 61, wherein:

(a) said at least one nucleic acid molecule configured for suspension conditions according to

(I), is as defined by any one of claims 47 to 51 and 56; and/or

(b) said at least one nucleic acid molecule configured for adherence conditions according to

(II), is as defined by any one of claims 52 to 56.

63. A cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s, the platform comprising at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and wherein at least one of:

(I) said at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell suspension conditions, wherein said transcription factor binding site comprising the nucleic acid motif:

(II) wherein said at least one synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell adherence conditions, wherein said transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO:83,; and/or any functional fragments thereof; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.

64. The cellular platform according to claim 63, comprising at least one cellular input-output unit or any nucleic acid cassette or vector comprising the same, said unit comprising:

(i) at least one nucleic acid molecule comprising at least one of said synthetic conditional nucleic acid sequence; and

65. The cellular platform according to any one of claims 63 and 64, wherein said nucleic acid molecule is as defined by any one of claims 47 to 56, said input-output unit is as defined by any one of claims 57 to 60, and wherein said cassette is as defined by any one of claims 61 to 62.

66. A cell or a population of said cells comprising and/or genetically engineered by, at least one synthetic conditional nucleic acid sequence, or a cellular input-output unit thereof, or any nucleic acid cassette or vector comprising the same, or at least one cellular platform configured for autonomously providing at least one desired phenotype upon sensing dynamic cellular- and/or environmental- state/s and/or condition/s, wherein said synthetic conditional nucleic acid sequence comprises at least two repeats of a transcription factor binding site, and wherein at least one of: (I) at least one of said synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing the cell suspension conditions, said transcription factor binding site comprises the nucleic acid motif: STTTCNNW, as denoted by SEQ ID NO: 75, and/or the reverse complement WNNGAAAS, as denoted by SEQ ID NO: 76; and/or any functional fragments thereof; wherein A is adenine, G is guanin, C is cytosine, T is thymine, W is adenine or thymine, S is guanin or cytosine, and N is any nucleic acid residue; and/or (II) at least one of said synthetic conditional nucleic acid sequence is configured for controlling the expression of at least one operably linked nucleic acid sequence of interest upon sensing cell adherence conditions, said transcription factor binding site comprises the nucleic acid motif: GGGRHDBHMY, as denoted by SEQ ID NO: 80, and/or the reverse complement RKDVADYCCC, as denoted by SEQ ID NO: 81, or the motif GGGRMWTYCC, as denoted by SEQID NO: 82, and/or the reverse complement GGRAWKYCCC, as denoted by SEQ ID NO: 83; wherein R is A or G, H is A, C or T, D is A, G or T, B is C, G or T, M is A or C, Y is C or T, W is A or T, V is G, C, or A and K is G or T.