Expression of Products Derived from Nucleic Acid Concatenamers

Abstract

Techniques are provided for producing expression products using one or more nucleic acid concatemers comprising tandem repeats of nucleic acid sequences encoding one or more expression products. In one embodiment, various expression products may be co-expressed using a concatemeric mixture of first and second nucleic acid concatemers having a predetermined ratio of each other.

Description

Technical Field

[0001] Sequence Listing This application includes a Sequence Listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The above ASCII copy, created on May 15, 2019, is named 324586-1_SL.txt and is 13,566 bytes in size.

[0002] The present disclosure generally relates to the expression of recombinant products derived from nucleic acid concatemers, and in certain embodiments, to the stoichiometric co-expression of recombinant products derived from nucleic acid concatemers. Stoichiometric co-expression has been demonstrated in both in vivo and cell-free expression systems.

Background Art

[0003] Recombinant products, including proteins, antibodies, and nucleic acids, are widely used in clinical applications. Such recombinant products can utilize the expression machinery of nucleic acids and / or proteins to generate products of interest with clinical utility. For example, in immunotherapy and other cell therapies, viral vectors are frequently utilized to deliver gene payloads to treat specific disease targets. In one example, viral vector delivery of a payload for genetic modification of cells having a chimeric antigen receptor for CAR-T immunotherapy can be used to treat certain types of blood cancers. Other applications of viral vectors include use in cancer vaccines, monogenic diseases, and infectious diseases.

[0004] Recombinant DNA technology is used in the production of recombinant products. However, certain desired products may contain multiple components that are combined or otherwise function together. The production of such multi-component expression products can be complex. A common approach to recombinant product production may include the step of separating the various components into multiple plasmids. For example, co-expression of multiple plasmid constructs can be performed in both in vivo and in vitro settings. In some examples, the multiple plasmid constructs may be transiently transfected into cultured cells, and such cultured cells are used to produce the desired recombinant product, such as a viral vector, protein, etc.

[0005] However, certain challenges are associated with the use of multiple plasmid constructs in cells. One of the challenges is to regulate the co-expression of multiple plasmid constructs in a stoichiometric manner. For example, when each plasmid construct encodes a different protein, co-expression of two (or more) proteins from the corresponding plasmids by a transfection procedure within a single cell has been reported to result in the expression of the proteins in a wide variety of ratios, and sub-populations of cells may express only one of the desired proteins.

[0006] Several approaches have been attempted to improve the regulation of protein stoichiometric co-expression and achieve the desired ratio of proteins in single living cells. For example, instead of using two or more separate plasmids, a multicistronic plasmid may be constructed. For example, a single dual-promoter plasmid with an internal ribosome entry site (IRES) and a viral 2A peptide can be used to cause co-expression of two or more proteins with reduced heterogeneity, but this requires the laborious cloning of each open reading frame into the multicistronic plasmid, and as a result, increases the size of the plasmid construct. In addition, since the plasmid contains additional sequences necessary for maintenance in bacteria, the use of plasmids requires post-generation purification and QC analysis to demonstrate the absence of bacterial contaminants in plasmid production, which is an important issue for cGMP (clinical good manufacturing) processes. The latter is a particularly difficult issue because the post-generation purification of plasmid DNA becomes more complicated as the size of the plasmid construct increases and it becomes difficult to distinguish from bacterial chromosomal DNA.

[0007] Therefore, there remains a need for systems and methods for reliable regulation of recombinant product expression in both in vitro and in vivo expression systems, particularly those with minimal labor for preparation.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0010] A summary of certain embodiments disclosed herein is set forth below. It should be understood that such aspects are presented merely to provide the reader with a concise summary of such certain embodiments and are not intended to limit the scope of the present disclosure. Indeed, the present disclosure may encompass various aspects not described below.

Means for Solving the Problems

[0011] In one embodiment, a step of preparing a concatemer mixture comprising at least a first nucleic acid concatemer and a second nucleic acid concatemer having a predetermined ratio to each other, wherein the first nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence and the second nucleic acid concatemer comprises tandem repeats of a second nucleic acid sequence; and a step of co-expressing the concatemer mixture to produce a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence are provided.

[0012] In another embodiment, a step of preparing a mixture comprising at least two nucleic acid concatemers in a predetermined ratio, wherein each of the nucleic acid concatemers comprises tandem repeats of two or more nucleic acid sequences; and a step of co-expressing the mixture to produce two or more expression products from each nucleic acid concatemer in the mixture are provided.

[0013] In another embodiment, at least one template comprising a first nucleic acid sequence is amplified using a strand displacement rolling circle amplification method to generate a first concatemer comprising tandem repeats of the first nucleic acid sequence; contacting the first concatemer with a second concatemer comprising tandem repeats of a second nucleic acid sequence to form a concatemer mixture having a predetermined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer; co-expressing the concatemer mixture to produce a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence, wherein the ratio of the first expression product to the second expression product is proportional to the predetermined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture.

[0014] In another embodiment, there is provided a method comprising producing a mixture comprising at least one nucleic acid concatemer and at least one plasmid having a predetermined ratio to each other, wherein the at least one nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence and the at least one plasmid comprises a second nucleic acid sequence; and co-expressing the mixture to produce a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence.

[0015] In another embodiment, the transfected cell is transiently transfected with a nucleic acid concatemer that is first provided and that comprises tandem repeats of a nucleic acid sequence and expresses at least one open reading frame within the nucleic acid sequence encoding the expression product in the transfected cell. In one embodiment, the transfected cell provided has already been transfected with one or more nucleic acid concatemers and / or one or more plasmids. In this method, the cell can be sequentially transfected with the nucleic acid concatemer. In an alternative embodiment, the transfected cell is transfected with the nucleic acid concatemer after stably expressing one or more desired products.

[0016] These and other features, aspects, and advantages of the present disclosure will be better understood by reference to the following detailed description when read in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout the several views.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] One or more specific embodiments are described below. In an attempt to provide a concise description of such embodiments, not all features of the implementation are described herein. It should be recognized that, as in any project of engineering or design, in the development of any such implementation, numerous implementation-specific decisions must be made in order to achieve the particular goals of the developers, which may vary according to each implementation, for example, to meet system- and business-related constraints. Moreover, such development efforts are complex and time-consuming, but nevertheless, it should be understood that they are routine business for those skilled in the art who will benefit from the present disclosure in their design, fabrication, and manufacture.

[0019] Expression of products derived from large-sized (i.e., high molecular weight) nucleic acids is associated with low efficiency. Such low efficiency is a result of the insufficient transfection efficiency of large nucleic acids, which in turn can lead to a decrease in the production of the desired final product and DNA sequence-based competition between nucleic acids for host factors required for transcription and translation. It has been shown that transfection efficiency decreases as a function of DNA size, and it has been shown that for plasmids larger than 10 kb, transfection efficiency decreases dramatically compared to smaller-sized plasmids. Provided herein is an expression technique using DNA concatemers or mixtures of DNA concatemers, wherein the concatemers contain tandem repeats of nucleic acid sequences encoding a desired expression product, such as a protein or nucleic acid expression product. In certain embodiments, the disclosed concatemers can be of a size larger than that which has been inefficient heretofore (e.g., at least 10 kb). However, as disclosed herein, two or more concatemers, when mixed, achieve high transfection efficiency despite their large size. Also, one or more concatemers mixed with smaller-sized plasmids achieve high transfection efficiency even though one of the two is of a large size. In one embodiment, the concatemers are generated using a strand displacement amplification method, such as rolling circle amplification (RCA), to generate multiple concatemers and contain tandem repeats of nucleic acid sequences encoding the desired expression product. That is, the number of tandem repeats in each individual concatemer nucleic acid molecule may be unknown. Nevertheless, high transfection efficiency and production of the desired expression product can be achieved even in situations where the concatemer sizes resulting from RCA are variable and / or mixed. Furthermore, robust production of the desired co-expression product and precise ratio-based regulation can be achieved herein even when the exact number of tandem repeats is unknown and without regulating sequence-based competition between nucleic acid sequences for host factors.

[0020] Furthermore, as disclosed herein, DNA concatemers can be used in combination with co-expression systems to co-express two or more expression products in a ratio-metrical manner as predicted. Two or more concatemers can be transfected and co-expressed to generate co-expression products. Ratio-metrical expression can be achieved using concatemers having tandem repeats of unknown numbers of expression sequences for a desired end product. In certain embodiments, at least one of the concatemers has a size greater than about 10 kb. Transfection and co-expression of two or more concatemers can be carried out in a single cell or cell-free expression system. This is an unexpected result according to the conventional theory regarding the relationship between size and transfection efficiency. It is also unexpected according to the unpredictable nature of competition based on the DNA sequences of host factors that control transcription and translation. For example, since nucleic acid concatemers are of large size (e.g., a typical nucleic acid concatemer can have a size greater than 10 kb), according to the conventional theory, in a DNA cocktail containing two or more different nucleic acid concatemers, especially when the nucleic acid concatemers are not processed (e.g., no further processing is performed after the generation of the nucleic acid concatemers), it is not expected to efficiently penetrate into a single cell and generate the desired expression products. In addition, the size of the concatemers typically cannot be accurately determined, and the size is non-uniform, making the design of such cocktails or mixtures (containing two or more concatemers mixed in a predetermined or defined ratio) an even more difficult task.

[0021] As disclosed herein, a nucleic acid concatemer mixture containing a first and a second concatemer and prepared using a defined ratio can be used to generate a co-expression product that co-expresses the concatemer mixture to produce a co-expression product containing at least a first and a second expression product resulting from the first nucleic acid concatemer and the second nucleic acid concatemer of the concatemer mixture, respectively. In certain embodiments, the ratio of the first and second expression products is proportional to the defined ratio of the first and second nucleic acid concatemers in the concatemer mixture.

[0022] The use of concatemers generated by RCA that express the desired final product provides advantages over other expression templates, such as plasmid constructs. In rolling circle amplification, rapid generation of specific DNA sequences (e.g., DNA minicircles) encoding a minimal viral vector with full relevance and bioactivity for the intended application, including proteins required for packaging, transduction, and expression elements, as well as virus production, is possible. This allows for an increase in the specific activity of the DNA (coding sections per DNA lump). In contrast, plasmids contain additional sequences that are necessary for maintenance in bacteria but unnecessary for the intended application. Since RCA concatemers are not produced in bacteria, the use of RCA avoids potential contamination of the final DNA product by exogenous bacterial components or purification reagents. The disclosed concatemer synthesis is performed without using bacteria and thus does not contain endotoxins derived from bacteria. Also, RCA concatemers eliminate or reduce the need for large-scale bacterial growth, DNA purification columns, endotoxin removal, and quality control associated with bacterial-derived products, such as QC analysis to demonstrate the absence of bacterial contaminants (genomic DNA and RNA) in plasmid products. In contrast, nucleic acid concatemers can be prepared and amplified without using bacteria, require minimal post-generation purification, and thus offer advantages in terms of cost and complexity for adaptation to cGMP manufacturing. Furthermore, concatemers generated by RCA can be used to encode products with bacterial toxicity that are difficult to scale up in bacteria-based systems. This is because it is difficult to maintain constructs that can be used to encode toxic products due to leakage expression of the products during bacterial growth, which causes loss of the constructs due to bacteria or death of the bacteria.

[0023] Moreover, the concatemers generated by RCA can enable the simplification of DNA generation and scale-up as needed. Milligram to gram amounts of rolling circle amplification DNA can be inexpensively generated from template DNA in less than a day using an isothermal amplification reaction. The total "hands-on" time to generate 5 mg of RCA DNA is less than 1 hour. In contrast, it takes approximately 8 hours for supercoiled plasmid isolated from the DH5 alpha strain of Escherichia coli (E. coli) using an endotoxin-free purification column. Therefore, the rolling circle amplification method is expected to provide a more convenient and low-cost manufacturing method compared to standard bacterium-derived plasmids.

[0024] Furthermore, the RCA method enables the insertion of modified nucleotides into amplified DNA, which may be restricted in plasmid-based systems of living cells. Modified nucleotides can contribute to the generation of functionalized DNA with enhanced nuclease resistance, enhanced stability, long-term gene expression, and improved genome integration efficiency. In one embodiment, transfection of RCA DNA modified with phosphorothioated nucleotides resulted in longer-term protein expression compared to RCA DNA containing only standard nucleotides. Therefore, in one embodiment, the present technology can be used to generate or produce expression products derived from concatemers incorporating modified nucleotides.

[0025] Figure 1 is a schematic diagram of the generation of a desired expression product using at least one concatemer provided herein. The expression array 12 within the circular template 14 is used to generate a concatemer 16 that includes tandem repeats of the expression array 12 using the rolling circle amplification method (RCA). Once generated, the concatemer 16, after purification or otherwise washing, can be used in an expression system 20 that acts on the concatemer 16 to express the expression array 12 to generate an expression product 26. However, in certain embodiments, the concatemer 16 can be applied in the expression system 20 without further processing. The expression system 20 can be a cell-based or cell-free expression system as disclosed herein. Once generated, the expression product 26 can be harvested for use in an appropriate therapeutic protocol or other application. The expression product 26 of the concatemer 16 can include one or more protein and / or nucleic acid expression products.

[0026] In certain embodiments, the concatemer 16 can be used alone, or in combination with other concatemers 16 (e.g., two or more concatemers 16) or other expression vectors of the expression system 20, for example, in a cell-free expression system or in a mixture with a plasmid that can be co-transfected in a cell-based system to co-express multiple expression products 26. In certain embodiments, a concatemer 16 having a size of about 10 kb or greater can be used in a cell-based expression system to generate an expression product 26. In contrast to other techniques, the present disclosure demonstrates that large-sized concatemers and concatemer mixtures do not similarly reduce transfection efficiency in a cell-based expression system and do not result in competition based on the DNA sequence for host factors that reduce co-expression efficiency in the expression system, as compared to plasmids of similar size.

[0027] Figure 2 is a schematic diagram of the co-expression of desired expression products using a mixture of multiple concatemers. The first concatemer 16a having a tandem repeat of the expression array 12a and generated from the template 14a by RCA can be mixed with the second concatemer 16b having a tandem repeat of the expression array 12b and generated from the template 14b by RCA as provided herein. The expression arrays 12a, 12b can be different such that a plurality of expression products 26a, 26b are generated by the expression system 20. That is, instead of generating various expression products 26a, 26b in different expression systems 20, various expression products 26a, 26b can be co-expressed in the same expression system 20, for example, generated simultaneously. Further, the ratio of the expression products 26a, 26b (and others) can be based on or proportional to the ratio of the concatemers 16a, 16b (and others) in the mixture.

[0028] The size of the recombinant plasmid can be easily determined based on the size of the plasmid construct together with any inserted expression array, but the size of the concatemer can vary. However, in embodiments of the present technology, rather than attempting to determine the exact number of tandem repeats of the expression array 12 within each concatemer 16, it includes the assumption that each concatemer 16 is a monomer. This assumption is used in determining the molar ratio of the concatemers 16 to each other. For example, the first concatemer 16a may have tandem repeats of an expression array 12a with a known base length of 1000, and the second concatemer 16b may have tandem repeats of an expression array 12b with a known base length of 1500. Thus, to achieve a molar ratio of the first concatemer 12a: the second concatemer 12b of 1:1, the total amount of the second concatemer 16b should be 1.5× that of the first concatemer 16a in the concatemer mixture used for co-expression. It should be understood that the concatemers 16 can be either both single-stranded, both double-stranded, or a mixture of single- and double-stranded. The molar ratio can also be determined for any additional concatemers 16. Table 1 summarizes examples of molar ratios used in a three-concatemer 16 co-expression system of adeno-associated virus (AAV) that uses the assumption that the molar ratio can be based on the size of the monomer of the expression array 12 (or expression array, e.g., plasmid / circular construct having a template 14).

[0029]

Table 1

[0030] The molar ratio can be estimated based on the estimated sizes and estimated concentrations of the concatemers 12a, 12b generated by RCA.

[0031] Thus, although co-expression of two or more proteins by transfection of separate plasmids into a single cell has been reported to occur at a wide variety of ratios of the expressed proteins (e.g., many cell subpopulations express only one of the two protein constructs), the present technique facilitates a programmable ratio of co-expression products that is proportional to the ratio of concatemers 16 used in expression system 20. Although concatemers have uncertainties regarding size variation, nevertheless, the present disclosure demonstrates that robust expression with a programmable ratio can be obtained by using concatemers 16 that co-express the desired expression product 26.

[0032] In the co-expression workflow, it should be understood that concatemers 16 can be mixed or made at a defined or predetermined ratio based on the desired ratio of each expression product 26. That is, to achieve the desired ratio of the first expression product 26a to the second expression product 26b, the concatemer mixture provided for expression system 20 can be made of concatemers 16a, 16b that are present in a predetermined ratio to each other in the mixture. For two concatemers 16, the ratio of the concatemers 16 can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc. For three or more concatemers 16, the ratio of the concatemers 16 is represented as C1:C2:C N where C1, C2 to C N each of which may be 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, etc., and not the same integer. In certain embodiments, the ratio is a molar ratio rather than a simple mass ratio. In one embodiment, a molar ratio of concatemers 16 of about 1:1 results in a ratio of each expression product 26 of about 1:1. Further, in a co-expression system comprising one or more DNA concatemers 16, instead of estimating or inferring the size of the concatemer, the exact length of the DNA can be measured to determine the appropriate ratio as disclosed.

[0033] In certain embodiments, the co-expression product is formed by a process of creating a concatenamer mixture comprising a first nucleic acid concatenamer and a second nucleic acid concatenamer in a defined ratio, and co-expressing the concatenamer mixture to generate at least a first expression product and a second expression product respectively resulting from the first nucleic acid concatenamer and the second nucleic acid concatenamer of the concatenamer mixture, wherein the ratio of the first expression product and the second expression product is proportional to the defined ratio of the first nucleic acid concatenamer and the second nucleic acid concatenamer in the concatenamer mixture. In certain embodiments, the ratio of the first and second expression products and the defined ratio of the first and second nucleic acid concatenamers are substantially the same.

[0034] Figures 3-5 are schematic diagrams of embodiments of co-expression of nucleic acid concatenamers to form a desired final product. One or more co-expression products can be in the form of non-complex co-expression products or complex co-expression products. In certain embodiments, a non-complex co-expression product can include two or more expression products that co-express and exist in a non-complex form. For example, as shown in Figure 3, the co-expression product can include a first expression product 30 and a second expression product 34 that do not form a complex with each other after co-expression. Each of the expression products can independently be a protein, an RNA, or a combination thereof. Additionally, each concatenamer 16 (16a, 16b, optionally 16c) can generate a plurality of expression products shown as expression products 32, 36, 38.

[0035] In certain embodiments, the complex co-expression product may include two or more expression products 40, 42 that co-express from respective concatemers 16a, 16b and then form a complex 50 with each other, as shown in FIG. 4. For example, the complex co-expression product may include a first expression product 40 and a second expression product 42 that co-express and form a complex 50 with each other. Each of the expression products can independently be a protein, RNA, or a combination thereof. Additionally, as shown, an expression product (e.g., expression product 46) that can also form part of the complex 50 by another concatemer 16c can be generated. Non-limiting examples of complex co-expression products can include antibodies (e.g., monoclonal antibodies (mAbs)), virus-like particles, CRISPR, or lentiviruses.

[0036] FIG. 5 is an embodiment in which the co-expression product can include a first expression product that acts on a molecule. The first expression product can be a complex co-expression product 56 formed from separate expression products 58, 60, 62. In one embodiment, the first expression product 56 can be an enzyme, and the molecule 64 acted upon can be a substrate on which the enzyme acts. In one embodiment, the molecule 64 is a concatemer. In one embodiment, an interaction of the molecule of the complex 56 results in a product 66, which can then form a complex 70 with the complex 56 and its components. In another embodiment, the first expression product can be a protein, and the second expression product can be DNA. Also, the expression system can include one or more additional concatemers (e.g., concatemer 16b) that form expression products (e.g., expression product 68) that are not part of the final complex 70. Non-limiting examples of co-expression products that act on a molecule and where the molecule is a concatemer include recombinant adeno-associated virus (AAV) products derived from DNA-transfected cells.

[0037] According to the present disclosure, a concatemer mixture is prepared that includes first and second nucleic acid concatemers in a defined ratio. The concatemer mixture is co-expressed to generate at least a first expression product and a second expression product resulting from the first and second nucleic acid concatemers of the concatemer mixture, respectively, where the ratio of the first expression product and the second expression product is proportional to the defined ratio of the first and second nucleic acid concatemers in the concatemer mixture.

[0038] In certain embodiments, at least one of the first and second nucleic acid concatemers includes a minimal expression sequence. The minimal expression sequence encodes one or more desired products that do not have foreign sequences required for DNA amplification in a host cell. The minimal expression sequence can be designed in silico and synthesized in vitro. In one embodiment, each of the expression sequences 12 can include an open reading frame (ORF) and a promoter operably linked to the open reading frame. In certain embodiments, at least one of the expression sequences can include one promoter operably linked to two or more ORFs. For example, one of the expression sequences can include a promoter operably linked to two different ORFs.

[0039] In embodiments using two or more concatemers 16, the concatemer mixtures provided herein may include a mixture of two or more concatemers 16, each of the concatemers 16 having different expression sequences 12 as compared to each other (i.e., nucleic acids having different sequences). The concatemer mixture may be formed from a first subset of a plurality of concatemers 16 of a first type (having tandem repeats of a first nucleic acid sequence) and a second subset of a plurality of concatemers 16 of a second type (having tandem repeats of a second nucleic acid sequence different from the first nucleic acid sequence), whereby it is to be understood that the mixture may include concatemers 16 of the first subset and the second subset. Further, in embodiments, the concatemer mixture may include a third subset of concatemers 16 of a third type (having tandem repeats of a third nucleic acid sequence different from the first and second nucleic acid sequences), and others. As described above, within each of the individual subsets, the size (base length) of the concatemers 16 and the number of their corresponding tandem repeats may vary.

[0040] Figure 6 is a schematic diagram of various runs of techniques for generating expression products according to embodiments of the present disclosure. In some embodiments, it may be desirable to generate a plurality of expression products to form a complex or interact with each other to ultimately form a desired final product. In an expression workflow, such expression products may be divided into various expression media (various concatemers 16 or in some embodiments, a mixture of one or more concatemers 16 and one or more plasmids, such as plasmid 80 or plasmid 82) depending on the type of expression product or the desired customization or diversity of the expression product.

[0041] For example, a particular type of expression product may include a customizable nucleic acid expression product 86. The customizable nucleic acid expression product 86 can be generated by using a template 14 having a customized or desired expression sequence. It can be difficult to predict a particular nucleic acid expression product 86 that may be of interest to an end user. Thus, rather than incorporating a nucleic acid sequence for the variable nucleic acid expression product 86 into an expression sequence 12 that includes other more constant or invariant desired end products, in the kits or expression product generation systems 87 provided herein, the customizable or more variable regions can be separated into separate templates 14 and concatemers 16. In this way, a more universal expression product 88 can be provided as separate templates 14 that are part of an assembly kit that incorporates expression sequences 12 for pre-generated or customizable expression products 86 into separate concatemers 16 or plasmids 82. In another embodiment, DNA concatemers (amplified from plasmids or minicircle templates) can be selected for variable or customizable components for each application, while plasmids can be selected for more constant or predictable components that do not vary by application. Examples of such separate constructs are considered below in view of FIGS. 7-10.

[0042] Lentivirus FIGS. 7-8 are schematic diagrams of a lentiviral vector generation system. The generation of lentiviral vectors can be useful for various gene therapy applications, including during the generation of CAR-T therapy cells. In particular, lentiviral vectors generated using the techniques disclosed herein can be used in the generation of CAR-T therapy cells. In one embodiment, the lentiviral transgene of a lentiviral vector generated by the disclosed techniques can encode a chimeric antigen receptor.

[0043] Lentiviruses have a linear single-stranded RNA (ssRNA) genome. In the embodiments depicted for the generation of second-generation lentiviral vectors, an ENV protein (e.g., VSV-G) may be encoded by the expression sequence of a first concatemer, and the desired transgene sequence adjacent to the long terminal repeat (LTR) sequence may be part of the expression sequence of a second concatemer, and the packaging proteins may be present on a third concatemer having open reading frames encoding the GAG, POL, REV, and TAT genes (Figure 7). In another arrangement for third-generation lentivirus generation (Figure 8), the packaging proteins are further split into two packaging concatemers having GAG and POL on one concatemer and REV on the other concatemer.

[0044] According to the present disclosure, efficient generation of functional lentiviruses was achieved by making and transfecting a mixture of RCA DNA (encoding VSV-G envelope, GAG / POL, REV, and GFP mRNA separately) in HEK293T cells in stoichiometric ratios. Plasmids encoding GAG / POL (pLP1, Invitrogen), REV (pLP2, Invitrogen), VSV-G envelope (pLP / VSVG, Invitrogen), and packaging GFP mRNA (pLenti-GFP, Cell Biolabs) were amplified by RCA to generate high molecular weight super-branched concatemers containing tandem repeats of each plasmid to evaluate good viral vector generation. Viral vectors can be generated from live cells or by cell-free expression.

[0045] According to the present disclosure, a concatemer mixture is made that contains multiple nucleic acid concatemers in defined ratios between each of the multiple nucleic acid concatemers. The concatemer mixture is co-expressed to generate multiple expression products each arising from a respective nucleic acid concatemer, where the ratio between each of the multiple expression products is proportional to the defined ratio between each respective nucleic acid concatemer in the concatemer mixture.

[0046] The disclosed embodiments relate to multiple concatemers used in the production of lentiviral vectors, although in other embodiments, mixtures of concatemers and plasmids are also contemplated. In one embodiment, only the transgene is expressed using a concatemer, while plasmid constructs encoding other components are co-transfected into a co-expression system together with the transgene concatemer. In another embodiment, the desired transgene and ENV protein are co-expressed from a mixture of two different concatemers, while a plasmid encoding other components is co-transfected into a co-expression system together with the concatemer mixture. The technology can include co-transfection of one or more concatemers, including lentiviral transgenes and any plasmid or concatemer having an open reading frame for other components of the viral vector (one or more packaging concatemers or plasmids, and one envelope concatemer or plasmid), into an expression system, such as HEK293T producer cells or A293T cells. Co-expression can be performed either cell-based or cell-free.

[0047] In certain embodiments, individual RCA products were mixed at a molar ratio of 1:1:1:1 and transfected into HEK293T cells. In certain embodiments, one or more RCA DNA concatemers can be replaced by plasmid DNA. Various ratios of RCA DNA to plasmid DNA can be used, for example, the molar ratios of pLenti-GFP:LP1:LP2:LP3 are 1:3:3:3 and 3:1:1:1, respectively. In either case, the concatemer RCA DNA worked efficiently with the plasmid DNA, resulting in the production of an equivalent amount of lentivirus.

[0048] Adeno-associated virus (AAV) Wild-type AAV has a linear single-stranded DNA (ssDNA) genome of approximately 4.7 kilobases (kb) with two inverted terminal repeats (ITRs) 145 nucleotides in length at its termini. The ITRs flank two viral genomes, Rep (replication) and Cap (capsid), which encode non-structural and structural proteins, respectively. The Rep gene encodes four regulatory proteins, Rep78, Rep68, Rep52, and Rep40. Such proteins are involved in the replication of the AAV genome. The Cap gene encodes three capsid proteins, VP1 (viral protein 1), VP2, and VP3. Among the AAV serotypes, AAV2 is the most widely used for gene delivery in vitro and in vivo.

[0049] The AAV ITR contains all the cis-acting elements involved in genome rescue, replication, and packaging and is separated from the trans-acting viral coding regions, i.e., the Rep and Cap gene regions. In the design of recombinant AAV (rAAV) vectors, the cis-acting viral DNA elements (e.g., ITRs) can be ligated to the sequence of interest, while the Rep and Cap gene regions can be placed in trans. Typically, rAAV particles are generated by transfecting producer cells with a plasmid (AAV cis plasmid) containing a clone of the recombinant AAV genome consisting of the DNA of interest flanked by AAV ITRs, as well as a separate plasmid that expresses the viral Rep and Cap genes in trans. Adenoviral helper factors, such as E1A, E1B, E2A, E4ORF6, and VA RNA, can be provided by either adenoviral infection or transfection of a third plasmid that provides such adenoviral helper factors into the producer cells. When HEK293 cells are used as AAV producer cells, the helper factors include E2A, E4ORF6, and VA RNA since HEK293 cells already contain the E1A / E1b genes.

[0050] One technique for the production of recombinant AAV, particularly recombinant AAV2, relies on the infection of a cell line of wild-type adenovirus containing the AAV Rep / Cap genes therein, as well as on AAV vector DNA. Another method, the helper-free method, is based on transient transfection without using adenovirus of all the elements required for AAV production in host cells, such as HEK293 cells. This involves co-transfecting AAV-producing cells with three plasmids: (1) an AAV transfer plasmid that places the gene of interest (e.g., the “transgene”) between two ITRs, (2) a plasmid carrying the AAV Rep-Cap genes, and (3) a helper plasmid that provides helper genes isolated from adenovirus. Such genes (E4, E2a, and VA) mediate the replication of AAV. The transfer plasmid, Rep / Cap, and helper plasmid are transfected into a host cell containing the E1A / E1b genes, such as HEK293 cells, to generate infectious AAV particles.

[0051] The method of using a wild-type adenovirus-induced AAV-producing cell line can expand the scale of culture and generate very high-titer AAV vectors, but it is a very difficult task to completely remove adenovirus from the AAV product, and the contamination of wild-type adenovirus is highly undesirable in terms of the safety and specificity of the vector. On the other hand, a high-titer AAV vector free of adenovirus is generated by the transient transfection method, but this method is very labor-intensive and expensive.

[0052] In addition, in the recombinant co-expression of AAV in single cells from separate plasmids by transfection procedures, expression has been reported to occur at a wide variety of ratios. The AAVcap gene encodes three structural proteins (VP1, VP2, and VP3) in a stoichiometry of approximately 1:1:10. To achieve this desirable stoichiometry, recombinant methods for generating AAV capsid proteins generally utilize complex promoter induction and / or combinations of low and high copy number plasmids to achieve, for example, a 1:1:10 expression ratio of VP1, VP2, and VP3 in yeast-based expression.

[0053] According to the present disclosure, stoichiometric expression of VP1, VP2, and VP3 capsid proteins has been demonstrated using one or more concatemers. Expression can be performed either cell-based or cell-free. In certain embodiments, mixing and supplementation of nucleic acid concatemers can be performed at a 1:1:10 ratio in a cell-free protein expression reaction.

[0054] According to the present disclosure, a concatemer mixture is made that comprises a plurality of nucleic acid concatemers at a defined ratio between each of the plurality of nucleic acid concatemers. The concatemer mixture is co-expressed to generate a plurality of expression products respectively resulting from each nucleic acid concatemer, wherein the ratio between each of the plurality of expression products is proportional to the defined ratio between each of the respective nucleic acid concatemers in the concatemer mixture.

[0055] In certain embodiments, each of the plurality of nucleic acid concatemers encodes, in addition to the desired transgene, important replication and viral capsid factors separately. In certain embodiments, at least one of the nucleic acid concatemers comprises a minimal expression sequence. The minimal expression sequence can be designed in silico and synthesized in vitro (e.g., SEQ ID NOs #1 - #3 listed below). In another embodiment, the transgene (e.g., the transgene payload) is present on a separate concatemer derived from one or more REP / CAP concatemers. Further, another concatemer can include one or more helper sequences depending on the desired expression system. The disclosed embodiments relate to a plurality of concatemers used for the production of AAV vectors, but in other embodiments, mixtures of concatemers and plasmids are also contemplated. In one embodiment, only the transgene is expressed using a concatemer, while plasmid constructs encoding other components are co-transfected into a co-expression system with the transgene concatemer. In another embodiment, the desired transgene and capsid protein are co-expressed from a mixture of two different DNA concatemers, while plasmids encoding other components are co-transfected into a co-expression system with the concatemer mixture. The technology can include co-transfection of one or more concatemers, including any plasmid or concatemer having an open reading frame for an AAV transgene and other components of the viral vector.

[0056] CRISPR Figure 10 is a schematic diagram of a co-expression system that results in CRISPR / Cas9 genome editing performed by a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, and tracrRNA along with an optional section of a DNA repair template to be utilized in either non-homologous end joining (NHEJ) or homologous recombination repair (HDR).

[0057] In CRISPR / Cas9, plasmids are often used to transfect target cells. The main components of the plasmid include crRNA, tracrRNA, sgRNA, Cas9 protein, and a repair template. crRNA contains a guide RNA that locates the correct section of the host DNA, along with a region that binds to tracrRNA. tracrRNA binds to crRNA to form an active complex. Single-guide RNA (sgRNA) is a complex RNA consisting of tracrRNA and at least one crRNA. Multiple crRNAs and tracrRNAs can be packaged together to form single-guide RNA (sgRNA). This sgRNA can be combined with the Cas9 gene to form a plasmid, which can then be transfected into cells. When these are constructed into a plasmid and transfected into cells, the Cas9 protein, with the help of crRNA, finds the correct sequence in the DNA of the host cell and, depending on the Cas9 variant, causes a single-stranded or double-stranded break in the DNA.

[0058] In addition, this technology is used to generate or produce a target CRISPR / Cas9 system using one or more concatemers 16 that replace conventional plasmid-based technologies. The first and second nucleic acid concatemers may each contain a first and a second expression sequence. One of the first and second expression sequences includes an open reading frame (ORF) encoding a protein of interest (e.g., Cas9) and a promoter operably linked to the open reading frame. The other expression sequence may include an open reading frame (ORF) encoding crRNA, tracrRNA, sgRNA, or a repair template.

[0059] antibody Immunoglobulins or antibodies characteristically contain heavy and light chains in stoichiometric amounts. For example, immunoglobulin G (IgG) contains two identical heavy chains and two identical light chains that are linked by disulfide bonds. Conventional recombinant methods for generating immunoglobulins may require complex design of a single vector expression cassette containing DNA, RNA, or protein sequence elements (e.g., various promoters, internal ribosome entry sites, post-translational cleavage sites, or polyadenylation signals of various strengths) to achieve the desired expression ratios of the heavy and light chains of the antibody, for example, in CHO cells. The expression ratio of the light chain to the heavy chain polypeptide is recognized as an important parameter for the recombinant yield of the desired antibody and for limiting undesirable aggregates and fragments.

[0060] Also, the present technology can be used to produce or manufacture antibodies that result in an expression ratio of the light chain to the heavy chain polypeptide in a more rational and robust manner by using a predetermined ratio of the first and second nucleic acid concatemers and then obtaining the desired ratio of the light chain to the heavy chain polypeptide. In one embodiment, the first and second nucleic acid concatemers each contain a first and a second expression sequence, and each expression sequence may contain an open reading frame (ORF) and a promoter operably linked to the open reading frame. In certain embodiments, the ORFs of the first and second expression sequences may each encode the heavy and light chains of an antibody, respectively. In certain embodiments, one of the expression sequences may contain one promoter operably linked to two or more ORFs. For example, one of the expression sequences may contain a promoter functionally linked to two different ORFs, where one ORF encodes the heavy chain of an antibody and the other ORF may encode the light chain. In another embodiment, the concatemer mixture may be made to express a bispecific antibody in which the first concatemer encodes a first expression sequence containing the light and heavy chain domains of a first antibody, while the second concatemer encodes a second expression sequence containing the light and heavy chain domains of a second antibody.

[0061] Virus-like particles Furthermore, the present technology can be used to generate or produce virus-like particles. Non-limiting examples of virus-like particles include HPV and Gardasil (quadrivalent human papillomavirus types 6, 11, 16, and 18) vaccine. HPV encodes two capsid proteins, L1 and L2. The major capsid protein L1 can spontaneously assemble to construct an icosahedral structure composed of 72 pentamers that closely resembles natural virions. The non-major capsid protein L2 is not required for capsid formation, but L2 can be present at an average of about 36 molecules per capsid (the estimated L1:L2 ratio is about 9:1 to 11:1). Gardasil (quadrivalent human papillomavirus types 6, 11, 16, and 18) vaccine is a recombinant VLP formulation of four different L1 capsomers each expressed individually from baker's yeast.

[0062] The first and second nucleic acid concatemers may each contain a first and a second expression sequence. The first and second expression sequences may each contain open reading frames (ORFs) encoding a first and a second protein of interest, such as the capsid proteins L1 and L2. Each of the expression sequences may further contain a promoter operably linked to the corresponding open reading frame. In certain embodiments, one of the expression sequences may contain one promoter operably linked to two or more ORFs. For example, one of the expression sequences may contain a promoter functionally linked to two different ORFs, where one ORF encodes the capsid protein L1 and the other ORF may encode the capsid protein L2.

Example

[0063] Unless otherwise specified, the components described in the examples are those commercially available from general chemical suppliers. Some abbreviations used in the examples section are described in more detail as follows: "mg": milligram, "ng": nanogram, "pg": picogram, "fg": femtogram, "mL": milliliter, "mg / mL": milligrams per milliliter, "mM": millimolar concentration, "mmol": millimole, "pM": picomolar concentration, "pmol": picomole, "μL": microliter, "min.": minute, and "h.": hour.

[0064] (Example 1) Stoichiometric expression of adeno-associated virus (AAV) capsid proteins from a 1:1:10 RCA mixture As shown in Figure 11, stoichiometric expression of VP1, VP2, and VP3 capsid proteins was demonstrated by mixing and supplementing RCA DNA in a 1:1:10 ratio in a cell-free protein expression reaction. Minimal expression sequences encoding VP1, VP2, and VP3 separately were designed in silico and synthesized in vitro (SEQ ID NOs #1-#3).

[0065] SEQ ID NO: 1

[0066] Array number 2

[0067] Array number 3

[0068] Each expression array contains a T7 promoter and a T7 gene 10 leader ribosome binding site for initiating cell-free mRNA transcription and protein translation, respectively. Double-stranded DNA encoding the minimal expression array was digested with BamHI and BglII to generate complementary overhangs, and after generating DNA minicircles by intramolecular ligation, exonucleolytic treatment (ExoI, ExoIII) was performed to digest any remaining non-circular DNA. Subsequently, rolling circle amplification (RCA) was used to generate high molecular weight, hyperbranched concatemers consisting essentially of tandem repeats of each minimal expression array. When provided herein, RCA may be performed as disclosed in U.S. Patent No. 10,077,459 and U.S. Patent No. 9,938,568, the disclosures of which are incorporated herein by reference for all purposes. RCA provides the advantages of an easy, economical, and robust option for performing in vitro transcription and translation-coupled reactions. RCA reagents containing water, reaction buffer, 40 μM primer, and 20 ng / μL phi29 DNA polymerase were pre-treated to remove contaminating DNA, and then ligated minicircle template and 400 μM dNTP were added. Amplification was performed using a hexamer primer having the sequence +N+N(atN)(atN)(atN)*N(AT hexamer), and 10 μM alphaS-dATP was included in the reaction to thioate the resulting RCA product. RCA products were quantified from 100 μL of the total RCA reaction volume using the Quant-It™ Picogreen® dsDNA Assay Kit (ThermoFisher Inc.) and then applied directly to the cell-free protein expression reaction without intermediate purification. To achieve stoichiometric expression of VP1, VP2, and VP3, individual RCA products were mixed in a mass ratio of 1:1:10 in Expressway™ extract (ThermoFisher Inc.) to give a total DNA content of 0.5 μg per 50 μL reaction. For control purposes, VP1, VP2, and VP3 (approximately 82 kD, 66 kD, and 60 kD, respectively) were expressed in separate cell-free reactions by adding 0.5 μg of their respective RCA DNA to 50 μL of ExpressWay reaction.FluoroTect(TM) GreenLYS in vitro Translation Label (Promega) was added to all cell-free expression reactions (via anticodon UUU tRNA) for randomly labeled nascent lysine residues with fluorescent BODIPY-FL label. All cell-free expression reactions were incubated at 30 °C for 6 h in an Eppendorf ThermoMixer (1200 rpm) and then analyzed by SDS-PAGE to visualize all BODIPY-labeled translation products using a Typhoon Variable Mode Imager (GE Healthcare) by fluorescence in-gel. The data presented in Figure 1 demonstrate that the stoichiometric expression of VP1, VP2, and VP3 capsid proteins was produced as made with a ratio of 1:1:10 of RCA DNA supplemented to the cell-free protein expression reaction. In certain embodiments, an RCA product encoding an assembly activation protein (AAP) and expressed at a ratio of 1:1:10 (relative to VP1, VP2, VP3) can be used to enhance the assembly and folding of the viral capsid. Alternatively, an RCA mixture encoding VP3 and AAP may be sufficient to cause stoichiometric expression of virus-like particles in vitro.

[0069] (Example 2) Stoichiometric expression of immunoglobulin chains from a 1:2 RCA mixture Stoichiometric expression of the heavy and light chains of immunoglobulin was demonstrated using two different RCA nucleic acid concatemer products. Minimal expression sequences encoding the IgG heavy and light chains separately were designed in silico and synthesized in vitro (SEQ ID NOs #4-#5).

[0070] SEQ ID NO:4

[0071] Array number 5

[0072] The expression array contained a T7 promoter and an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV) for initiating cell-free mRNA transcription and protein translation, respectively. The double-stranded DNA encoding the minimal expression array was digested with BamHI and BglII to generate complementary overhangs, and DNA minicircles were generated by intramolecular ligation. After that, exonucleolytic treatment (ExoI, ExoIII) was performed to digest any remaining circular DNA. Subsequently, rolling circle amplification (RCA) was used to generate high-molecular-weight, hyperbranched concatemers consisting essentially of tandem repeats of each minimal expression array. RCA and quantification were performed as disclosed in Example 1. The RCA products were quantified from 100 μL of the total RCA reaction volume using the Quant-It™ Picogreen® dsDNA Assay Kit (ThermoFisher Inc.) and then purified by ethanol precipitation. To achieve stoichiometric expression, the individual RCA products encoding the light and heavy chains were mixed at a mass ratio of 1:2 in 1-Step Human Coupled IVT Extract (ThermoFisher Inc.) such that the total DNA content was 125 ng per 25 μL reaction. For control purposes, the individual light and heavy chains (approximately 26 kDa and 52 kDa, respectively) were expressed in separate cell-free reactions by adding 125 ng of the respective RCA DNA to 25 μL of the 1-Step reaction. FluoroTect™ GreenLYS in vitro Translation Label (Promega) was added to all cell-free expression reactions for (via anticodon UUU tRNA) random labeling of nascent lysine residues with fluorescent BODIPY-FL. The data presented in FIG. 12 demonstrate stoichiometric expression of the light and heavy chain polypeptides as defined by the ratio of RCA DNA supplemented in the cell-free protein expression reaction. The cell-free protein expression reaction may be further expanded by increasing the microsomal content of the extract that promotes signal peptide processing and disulfide bond formation of the final IgG molecule.

[0073] (Example 3) Preparation of RCA mixture for functional AAV virus production The production of recombinant AAV generally involves the co - transfection of three different plasmids (Rep / Cap, helper, and packaged DNA transgene) into HEK293T producer cells. The performance of transient transfection is used when manufacturing viral vectors for clinical trials because the therapeutic virus can be produced rapidly, in high yield, and generally at high titers (without significant preliminary optimization) from adherent - dependent producer cells compared to suspension - compatible cells. However, the scale - up of AAV production is limited by the lead time and cost of plasmid DNA production. Here, we demonstrated the efficient generation of functional AAV viral vectors by preparing a mixture of RCA DNA (encoding Rep / Cap, helper, and GFP transgene) at a ratio of 1:1:1 and transfecting HEK293T cells. Plasmids encoding Rep / Cap (pAAV - RC6, Agilent Genomics), helper (pHelper, Agilent Genomics), and packaged DNA transgene (pscAAV - GFP, Cell Biolabs) were amplified using RCA and carried out and quantified as disclosed herein to generate high - molecular - weight super - branched concatemers containing tandem repeats of each plasmid. To generate AAV particles, individual RCA products were mixed at a mass or molar ratio of 1:1:1, and 4 μg of total DNA was transfected into HEK293T cells using DharmaFECT kB reagent. For control purposes, 4 μg of plasmid DNA or linearized digests of RCA DNA (after ScaI endonuclease cleavage of concatemers into double - stranded monomers) were transfected into HEK293T cells at the same ratio of 1:1:1. The DNA mixture was pre - incubated with DharmaFECT for 10 minutes at room temperature and then incubated with HEK293T cells for about 20 hours, after which fresh medium was given to the transfected cells. Three days after transfection, the cells were harvested and washed, and then AAV crude lysates were collected by performing 4 cycles of freeze - thaw (by warming in a 37 °C water bath after a dry ice / ethanol bath at about - 72 °C).Cell pellets were removed by centrifugation (10,000 g × 10 min), and AAV crude lysates at two different final concentrations (18% and 2%) were incubated with naive HEK293T cells in fresh medium. Three days after transduction, GFP-positive cells were quantified by flow cytometry relative to appropriate controls for proper gating. The data summarized in Figure 13 demonstrate equivalent AAV production from intact RCA concatemers compared to plasmid DNA. Using both concatemeric and digested forms, less virus was produced by an RCA DNA mass ratio of 1:1:1 compared to an RCA DNA molar ratio of 1:1:1. Ratio metric comparison revealed that at a DNA mass ratio of 1:1:1, the relative amount of the longest coding DNA in the DNA mixture decreased, suggesting that virus production may be limited by pHelper DNA. In subsequent experiments, individual RCA DNAs were mixed with individual plasmids, and a molar concentration combination of 1:1:1 was evaluated by transfection. In all cases, as shown in Figure 14, concatemeric RCA DNA worked efficiently with plasmid DNA to produce equivalent amounts of AAV virus.

[0074] (Example 4) Preparation of RCA Mixtures for Functional Lentivirus Production By preparing a mixture of RCA DNA (encoding VSV-G envelope, Gag / Pol, Rev, and GFP mRNA separately) in stoichiometric ratios and transfecting HEK293T cells, efficient generation of functional lentiviruses was demonstrated. Plasmids encoding Gag / Pol (pLP1, Invitrogen), Rev (pLP2, Invitrogen), VSV-G envelope (pLP3, Invitrogen), and packaging GFP mRNA (pLenti-GFP, Cell Biolabs) were amplified using RCA, carried out and quantified as disclosed herein to generate high-molecular-weight, super-branched concatemers containing tandem repeats of each plasmid. Amplification was performed using a hexamer primer with the sequence +N+N(atN)(atN)(atN)*N (AT hexamer), and 10 μM alphaS-dATP was optionally included in the pLenti-GFP reaction that thioates the resulting RCA product. To generate lentiviruses, individual RCA products were mixed at a molar ratio of 1:1:1:1, and approximately 3 μg of total DNA was transfected into HEK293T cells using DharmaFECT kB reagent. For control purposes, approximately 3 μg of circular plasmid or linearized digest of the plasmid (after digestion with ScaI or PvuI endonucleases) was transfected into HEK293T cells at the same ratio of 1:1:1:1. The DNA mixture was pre-incubated with DharmaFECT for 10 minutes at room temperature and then incubated with HEK293T cells for approximately 20 hours, after which it was replaced with fresh medium. Three days after transfection, the used medium (containing lentiviruses) was collected, filtered through a 0.2 μm syringe filter, and naive HEK293T cells were transduced in the presence of 5 μg / mL polybrene. Approximately 24 hours after virus transduction, the cells were given fresh medium and incubated for an additional 2 - 3 days. The cells were then harvested (3 - 4 days after transduction), and GFP-positive cells were quantified by flow cytometry relative to appropriate gating controls. The data summarized in Figure 15 demonstrate non-optimized lentivirus generation from intact RCA concatemers.A specific stoichiometric mixture of RCA products or plasmids is an example, and lentiviruses can also be generated using alternative stoichiometric mixtures. For example, mixtures with a high ratio of plasmid DNA to RCA DNA were tested, particularly the molar concentration combinations of pLenti-GFP:LP1:LP2:LP3 at 1:3:3:3 and 3:1:1:1, respectively. In both cases, as shown in Figure 15, concatemeric RCA DNA worked efficiently with plasmid DNA, thereby generating an equivalent amount of lentivirus.

[0075] Concatemer generation and expression products of concatemers Techniques are provided herein for generating desired expression products that may contain one or more proteins or nucleic acids using a concatemer (e.g., concatemer 16). The generated expression products can be harvested (e.g., purified, recovered) after expression and used as part of a treatment or therapy protocol for a subject. Embodiments of the present disclosure include the step of generating one or more expression products from the concatemers provided herein and the step of treating a subject using the generated expression products. For example, the disclosed techniques can be used to generate viral vectors encoding a desired transgene. Thus, the expression products provided herein may be part of a viral vector comprising a nucleic acid having an expression sequence encoding a desired transgene, and moreover, may be used as part of a gene therapy protocol for treating a subject with the viral vector. Since the transgene is selected based on the subject's need for therapy, the transgene may be a variable or customizable component of the viral vector generation system, while other components of the viral vector generation system are either constant or not very variable. Additionally, specific components of the viral vector can be selected based on the desired tropism or patient characteristics. Accordingly, kits are provided herein that include a pre-made or pre-generated portion of the viral vector generation system that is either constant or selected from a small number of possible options as well as reagents and precursor molecules, enabling the user to generate a customizable portion of the system.

[0076] In another embodiment, concatenamer expression products can be used to generate custom antibodies. In another embodiment, concatenamer expression products can be used to generate CRISPR / Cas9 systems. In another embodiment, concatenamer expression products can be used to generate virus-like particles. Specific embodiments are disclosed that include gene editing, gene therapy, and the expression of products related to custom molecules, but it should be understood that the disclosed techniques can be used to generate expression products suitable for other applications. Such things include multi-subunit protein complexes (e.g., human DNA polymerase alpha is a complex of four different subunits in a ratio of 1:1:1:1) and combinations of enzymes capable of performing various steps of biochemical pathways.

[0077] Throughout the specification, examples of specific terms should be considered as non-limiting examples. The singular forms of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Approximating language, as used in this invention throughout the specification and claims, may apply to modify any quantitative expression that can vary within a range without causing a change in the relevant basic function.

[0078] In the present disclosure, nucleic acid amplification techniques can be used as part of a workflow to generate expression products. As used herein, the term "primer" refers to a short, linear oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to stimulate a nucleic acid synthesis reaction. The primer can be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. The primer can include natural, synthetic, or modified nucleotides. Both the upper and lower limits of the primer length are determined empirically. The lower limit of the primer length is the minimum length required to form a stable double-strand upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 nucleotide lengths) do not form a thermodynamically stable double-strand with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of forming a double-strand in regions other than a given nucleic acid sequence of the target nucleic acid. Generally, a suitable primer length ranges from about 3 nucleotide lengths to about 40 nucleotide lengths.

[0079] As used herein, the term "random primer" refers to a mixture of primer sequences generated by randomizing the nucleotides at any given position in an oligonucleotide sequence such that a given position can consist of either a possible nucleotide or an analog thereof (fully randomized). Thus, a random primer is a random mixture of oligonucleotide sequences consisting of all possible combinations of nucleotides within the sequence. For example, a hexamer random primer can be represented by the sequence NNNNNN or (N)6. A hexamer random DNA primer consists of all possible combinations of hexamers of the four DNA nucleotides A, C, G, and T, 4 6A random mixture is generated that contains (4,096) species of unique hexamer DNA oligonucleotide sequences. Random primers can be used efficiently to stimulate nucleic acid synthesis reactions when the target nucleic acid sequence is unknown or for performing whole genome amplification reactions. Also, random primers can be effective in stimulating and generating double-stranded RCA products rather than single-stranded RCA products, depending on the concentration of the primer.

[0080] As used herein, the term "nucleotide analog" refers to a compound that is structurally similar to a naturally occurring nucleotide. A nucleotide analog can have an altered phosphate backbone, sugar moiety, nucleobase, or a combination thereof. A nucleotide analog can be a natural nucleotide, a synthetic nucleotide, a modified nucleotide, or an alternative substituent (e.g., inosine). Generally, nucleotide analogs with altered nucleobases confer, inter alia, various base pairing and base stacking properties. As used herein, the term "LNA (locked nucleic acid) nucleotide" refers to a nucleotide analog, where the sugar moiety of the nucleotide contains a bicyclic furanose unit that is locked in a ribonucleic acid (RNA) mimetic sugar conformation. The structural change from a deoxyribonucleotide (or ribonucleotide) to an LNA nucleotide, i.e., the introduction of an additional bond between carbon atoms at the 2'- and 4'-positions (e.g., 2'-C, 4'-C-oxymethylene bond, see, e.g., Singh, S. K. et al., Chem. Comm., 4, 455 - 456, 1998 or Koshkin, A. A. et al., Tetrahedron, 54, 3607 - 3630, 1998) is restricted from a chemical perspective. The 2'- and 4'-positions of the furanose unit in an LNA nucleotide can be linked by, for example, O-methylen (e.g., oxy-LNA: 2'-O,4'-C-methylen-β-D-ribofuranosyl nucleotide), S-methylen (thio-LNA), or NH-methylen moieties (amino-LNA). Such linkages restrict the conformational freedom of the furanose ring. LNA oligonucleotides exhibit enhanced hybridization affinity for complementary single-stranded RNA and complementary single- or double-stranded DNA. LNA oligonucleotides can induce an A-type (RNA-like) double-stranded conformation. Nucleotide analogs with an altered phosphate-sugar backbone (e.g., PNA, LNA) often modify, inter alia, strand properties such as secondary structure formation. An asterisk (*) preceding a letter designation means that the nucleotide represented by the letter is a phosphorothioate-modified nucleotide. For example, *N represents a phosphorothioate-modified random nucleotide.A plus (+) sign preceding a text representation means that the nucleotide represented by the text is an LNA nucleotide. For example, +A represents an adenosine LNA nucleotide and +N represents a locked random nucleotide (i.e., a random LNA nucleotide). As used herein, the terms "phosphorothioated nucleotide" or "thioated nucleotide" refer to a nucleotide in which the phosphate backbone is modified, where the sugar moiety is linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, a phosphorothioate bond contains a sulfur atom in place of a non-bridging oxygen atom. This modification can render the nucleotide-nucleotide bond resistant to nuclease degradation. Thioated nucleotides (thioated dNTPs) can include, but are not limited to, α-S-dGTP, α-S-dCTP, α-S-dATP or α-S-dTTP.

[0081] As used herein, the term "rolling circle amplification (RCA)" refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., single / double-stranded DNA circle) by a rolling circle mechanism. The rolling circle amplification reaction is initiated by hybridization of a primer to a nucleic acid template that is often circular and single-stranded. Subsequently, a nucleic acid polymerase continuously proceeds around the circular nucleic acid template to repeatedly replicate the sequence of the nucleic acid template many times (rolling circle mechanism), thereby extending the primer hybridized to the circular nucleic acid template. The rolling circle amplification method typically generates concatemers containing tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification method can be linear RCA (LRCA) showing a linear amplification kinetics (e.g., RCA using a single-stranded specific primer), or exponential RCA (ERCA) showing an exponential amplification kinetics. Also, the rolling circle amplification method can be performed using multiple primers (rolling circle amplification method with composite stimulation or MPRCA), resulting in hyperbranched concatemers. For example, in double-primed RCA, one primer can be complementary to the circular nucleic acid template like linear RCA, while the other can be complementary to the tandem repeat unit nucleic acid sequence of the RCA product. Thus, double-primed RCA can proceed as a chain reaction having an exponential amplification kinetics that characterizes a series of multiple hybridizations, primer extensions, and cascades in a strand displacement phenomenon involving both of these primers and both strands. This often results in the generation of separate sets of double-stranded nucleic acid amplification products of the concatemer. RCA can be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase, e.g., Phi29 DNA polymerase. Suitable polymerases have strand displacement DNA synthesis ability. In certain embodiments, the rolling circle amplification method can be performed using a random primer mixture containing nucleotide analogs.

[0082] As used herein, the terms "rolling circle amplification (RCA) product" or "RCA-generated DNA" refer to nucleic acid amplification products, where circular nucleic acid templates (e.g., single / double-stranded DNA circles) are amplified by a rolling circle amplification reaction mechanism. The template is of a smaller size compared to the RCA product. Typically, a concatemer containing tandem repeat units of the circular nucleic acid template sequence is generated by the rolling circle amplification method. RCA-generated DNA can be generated by linear RCA (LRCA) showing a linear amplification kinetics (e.g., RCA using a single-stranded specific primer), or by exponential RCA (ERCA) showing an exponential amplification kinetics. Also, RCA-generated DNA may be generated by using multiple primers (multiple primer rolling circle amplification method or MPRCA), where the RCA-generated DNA is a hyper-branched concatemer. In double primer RCA, one primer may be complementary to the circular nucleic acid template as in linear RCA, while the other may be complementary to the tandem repeat unit nucleic acid sequence of the RCA-generated DNA. RCA-generated DNA can be generated by in vitro RCA under isothermal conditions using a suitable nucleic acid polymerase, e.g., Phi29 DNA polymerase.

[0083] DNA amplification techniques, such as the rolling circle amplification method (RCA), can be utilized to generate large amounts of high-quality DNA starting from circular nucleic acid templates. By the rolling circle amplification method, a nucleic acid concatemer containing tandem repeat units of the corresponding circular nucleic acid template can be generated. The nucleic acid concatemer can be a linear or branched concatemer.

[0084] As used herein, the term "nucleic acid concatemer" (e.g., concatemer 16 disclosed herein) refers to a nucleic acid molecule having tandem repeats or tandem repeat units of a nucleic acid sequence (e.g., expression sequence 12). The terms "concatemer", "nucleic acid concatemer" and "DNA concatemer" may be used interchangeably throughout the present disclosure. The concatemer can be single-stranded or double-stranded. As used herein, the term "double-stranded concatemer DNA" refers to a double-stranded DNA molecule containing multiple copies of the same DNA sequence joined in series. Concatemers generated by RCA can be larger than 1 kilobase (kb), larger than 10 kb, and larger than 150 kb. In one embodiment, the concatemer can be in the range of 50 kb to 150 kb. The size of the concatemer is related to the size of the starting template (e.g., template 14) and the number of tandem repeats, which can vary. Thus, RCA performed on a template solution can generate a pool of concatemers having tandem repeats of the same sequence but a variable number of tandem repeats and thus variable lengths. In certain embodiments, the nucleic acid concatemer may contain multiple tandem repeat sequences, where each of the multiple tandem repeat sequences contains an expression sequence encoding an expression product.

[0085] In certain embodiments, the nucleic acid concatemer is a DNA concatemer. The DNA concatemer may be generated using a DNA minicircle as a template, where the DNA minicircle consists essentially of a minimal expression sequence. The resulting concatemer contains tandem repeats of the minimal expression sequence derived from the DNA minicircle. In certain embodiments, the minimal expression sequence consists essentially of a promoter, a cap-independent translation element, and an open reading frame. In certain embodiments, the nucleic acid concatemer contains modified nucleotides, nucleotide analogs, or combinations thereof.

[0086] As used herein, the term "expression sequence" or "repeat unit of an expression sequence" (e.g., expression sequence 12) refers to a DNA sequence having the ability to express RNA and / or protein. Thus, the expression product includes proteins, RNAs, or mixtures thereof.

[0087] In certain embodiments where protein expression is sought, the expression sequence may include an expression competent unit that includes an open reading frame (ORF) and a promoter operably linked to the open reading frame. In one embodiment, the ORF may encode one or more proteins. The encoded proteins may be the same or different. In some embodiments, the expression sequence may include one promoter operably linked to two or more ORFs.

[0088] In certain embodiments where RNA expression is sought, the expression sequence may include an RNA expression competent unit that includes at least one promoter and a transcription termination sequence.

[0089] The repeating unit of the concatemer sequence may include a promoter, an open reading frame, a ribosome binding site, and a translation termination sequence. This may additionally include sequences that do not substantially affect in vitro transcription and / or translation of the RCA product. For example, this may further include sequences such as translation enhancing sequences, insulator sequences, or transcription termination sequences.

[0090] Numerous examples of suitable promoters are known in the art and include, for example, the T7 RNA polymerase promoter sequence or promoter sequences derived from viruses such as CMV or SV40.

[0091] Similarly, numerous examples of suitable ribosome binding sites are known in the art, including, for example, internal ribosome entry sites (IRES), polyA tracts, species-independent translation leaders (SITS), Kozak consensus sequences, and Shine-Dalgarno sequences. The efficiency of ribosome binding or translation initiation is generally enhanced by insulator sequences. Numerous examples of suitable insulator sequences exist in the art, including, for example, sequences encoding poly-histidine tracts. In some embodiments, the insulator sequence can be determined empirically by inserting a spacer sequence around the ribosome binding site or by optimizing or inserting codons at the N-terminus of the expressed protein. In certain embodiments, the expression sequence can include a polyA sequence, a transcription termination sequence, an insulator sequence, or a combination thereof.

[0092] In certain embodiments, the open reading frame of the expression sequence can include a codon optimization sequence, a purification tag sequence, an amino-terminal peptide fusion sequence derived from an IRES, a sequence for protease cleavage or nucleotide cleavage, or a combination thereof. In some embodiments, the expression sequence includes both coding and non-coding sequences.

[0093] The codon optimization sequence of the open reading frame can enhance the rate and quality of translation of the RCA product. By codon optimization, protein expression generally increases due to improved translation efficiency of the gene of interest. Also, the functionality of a gene can be improved by optimizing codon usage in a custom-designed gene. In codon optimization embodiments, low-frequency codons in a species can be replaced with high-frequency codons; for example, the low-frequency codon UUA can be replaced with the high-frequency codon CUG for leucine. Codon optimization improves mRNA stability, and thus the rate of protein translation or protein folding can be modified. Further, codon optimization can customize the regulation of transcription and translation, modify the ribosome binding site, or stabilize the mRNA degradation site.

[0094] The open reading frame of the expression array may include a purification tag sequence for the purification of the expressed product (e.g., the expressed protein). The tag sequence can be an affinity tag, a tag for protease cleavage, or a combination thereof. The affinity tag can be used for the rapid purification and detection of recombinant proteins.

[0095] The open reading frame of the expression array may include an amino-terminal peptide fusion sequence derived from an IRES for enhancing ribosome recognition.

[0096] In some embodiments, the expression array includes a coding sequence, where the coding sequence encodes or generates a desired protein expression product in a eukaryotic cell. The coding sequence is a nucleic acid sequence containing a specific target gene. Generally, the coding sequence includes a promoter and an open reading frame (ORF). Optionally, the coding sequence may include a cap-independent translation element (CITE). In some embodiments, the coding sequence further includes a ribosome binding site. The coding sequence may include a transcription termination sequence that is outside the open reading frame but located within the expression array.

[0097] In one or more embodiments, each of the plurality of tandem repeat arrays includes at least one expression array. In some embodiments, the at least one expression array includes at least one coding sequence. In such embodiments, the at least one coding sequence of the at least one expression array includes at least one promoter and at least one open reading frame. In some embodiments, each of the plurality of tandem repeat arrays includes two or more expression arrays. The two or more expression arrays including coding sequences can encode the same protein or different proteins. In some embodiments, the expression array includes at least one promoter that is operably linked to at least one open reading frame. For example, in one aspect, in the expression array, one promoter is operably linked to one open reading frame. In another aspect, in the expression array, one promoter is operably linked to two different open reading frames. In some embodiments, the expression array can include two or more promoters that are operably linked to two or more open reading frames.

[0098] The expression array may include a promoter operably linked to two different open reading frames, for example, a first open reading frame and a second open reading frame, each of which encodes a different protein. In this example, a single promoter is functionally linked to the two open reading frames via a cap-independent translation element. Each of the open reading frames includes a translation start and a translation termination sequence. A translation termination or stop sequence is required for the expression array, otherwise an infinite polyprotein could be synthesized, which is not desirable. However, a transcription termination codon may be optional for the first open reading frame that results in the production of a polycistronic mRNA during transcription. In such a case, an intervening sequence between the first and second open reading frames may be selected such that a single polycistronic mRNA can be translated into two different proteins when the protein is expressed in vivo. After the synthesis of the first protein by translation of the first open reading frame, ribosome slippage to the second translation start sequence of the second open reading frame may occur, and the synthesis of the second protein from the second open reading frame may be initiated. This can be achieved by incorporating a "self-cleaving sequence" between the first and second open reading frames. Suitable self-cleaving sequences, such as the viral P2A motif, facilitate the production of two or more proteins from one single mRNA.

[0099] In some embodiments, the expression array includes a non-coding array, where the desired RNA expression product is generated by the non-coding array. Such an expression array does not include any coding array. The non-coding array includes a promoter and a transcription termination sequence. The non-coding array generally lacks an open reading frame. Also, the expression array containing the non-coding array is called an RNA expression array. In some embodiments, the expression array consists essentially of a non-coding array. In some other embodiments, the expression array includes both coding and non-coding arrays, where the RNA can be generated from the non-coding array of the expression array. In such embodiments, the desired protein can also be subsequently generated from the coding array of the same expression array. In some embodiments, the generated RNA can be extracted from eukaryotic cells for various downstream applications. In one embodiment, the extracted RNA can be subsequently packaged into a lentiviral system and delivered to another cell. The non-coding array can include, but is not limited to, sequences for antisense RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), microRNA mimics, transfer RNA (tRNA), ribosomal RNA (rRNA), or combinations thereof. Also, the non-coding array can include CRISPR RNA (tracrRNA, crRNA, sgRNA, or gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), telomerase RNA, spliceosome RNA, enhancer RNA, retrotransposon, X-inactive specific transcript (Xist), RNA encoded by RNA polymerase I and RNA polymerase III, or combinations thereof.

[0100] As described above, both the coding and non-coding sequences include a promoter. For example, any suitable promoter in the art, including, for example, the T7 RNA polymerase or CMV promoter sequence, can be used in the methods described herein. Similarly, any suitable ribosome binding site in the art can be used, including, but not limited to, an IRES, a polyA tract, a species-dependent translational leader (SITS), a Kozak consensus sequence, and a Shine-Dalgarno sequence.

[0101] The open reading frame includes a translation start and a translation stop sequence. In some embodiments, the open reading frame includes a codon optimization sequence for translation enhancement. The open reading frame may include an amino-terminal peptide fusion sequence derived from an internal ribosome entry site (IRES) for enhanced ribosome recognition, a tag sequence for purification of the desired protein, or a combination thereof. The CITE may include an IRES, a translation enhancement element (TEE), or a combination thereof.

[0102] The desired protein may be purified by a tag array, where the tag array can be a fusion tag for affinity purification, a tag for protease cleavage, or a combination thereof. The fusion tag for affinity purification can be used for rapid purification and detection of the expressed protein. Also, such a tag is called an affinity tag. Affinity tags can include a polyhistidine tag, a glutathione S-transferase tag (GST), hemagglutinin (HA), myc (derived from the c-myc gene product), FLAG (consisting of eight amino acids Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys including an enterokinase cleavage site), or a combination thereof. The fusion tag is useful in the rapid purification and detection of the desired protein, but the tag may not be considered a permanent attachment or domain of the recombinant protein. Therefore, removal of the fusion tag is often required for high-level analytical studies of recombinant protein structure and function. The tag for purification can be removed from the protein by using another type of tag, for example, a protease cleavage tag. Protease cleavage tags can be used to cleave characteristic peptide bonds within specific protein or peptide sequences. Protease cleavage tags can include, for example, a PreScission (trademark) Protease tag (GE Healthcare Life Sciences) or a thrombin protease tag (GE Healthcare Life Sciences).

[0103] As described above, the open reading frame of the coding sequence may include a codon-optimized sequence, where the codon-optimized sequence is generated by considering various factors such as codon bias, context-dependent codon preference, and / or individual codon preference. The codon-optimized sequence of the open reading frame can enhance the translation speed and quality of the RCA product. By codon optimization, the translation efficiency of the gene of interest is improved, generally increasing protein expression from the coding sequence. Also, the functionality of the gene can be enhanced by optimizing codon usage in custom-designed genes. In codon-optimization embodiments, low-frequency codons in a species may be replaced with high-frequency codons; for example, the low-frequency codon UUA may be replaced with the high-frequency codon CUG for leucine. Codon optimization improves mRNA stability, and thus the rate of protein translation or protein folding can be modified. Further, codon optimization can customize transcriptional and translational regulation, modify ribosome binding sites, or stabilize mRNA degradation sites.

[0104] The transcription termination sequence is generally located at the 3' end of the gene in the DNA template. The transcription termination sequence provides a signal to initiate the process of releasing the mRNA from the transcription complex in the newly synthesized mRNA, which can also be useful for the efficient translation of the desired protein product. The insulator sequence generally enhances the efficiency of ribosome binding or translation initiation. A number of suitable insulator sequence examples existing in the art may be used, such as, for example, a sequence encoding a polyhistidine tract. In some embodiments, the insulator sequence can be determined empirically by inserting a spacer sequence around the ribosome binding site or by optimizing or inserting codons at the N-terminus of the expressed protein.

[0105] In some embodiments, the expression array includes a coding sequence, a non-coding sequence, or a combination thereof. The coding sequence includes a promoter, an open reading frame, and optionally, a cap-independent translation element (CITE). The cap-independent translation element (CITE) of the coding sequence can be an internal ribosome entry site (IRES), a translation enhancing element (TEE), or a combination thereof. The open reading frame of the coding sequence can be codon-optimized to enhance translation. The open reading frame can further include a tag sequence for purification of the desired protein, an amino-terminal peptide fusion sequence derived from an IRES for enhancing ribosome recognition, or a combination thereof. The expression array further includes a polyA sequence, a transcription termination sequence, an insulator sequence, or a combination thereof.

[0106] In some embodiments, the expression array is a minimal expression array that lacks any exogenous sequences required for propagation of the plasmid in the host cell. The minimal expression array for expressing a desired protein includes at least a promoter, a ribosome binding site, and a translation termination sequence. The minimal expression array for expressing a desired RNA includes at least a promoter, a ribosome binding site, and a translation termination sequence. In some embodiments, the double-stranded RCA generating DNA consists essentially of tandem repeats of the minimal expression array. In such embodiments, the expression array can further include sequences that do not substantially affect in vivo protein expression or RNA expression using the RCA generating DNA as a template. For example, this can further include sequences such as a translation enhancing sequence, an insulator sequence, or a transcription termination sequence. The minimal expression array of the RCA generating DNA excludes any exogenous sequences such as an antibiotic selection gene, or any other accessory sequences required for cloning, selection, screening, and / or replication in the host cell. The RCA product can be a linear or branched concatemer that includes tandem repeats of the minimal expression array. The minimal expression array of the RCA generating DNA can be derived from a DNA minicircle that includes only the minimal expression array.

[0107] The two-strand concatemer DNA may further contain inosine-containing nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, polycationic nucleotides, or combinations thereof. In some embodiments, modified nucleotides, such as inosine-containing nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, 2-amino-deoxyadenosine, 2-thio-deoxythymidine, polycationic nucleotides, are part of the primer sequences utilized in the rolling circle amplification method.

[0108] DNA minicircle templates for use by the disclosed techniques can be prepared using a variety of methods. In some embodiments, a linear DNA template can be circularized to generate a DNA minicircle template. In one exemplary embodiment, the circularization of the linear DNA template can occur by an enzymatic reaction, such as incubation with a ligation enzyme, such as DNA ligase. In some embodiments, both ends of the linear DNA template are hybridized to nucleic acid sequences to bring the two ends very close together. Then, incubation with a ligation enzyme causes circularization of the hybridized linear DNA template, generating a DNA minicircle. Also, a suitable DNA minicircle template can be generated by circularization of a PCR product after PCR amplification of a portion of a large DNA (e.g., genomic DNA or DNA from a DNA library) using appropriate PCR primers. Additionally, DNA minicircles can be generated by circularization of a synthesized oligonucleotide after chemical synthesis of a suitable linear oligonucleotide. In some embodiments, the synthesized linear oligonucleotide consists essentially of a minimal expression sequence and achieves circularization by DNA ligase to generate a DNA minicircle.

[0109] Provided herein is an expression system (e.g., expression system 20) that can be used to generate an expression product (e.g., expression product 26). Expression system 20 can be a cell-based expression system (e.g., mammalian, insect) or a cell-free expression system. Examples of cell-based expression systems include expression systems based on CHO, NIH3T3, BHK, HepG2, and HEK293 cells.

[0110] In certain embodiments, a "cell-free expression system" refers to an in vitro transcription and translation system. Cell-free expression generally encompasses two forms: (1) generating mRNA and protein in a single reaction, or (2) generating mRNA in a first reaction and adding the resulting mRNA product to a second separate translation reaction. RCA products derived from DNA circles can be utilized in either form (1) or (2). For example, in one embodiment, the RCA product may be provided for an "in vitro transcription-translation coupled reaction", where the RCA-generated DNA is converted to mRNA, and the mRNA simultaneously expresses protein in a single reaction mixture having the ability to generate both RNA and protein. In another embodiment, the RCA product may be provided for a "transcription-translation related reaction", where the RCA-generated DNA is first converted to mRNA, and the mRNA is separately added to a translation reaction mixture to express protein. In certain embodiments, the concatemer is provided to an unprocessed (or without any further processing) cell-free expression system. In one embodiment, the concatemer is added to the cell-free system immediately after amplification such as RCA. The term "further processing" means including the actions of restriction digestion, ligation of the concatemer, or combinations thereof. However, in some embodiments, the RCA product can be separated (e.g., by precipitation) to remove salts or other contaminants, such as primers or small fragmented DNA, from the reaction medium, and then cell-free expression can proceed, for example, using a eukaryotic cell extract.

[0111] A cell-free expression system can be an in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis system. Non-limiting examples of cell-free expression systems are eukaryotic cell-free expression systems. In certain embodiments, concatemer DNA, after being immobilized on a substrate, can be subjected to a cell-free expression system. The concatemer DNA immobilized on the substrate can be recovered from the cell-free expression system after in vitro expression of a recombinant product and reused for subsequent in vitro transcription and translation reactions.

[0112] Concatemer DNA (e.g., double-stranded concatemer DNA) can be delivered to a eukaryotic cell expression system by any method including, but not limited to, electroporation, sonoporation, impalefection, transduction, optical transfection, magnetofection, nucleofection, hydrodynamic delivery, heat shock-mediated gene delivery, nanoparticle-mediated gene gun delivery, calcium phosphate-mediated delivery, cationic polymer-mediated delivery, or liposome-mediated delivery.

[0113] In some embodiments, eukaryotic cells are provided that contain exogenous double-stranded concatemer DNA containing multiple tandem repeats. In one embodiment, each of the multiple tandem repeats contains phosphorothioated nucleotides, where the ratio of phosphorothioated nucleotides to total nucleotides is at least 1:1600. The exogenous double-stranded concatemer DNA used for transfection into eukaryotic cells to generate these cells can be unprocessed or processed RCA-generated DNA. The eukaryotic cells can be protozoa, yeast cells, insect cells, or mammalian cells.

[0114] The technical effects of the disclosed embodiments include improving the expression of a desired expression product using nucleic acid concatemers having sizes that are typically not associated with robust transfection and the production of the desired expression product. Further, the concatemers may contain an unknown or variable number of tandem repeats of the expression sequences, yet various concatemers can be co-expressed stoichiometrically, e.g., based on molar ratios, to produce a production expression product in a robust ratio based on evidence. In the present disclosure, it is demonstrated that concatemers having nucleic acid expression sequences for one or more desired expression products can be used in place of and / or in combination with traditional expression techniques, e.g., plasmids, to produce a desired expression product having increased purity and robust co-expression. Further, nucleic acid concatemers can be used to generate more complex structures or assemblies where multiple co-expression products are related to each other.

[0115] The foregoing examples are illustrative of some of the features of the present disclosure and are embodiments selected from among a number of possible embodiments. Only specific features are illustrated and described herein, but those skilled in the art can make modifications / changes to optimize the parameters in view of the advantages of the present disclosure. Accordingly, the foregoing embodiments are considered to be illustrative rather than limiting of the embodiments described herein in any way. When ranges are given, these ranges include all sub-ranges there between.

[0116] In the description of the present disclosure, examples are used that include the best mode and that enable those skilled in the art to make and use the devices or systems and to practice any methods incorporated. The scope of patentability is defined by the claims and may include other examples found by those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims or if they include equivalent structural elements that have non-substantive differences from the literal language of the claims.

Claims

**Claim 1** A step of producing a concatemer mixture comprising at least a first nucleic acid concatemer and a second nucleic acid concatemer having a predetermined ratio to each other, wherein the first nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence, and the second nucleic acid concatemer comprises tandem repeats of a second nucleic acid sequence, A step of co-expressing the concatemer mixture to produce a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence A method for generating a viral vector or virus-like particle, comprising: **Claim 2** The method according to claim 1, comprising a step of generating a first nucleic acid concatemer and a second nucleic acid concatemer using a rolling circle amplification method. **Claim 3** The method according to claim 2, comprising a step of amplifying circular or plasmid DNA using a rolling circle amplification method to generate a first nucleic acid concatemer and / or a second nucleic acid concatemer. **Claim 4** The method according to any one of claims 1 to 3, wherein the first tandem repeat and / or the second tandem repeat is a tandem repeat of at least a part of circular or plasmid DNA. **Claim 5** The method according to any one of claims 1 to 4, comprising a step of transfecting a concatemer mixture into cultured cells to cause co-expression, or causing co-expression in a cell-free expression system. **Claim 6** The method according to any one of claims 1 to 5, wherein the first nucleic acid concatemer, the second nucleic acid concatemer, or both have a length exceeding 10 kb. **Claim 7** The method according to any one of claims 1 to 6, comprising a step of collecting the first expression product and the second expression product. **Claim 8** The method according to any one of claims 1 to 7, wherein one or both of the first nucleic acid concatemer or the second nucleic acid concatemer are not processed before co-expression. **Claim 9** The method according to any one of claims 1 to 8, wherein the first expression product is a viral envelope or packaging protein, the second expression product comprises the transgene of the virus, and the first expression product and the second expression product form a viral vector for delivering the transgene. **Claim 10** The method according to any one of claims 1 to 9, wherein the first expression product is a nucleic acid product and the second expression product is a protein product. **Claim 11** The method according to claim 10, wherein the first expression product forms a complex with the second expression product.

12. The method according to any one of claims 1 to 11, wherein the first expression product acts on a third nucleic acid concatemer in a concatemer mixture.

13. A step of preparing a mixture containing at least two nucleic acid concatemers in a predetermined ratio, wherein each of the nucleic acid concatemers contains a tandem repeat of two or more nucleic acid sequences. A step of co-expressing the concatemer mixture to generate two or more expression products from each nucleic acid concatemer in the mixture. A method for generating a viral vector or virus-like particle, comprising:

14. The method according to claim 13, wherein the nucleic acid sequences of the individual concatemers in the concatemer mixture contain a plurality of expression sequences that generate a plurality of proteins when expressed to generate two or more expression products.

15. The method according to claim 13, wherein the nucleic acid sequences of the individual concatemers in the concatemer mixture contain a plurality of expression sequences that generate a mixture containing at least one protein expression product and at least one nucleic acid expression product when expressed to generate two or more expression products.

16. A step of amplifying at least one template containing a first nucleic acid sequence using a strand displacement rolling circle amplification method to generate a first concatemer containing a tandem repeat of the first nucleic acid sequence. A step of contacting the first concatemer with a second concatemer containing a tandem repeat of a second nucleic acid sequence to form a concatemer mixture in which the first nucleic acid concatemer is present in a predetermined ratio to the second nucleic acid concatemer. A step of co-expressing the concatemer mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence, wherein the ratio of the first expression product to the second expression product is proportional to the predetermined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture. A method for generating a viral vector or virus-like particle, comprising:

17. The method according to claim 16, comprising a step in which the first expression product can form a complex with the second expression product.

18. The method according to claim 16 or 17, wherein the first expression product and the second expression product comprise various viral products derived from the same virus, such as adeno-associated virus or lentivirus.

19. The method according to any one of claims 16 to 18, wherein the first expression product comprises viral mRNA and the second expression product comprises a plurality of viral packaging proteins.

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