Expression of products from nucleic acid concatemers

Abstract

To provide systems and methods for reliably controlled expression of recombinant products in both in vitro and in vivo expression systems, especially with minimal labor for preparation.SOLUTION: Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding one or more expression products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein 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 relates generally to expression of recombinant products from nucleic acid concatemers, and in particular embodiments, to ratiometric co-expression of recombinant products from nucleic acid concatemers. Ratiometric co-expression has been demonstrated in both in vivo and cell-free expression systems. [Background technology]

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

[0004] Recombinant DNA technology has been used in the production of recombinant products. However, a particular desired product may contain multiple components that are combined or otherwise function together. The production of such multi-component expression products can be complex. One common approach to recombinant product production may involve separating the various components into multiple plasmids. For example, co-expression of multiple plasmid constructs may be performed in both in vivo and in vitro settings. In some cases, multiple plasmid constructs may be transiently transfected into cultured cells, and such cultured cells are used to produce the desired recombinant product, e.g., viral vectors, proteins, 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 ratiometrically. For example, when each plasmid construct encodes a separate protein, it has been reported that the co-expression of two (or more) proteins from corresponding plasmids in a single cell by transfection procedure results in the proteins being expressed in a wide variety of ratios, and a subpopulation of cells may only express one of the desired proteins.

[0006] Several approaches have been attempted to improve the regulation of ratiometric co-expression of proteins and achieve the desired ratio of proteins in a single living cell. 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 laborious cloning of each open reading frame into the multicistronic plasmid, which in turn increases the size of the plasmid construct. In addition, because the plasmid contains additional sequences required for maintenance in bacteria, the use of plasmids requires post-production purification and QC analysis to prove the absence of bacterial contaminants in the plasmid production, which poses a significant challenge to cGMP (clinical good manufacturing) processes. The latter is particularly challenging as the post-production purification of plasmid DNA becomes complicated as the size of the plasmid construct increases and becomes difficult to distinguish from bacterial chromosomal DNA.

[0007] Thus, 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 that require minimal preparation effort. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 10,077,459 [Patent Document 2] U.S. Patent No. 9,938,568 [Non-patent literature]

[0009] [Non-Patent Document 1] Singh, SK et al., Chem. Comm., 4, 455-456, 1998 [Non-Patent Document 2] Koshkin, AA et al., Tetrahedron, 54, 3607-3630, 1998 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 a variety of aspects that are not set forth below. [Means for solving the problem]

[0011] In one embodiment, a method is provided that includes the steps of: generating 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 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.

[0012] In another embodiment, a method is provided that includes creating a mixture comprising at least two nucleic acid concatemers in a predetermined ratio, each of the nucleic acid concatemers comprising tandem repeats of two or more nucleic acid sequences, and co-expressing the mixture to produce two or more expression products from each nucleic acid concatemer in the mixture.

[0013] In another embodiment, a method is provided that includes amplifying at least one template comprising a first nucleic acid sequence using strand displacement rolling circle amplification to produce first concatemers comprising tandem repeats of the first nucleic acid sequence; contacting the first concatemers with second concatemers comprising tandem repeats of a second nucleic acid sequence to form a concatemer mixture having a predetermined ratio of first nucleic acid concatemers to second nucleic acid concatemers; and 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 concatemers to the second nucleic acid concatemers in the concatemer mixture.

[0014] In another embodiment, a method is provided that includes generating 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 first provided and transiently transfected with a nucleic acid concatemer, which comprises a tandem repeat of a nucleic acid sequence and expresses at least one open reading frame in the nucleic acid sequence that encodes an expression product in the transfected cell. In one embodiment, the transfected cell provided is already transfected with one or more nucleic acid concatemers and / or one or more plasmids. In this method, the cell can be transfected with the nucleic acid concatemers sequentially. In an alternative embodiment, the transfected cell is transfected with the nucleic acid concatemers after stably expressing one or more desired products.

[0016] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read in conjunction with the accompanying drawings, in which like characters represent like parts throughout. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of the generation of concatemers for use in an expression system to produce a desired expression product according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram of the generation of two or more concatemers for use in a co-expression system to produce one or more desired expression products according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 is a schematic diagram of a potential end product of a co-expression system according to an embodiment of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram of potential combined end products of a co-expression system according to an embodiment of the present disclosure. [Diagram 5] FIG. 1 is a schematic diagram of potential end products of a co-expression system according to embodiments of the present disclosure acting on each other or on another nucleic acid or protein. [Figure 6] FIG. 1 is a schematic diagram of a mixed expression strategy in which all or part of a desired expression product in a multi-producing system is expressed using concatemers according to an embodiment of the present disclosure. [Figure 7] FIG. 1 is a schematic diagram of an embodiment of lentiviral vector product expression according to an embodiment of the present disclosure. [Figure 8] FIG. 1 is a schematic diagram of an embodiment of lentiviral vector product expression according to an embodiment of the present disclosure. [Figure 9] FIG. 1 is a schematic diagram of an embodiment of an adeno-associated viral vector product expression according to an embodiment of the present disclosure. [Figure 10] FIG. 1 is a schematic diagram of an embodiment of CRISPR product expression according to an embodiment of the present disclosure. [Figure 11] 1 is an agarose gel photograph showing stoichiometric expression of adeno-associated virus (AAV) capsid proteins according to embodiments of the present disclosure. [Figure 12]1 is a photograph of an agarose gel showing stoichiometric expression of light and heavy chain polypeptides according to embodiments of the present disclosure. [Figure 13] 1 is a bar graph showing AAV production according to embodiments of the present disclosure from intact rolling circle amplification-produced concatemers, rolling circle amplification-produced linear digests, and plasmids transfected at various concentrations (18% and 2%) into naive cells. [Figure 14] 1 is a bar graph showing AAV production from mixed concatemer and plasmid expression sources according to embodiments of the present disclosure. [Figure 15] 1 is a bar graph showing lentivirus production according to embodiments of the present disclosure from mixed concatemer and plasmid expression sources. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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. As in any engineering or design project, it should be recognized that in the development of any such implementation, numerous implementation-specific decisions must be made to achieve the developer's particular goals, e.g., compliance with system and commercial related constraints, which may vary with each implementation. Moreover, it should be understood that such development efforts are complex and may take time, but are nevertheless a routine task for those skilled in the art who have the benefit of this disclosure to design, fabricate, and manufacture.

[0019] Expression of products from nucleic acids of large size (i.e., high molecular weight) is associated with low efficiency. Such low efficiency is the result of poor transfection efficiency of large nucleic acids, which in turn can result in reduced production of the desired end product, as well as DNA sequence-based competition between nucleic acids for host factors required for transcription and translation. Transfection efficiency has previously been shown to decrease as a function of DNA size, with plasmids over 10 kb in size showing dramatically reduced transfection efficiency compared to smaller sized plasmids. Provided herein are expression techniques that use DNA concatemers or DNA concatemer mixtures, whereby the concatemers contain tandem repeats of nucleic acid sequences that code for the desired expression product, e.g., a protein or nucleic acid expression product. In certain embodiments, the disclosed concatemers can be of a size (e.g., at least 10 kb) larger than previously inefficient sizes. 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 a smaller size plasmid achieve high transfection efficiency, despite the larger size of one of the two. In one embodiment, the concatemers are generated using strand displacement amplification, e.g., rolling circle amplification (RCA), to generate multiple concatemers containing tandem repeats of a nucleic acid sequence encoding a 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 a desired expression product may be achieved even in situations where the concatemer size resulting from RCA is variable and / or mixed. Furthermore, robust production of a desired co-expression product and desired ratiometric control may be achieved even when the exact number of tandem repeats provided herein is unknown, without controlling for sequence-based competition between nucleic acid sequences for host factors.

[0020] Furthermore, as disclosed herein, DNA concatemers can be used in conjunction with co-expression systems to predictably co-express two or more expression products in a ratiometric manner. Two or more concatemers can be transfected and co-expressed to generate co-expression products. Ratiometric expression can occur using concatemers with an unknown number of tandem repeats 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 occur in a single cell or cell-free expression system. This is an unexpected result according to conventional wisdom regarding the relationship between size and transfection efficiency. This is also unexpected due to the unpredictable nature of DNA sequence-based competition for host factors that control transcription and translation. For example, due to the large size of nucleic acid concatemers (e.g., a typical nucleic acid concatemer may have a size of more than 10 kb), conventional wisdom holds that a DNA cocktail containing two or more different nucleic acid concatemers is not expected to efficiently enter a single cell and produce a desired expression product, especially if the nucleic acid concatemers are not processed (e.g., no further processing is performed after the nucleic acid concatemers are produced). In addition, the size of the concatemers typically cannot be precisely determined, and the size is heterogeneous, making the design of such cocktails or mixtures (containing two or more concatemers mixed in a predetermined or defined ratio) even more challenging.

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

[0022] The use of RCA-generated concatemers expressing the desired final product offers advantages over other expression templates, such as plasmid constructs. Rolling circle amplification allows for the rapid generation of specific DNA sequences (e.g., DNA minicircles) that are fully relevant and biologically active for the intended application, and that code for minimal viral vectors, including packaging, transduction and expression elements and proteins required for virus production. This allows for increased specific activity of DNA (coding sections per piece of DNA). In contrast, plasmids contain additional sequences that are necessary for maintenance in bacteria but not for the intended application. Because RCA concatemers are not produced in bacteria, the use of RCA avoids potential contamination of the final DNA product with adventitious bacterial components or purification reagents. The disclosed concatemer synthesis is performed without the use of bacteria and is therefore free of bacterial endotoxins. RCA concatemers also 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 verify the absence of bacterial contaminants (genomic DNA and RNA) in the plasmid product. In contrast, nucleic acid concatemers can be prepared and propagated without the use of bacteria, and require minimal post-production purification, thus providing the cost and complexity advantages of being compatible with cGMP manufacturing. Furthermore, RCA-generated concatemers can be used to encode products with bacterial toxicity that are difficult to scale up in bacterial-based systems. This is because constructs that can be used to encode toxic products are difficult to maintain due to leaky expression of the product during bacterial growth, resulting in loss of the construct due to bacteria or bacterial death.

[0023] Moreover, RCA-generated concatemers can allow for easy DNA production and scale-up when needed. Milligram-gram quantities of rolling circle amplified DNA can be inexpensively produced from template DNA in less than a day using isothermal amplification reactions. The total "hands-on" time to produce 5 mg of RCA DNA is less than 1 hour. In contrast, approximately 8 hours are required for supercoiled plasmids isolated from the DH5 alpha strain of E. coli using endotoxin-free purification columns. Thus, the rolling circle amplification method is also expected to provide a more convenient and less expensive method of production compared to standard bacterial-derived plasmids.

[0024] Furthermore, the RCA method allows the insertion of modified nucleotides into the amplified DNA, which may be limited in plasmid-based systems in living cells. Modified nucleotides may 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 protein expression compared to RCA DNA containing only standard nucleotides. Thus, in one embodiment, the present technology may be used to generate or manufacture expression products from concatemers incorporating modified nucleotides.

[0025] FIG. 1 is a schematic diagram of the production of a desired expression product using at least one concatemer provided herein. The expression sequences 12 in a circular template 14 are used to generate concatemers 16 that contain tandem repeats of the expression sequences 12 using rolling circle amplification (RCA). Once generated, the concatemers 16 may be purified or otherwise cleaned and then used in an expression system 20 that operates on the concatemers 16 to express the expression sequences 12 to generate an expression product 26. However, in certain embodiments, the concatemers 16 may be applied in the expression system 20 without the need for further processing. The expression system 20 may be a cell-based or cell-free expression system as disclosed herein. Once generated, the expression product 26 may be harvested for use in an appropriate therapeutic protocol or other application. The expression product 26 of the concatemers 16 may include one or more protein and / or nucleic acid expression products.

[0026] In certain embodiments, the concatemers 16 may be used alone or in combination with other concatemers 16 (e.g., two or more concatemers 16) or other expression vectors in the expression system 20, for example, in a cell-free expression system or in a mixture with a plasmid that may be co-transfected in a cell-based system to co-express multiple expression products 26. In certain embodiments, concatemers 16 of about 10 kb or larger in size may be used in a cell-based expression system to generate expression products 26. In contrast to other techniques, the present disclosure demonstrates that large size concatemers and concatemer mixtures do not similarly reduce transfection efficiency in cell-based expression systems compared to similarly sized plasmids, nor do they result in DNA sequence-based competition for host factors that reduce co-expression efficiency in the expression system.

[0027] 2 is a schematic diagram of co-expression of a desired expression product using a mixture of multiple concatemers. A first concatemer 16a having a tandem repeat of an expression sequence 12a and generated from a template 14a by RCA can be mixed with a second concatemer 16b having a tandem repeat of an expression sequence 12b and generated from a template 14b by RCA as provided herein. The expression sequences 12a, 12b can be different such that multiple expression products 26a, 26b are generated by an expression system 20. That is, rather than generating the various expression products 26a, 26b in different expression systems 20, the various expression products 26a, 26b can be co-expressed in the same expression system 20, for example, generated at the same time. Furthermore, 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 with any inserted expression sequences, but the size of the concatemers can vary. However, in an embodiment of the present technology, rather than attempting to determine the exact number of tandem repeats of the expression sequences 12 in each concatemer 16, the assumption is included 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 a tandem repeat of the expression sequence 12a of known base length 1000, and the second concatemer 16b may have a tandem repeat of the expression sequence 12b of known base length 1500. Thus, to achieve a molar ratio of the first concatemer 12a:second concatemer 12b of 1:1, the total amount of the second concatemer 16b should be 1.5x that of the first concatemer 16a in the concatemer mixture used for co-expression. It should be understood that the concatemers 16 can be both single-stranded, both double-stranded, or a mixture of single and double stranded. Any additional concatemers 16 can also determine the molar ratio. Table 1 summarizes examples of molar ratios used for three concatemers 16 co-expression systems of adeno-associated virus (AAV), using the assumption that the molar ratio can be based on the size of the monomers of the expression sequence 12 (or the expression sequence, e.g., the plasmid / circular construct with template 14).

[0029] [Table 1]

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

[0031] Thus, while co-expression of two or more proteins by transfection of separate plasmids into a single cell has been reported to result in widely varying ratios of expressed proteins (e.g., many cell subpopulations express only one of the two protein constructs), the present technology facilitates a programmable ratio of co-expressed products that is proportional to the ratio of concatemers 16 used in the expression system 20. Although concatemers have uncertainties regarding size variation, the present disclosure nevertheless demonstrates that the use of concatemers 16 that co-express desired expression products 26 results in robust expression with programmable ratios.

[0032] It should be understood that in a co-expression workflow, the concatemers 16 may be mixed or created in a defined or predetermined ratio based on the desired ratio of each expression product 26. That is, to achieve a desired ratio of a first expression product 26a to a second expression product 26b, the concatemer mixture provided for the expression system 20 may be created with the concatemers 16a, 16b present in the mixture in a predetermined ratio to each other. With two concatemers 16, the ratio of the concatemers 16 may 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. With three or more concatemers 16, the ratio of the concatemers 16 may 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. N where C1, C2 to C N may be 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, etc., and may not be 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. Furthermore, in a co-expression system that includes one or more DNA concatemers 16, the exact length of the DNA can be measured to determine the appropriate ratio, as disclosed, rather than guessing or estimating the size of the concatemers.

[0033] In certain embodiments, the co-expression product may be formed by preparing a concatemer mixture comprising a first nucleic acid concatemer and a second nucleic acid concatemer in a defined ratio, and co-expressing the concatemer mixture to produce at least a first expression product and a second expression product, respectively, resulting from the first nucleic acid concatemer and the second nucleic acid concatemer of the concatemer mixture, where the ratio of the first expression product and the second expression product is proportional to the defined ratio of the first nucleic acid concatemer and the second nucleic acid concatemer in the concatemer mixture. In certain embodiments, the ratio of the first and second expression products and the defined ratio of the first and second nucleic acid concatemers are substantially identical.

[0034] 3-5 are schematic diagrams of embodiments of co-expression of nucleic acid concatemers to form a desired end product. One or more co-expression products may be in the form of a non-composite co-expression product or a composite co-expression product. In certain embodiments, a non-composite co-expression product may include two or more expression products that are co-expressed and exist in a non-composite form. For example, the co-expression product may include a first expression product 30 and a second expression product 34 that are not complexed with each other after co-expression, as shown in FIG. 3. Each of the expression products may independently be a protein, an RNA, or a combination thereof. Additionally, each concatemer 16 (16a, 16b, and optionally 16c) may produce multiple expression products, shown as expression products 32, 36, 38.

[0035] In certain embodiments, a composite co-expression product may include two or more expression products 40, 42 that form a complex 50 with each other after being co-expressed from each of the concatemers 16a, 16b, as shown in FIG. 4. For example, a composite co-expression product may include a first expression product 40 and a second expression product 42 that are co-expressed to form a complex 50 with each other. Each of the expression products may be, independently, a protein, an RNA, or a combination thereof. In addition, as shown, an expression product (e.g., expression product 46) may also be generated by another concatemer 16c that may also form part of the complex 50. Non-limiting examples of composite co-expression products may include an antibody (e.g., a monoclonal antibody (mAb)), a virus-like particle, a CRISPR, or a lentivirus.

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

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

[0038] In certain embodiments, at least one of the first and second nucleic acid concatemers comprises a minimal expression sequence. The minimal expression sequence encodes one or more desired products without any extraneous sequences required for DNA propagation in a host cell. The minimal expression sequence may be designed in silico and synthesized in vitro. In one embodiment, each of the expression sequences 12 may comprise 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 may comprise a promoter operably linked to two or more ORFs. For example, one of the expression sequences may comprise a promoter operably linked to two different ORFs.

[0039] In embodiments using two or more concatemers 16, the concatemer mixture provided herein may include a mixture of two or more concatemers 16, each of which has a different expressed sequence 12 compared to each other (i.e., a nucleic acid having a different sequence). It should be understood that 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), such that the mixture includes the first and second subsets of concatemers 16. Additionally, in embodiments, the concatemer mixture may include a third subset of a third type of concatemers 16 (having tandem repeats of a third nucleic acid sequence different from the first and second nucleic acid sequences), and so on. As mentioned above, in each individual subset, the size (base length) of the concatemers 16 and the number of their corresponding tandem repeats can vary.

[0040] 6 is a schematic diagram of various implementations of the technique for generating expression products according to embodiments of the present disclosure. In some embodiments, it may be desired to generate multiple expression products to form a complex or to interact with each other to ultimately form a desired end product. In an expression workflow, such expression products may be split into various expression vehicles (various concatemers 16 or, in some embodiments, a mixture of one or more concatemers 16 and one or more plasmids, e.g., 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 may be generated by using a template 14 with a customized or desired expression sequence. It may be difficult to predict the particular nucleic acid expression product 86 that may be of interest to the end user. Thus, rather than incorporating the nucleic acid sequence for the variable nucleic acid expression product 86 into an expression sequence 12 that contains an otherwise more constant or invariant desired end product, the kit or expression product generation system 87 provided herein may separate the customizable or more variable regions into separate templates 14 and concatemers 16. In this manner, a more universal expression product 88 may be provided as a separate template 14 that is pre-generated or part of an assembled kit that incorporates the expression sequence 12 for the customizable expression product 86 into separate concatemers 16 or plasmids 82. In another embodiment, the DNA concatemers (amplified from plasmids or minicircle templates) may be selected for variable or customizable components for each application, while the plasmids may be selected for more constant or predictable components that do not change with application. Examples of such separate constructs are discussed below in conjunction with Figures 7-10.

[0042] Lentivirus 7-8 are schematic diagrams of lentiviral virus vector generation systems. The generation of lentiviral virus vectors can be useful in a variety of gene therapy applications, including in 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 the lentiviral vector generated by the disclosed techniques can encode a chimeric antigen receptor.

[0043] Lentiviruses have a linear single-stranded RNA (ssRNA) genome. In an embodiment depicted for second generation lentiviral vector production, the ENV protein (e.g., VSV-G) may be encoded by the expression sequence of the first concatemer, the desired transgene sequence flanked by long terminal repeat (LTR) sequences may be part of the expression sequence of the second concatemer, and the packaging protein may be present on the third concatemer with ORFs encoding GAG, POL, REV and TAT genes (Figure 7). In another arrangement for third generation lentivirus production (Figure 8), the packaging protein is further split into two packaging concatemers with GAG, POL present on one concatemer and REV present on the other concatemer.

[0044] According to the current disclosure, efficient generation of functional lentivirus was achieved by producing and transfecting a mixture of RCA DNA (separately encoding VSV-G envelope, GAG / POL, REV and GFP mRNA) in stoichiometric ratios in HEK293T cells. 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 hyperbranched concatemers containing tandem repeats of each plasmid to assess successful viral vector generation. Viral vectors can be generated from living cells or by cell-free expression.

[0045] According to the current disclosure, a concatemer mixture is created that includes a plurality of nucleic acid concatemers with a defined ratio between each of the plurality of nucleic acid concatemers. The concatemer mixture is co-expressed to produce a plurality of expression products each arising from each of the nucleic acid concatemers, where 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.

[0046] Although the disclosed embodiments relate to multiple concatemers used to generate lentiviral vectors, other embodiments also contemplate a mixture of concatemers and plasmids. In one embodiment, only the transgene is expressed using the concatemers, while a plasmid construct encoding the other components is co-transfected with the transgene concatemers into a co-expression system. In another embodiment, the desired transgene and ENV protein are co-expressed from a mixture of two different concatemers, while a plasmid encoding the other components is co-transfected with the concatemer mixture into a co-expression system. The technology may include co-transfection of one or more concatemers containing the lentiviral transgene and any plasmid or concatemer (one or more packaging concatemers or plasmids and one envelope concatemer or plasmid) carrying an open reading frame for the other components of the viral vector into an expression system, for example, HEK293T producer cells or A293T cells. Co-expression may be performed by cells or a cell-free system.

[0047] In certain embodiments, the individual RCA products were mixed in a molar ratio of 1:1:1:1 and transfected into HEK293T cells. In certain embodiments, one or more RCA DNA concatemers may be replaced by plasmid DNA. Various ratios of RCA DNA to plasmid DNA may 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 both cases, the concatemeric RCA DNA worked efficiently with the plasmid DNA, which produced comparable amounts of lentivirus.

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

[0049] AAV ITRs contain all cis-acting elements involved in genome rescue, replication and packaging, and are 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 linked to a 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) that contains a clone of a recombinant AAV genome consisting of a DNA of interest flanked by AAV ITRs, and a separate plasmid expressing 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 the producer cells with a third plasmid that provides such adenoviral helper factors. When HEK293 cells are used as AAV production 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 cell lines with wild-type adenovirus harboring the AAV Rep / Cap genes, as well as AAV vector DNA. Another method, the helper-free method, is based on adenovirus-free transient transfection of all elements required for AAV production in host cells, such as HEK293 cells. It involves co-transfecting AAV-producing cells with three plasmids: (1) an AAV transfer plasmid that places the gene of interest (e.g., "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 host cells, such as HEK293 cells, that contain the E1A / E1b genes, to generate infectious AAV particles.

[0051] Although the method using wild-type adenovirus-induced AAV-producing cell lines can scale up the culture and produce very high titer AAV vectors, it is very difficult to completely remove adenovirus from AAV products, and contamination with wild-type adenovirus is highly undesirable in terms of vector safety and specificity. On the other hand, the transient transfection method produces high titer adenovirus-free AAV vectors, but this method is very laborious and expensive.

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

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

[0054] According to the current disclosure, a concatemer mixture is created that includes a plurality of nucleic acid concatemers with a defined ratio between each of the plurality of nucleic acid concatemers. The concatemer mixture is co-expressed to produce a plurality of expression products each arising from each of the nucleic acid concatemers, where 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 multiple nucleic acid concatemers separately encodes important replication and viral encapsidation factors in addition to the desired transgene. In certain embodiments, at least one of the nucleic acid concatemers includes a minimal expression sequence. The minimal expression sequence may be designed in silico and synthesized in vitro (e.g., SEQ ID NOs: #1-#3 listed below). In another embodiment, the transgene (e.g., transgene payload) is present on a separate concatemer from one or more REP / CAP concatemers. Additionally, the separate concatemers may include one or more helper sequences depending on the desired expression system. Although the disclosed embodiments relate to multiple concatemers for use in generating AAV vectors, other embodiments also contemplate mixing of concatemers and plasmids. In one embodiment, only the transgene is expressed using the concatemer, while a plasmid construct encoding the other components is co-transfected with the transgene concatemer into a co-expression system. In another embodiment, the desired transgene and capsid protein are co-expressed from a mixture of two different DNA concatemers, while a plasmid encoding the other components is co-transfected with the concatemer mixture into a co-expression system. This technique can involve the co-transfection of one or more concatemers, including the AAV transgene, as well as any plasmid or concatemer carrying an open reading frame for the 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 by a Type II CRISPR system. When used for genome editing, this system includes Cas9, crRNA, tracrRNA, along with an optional section of DNA repair template for use in either non-homologous end joining (NHEJ) or homology directed repair (HDR).

[0057] CRISPR / Cas9 often utilizes a plasmid to transfect target cells. The main components of the plasmid include crRNA, tracrRNA, sgRNA, Cas9 protein, and repair template. The crRNA contains a guide RNA that locates the correct section of the host DNA, along with a region that binds to the tracrRNA. The tracrRNA binds to the crRNA and forms an active complex. A single guide RNA (sgRNA) is a complex RNA consisting of a tracrRNA and at least one crRNA. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be combined with the Cas9 gene into a plasmid that can be transfected into cells. When assembled into a plasmid and transfected into cells, the Cas9 protein, with the help of the crRNA, finds the correct sequence in the host cell's DNA and creates a single or double strand break in the DNA, depending on the Cas9 variant.

[0058] The technology is also used to generate or manufacture targeted CRISPR / Cas9 systems using one or more concatemers 16, instead of traditional plasmid-based technology. The first and second nucleic acid concatemers may each include a first and 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 a crRNA, tracrRNA, sgRNA, or repair template.

[0059] antibody Immunoglobulins or antibodies contain heavy and light chains with a characteristic stoichiometry. For example, immunoglobulin G (IgG) contains two identical heavy chains and two identical light chains linked by disulfide bonds. Conventional recombinant methods for producing 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 different strengths) to achieve the desired expression ratio of antibody heavy and light chains, for example in CHO cells. The expression ratio of light to heavy chain polypeptides is recognized as an important parameter for the recombinant yield of the desired antibody and the limitation of undesired aggregates and fragments.

[0060] The present technology may also be used to generate or manufacture antibodies that provide a light to heavy chain polypeptide expression ratio in a more rational and robust manner by using a predefined ratio of first and second nucleic acid concatemers and then obtaining a desired ratio of light to heavy chain polypeptides. In one embodiment, the first and second nucleic acid concatemers each comprise a first and second expression sequence, each of which may comprise 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 comprise a promoter operably linked to two or more ORFs. For example, one of the expression sequences may comprise a promoter operably linked to two different ORFs, one ORF may encode the heavy chain of an antibody and the other ORF may encode the light chain. In another embodiment, a concatemer mixture may be generated to express a bispecific antibody, where a first concatemer encodes a first expressed sequence comprising the light and heavy chain domains of a first antibody, while a second concatemer encodes a second expressed sequence comprising the light and heavy chain domains of a second antibody.

[0061] Virus-like particles The technology may also be used to generate or manufacture virus-like particles. Non-limiting examples of virus-like particles include HPV and Gardasil (tetravalent human papillomavirus types 6, 11, 16 and 18) vaccines. HPV encodes two capsid proteins, L1 and L2. The major capsid protein L1 can spontaneously assemble into an icosahedral structure of 72 pentamers, closely resembling the natural virion. The minor capsid protein L2 is not required for capsid formation, but there may be an average of about 36 molecules of L2 per capsid (estimated L1:L2 ratio of about 9:1 to 11:1). The Gardasil (tetravalent human papillomavirus types 6, 11, 16 and 18) vaccine is a recombinant VLP formulation of four different L1 capsomeres, each individually expressed from baker's yeast.

[0062] The first and second nucleic acid concatemers may each comprise a first and a second expression sequence. The first and second expression sequences may each comprise an open reading frame (ORF) encoding a first and a second protein of interest, for example, capsid protein L1 and L2, respectively. Each of the expression sequences may further comprise a promoter operably linked to the corresponding open reading frame. In certain embodiments, one of the expression sequences may comprise a promoter operably linked to two or more ORFs. For example, one of the expression sequences may comprise a promoter operably linked to two different ORFs, one ORF encoding capsid protein L1 and the other ORF encoding capsid protein L2. EXAMPLES

[0063] Unless otherwise indicated, ingredients described in the examples are commercially available from common chemical suppliers. Some abbreviations used in the Examples section are further detailed as follows: "mg": milligram, "ng": nanogram, "pg": picogram, "fg": femtogram, "mL": milliliter, "mg / mL": milligram per milliliter, "mM": millimolar, "mmol": millimole, "pM": picomolar, "pmol": picomole, "μL": microliter, "min.": minutes and "h.": hours.

[0064] Example 1 Stoichiometric expression of adeno-associated virus (AAV) capsid proteins from a 1:1:10 RCA mixture Stoichiometric expression of VP1, VP2 and VP3 capsid proteins was demonstrated by mixing and supplementing RCA DNA at a ratio of 1:1:10 in cell-free protein expression reactions, as shown in Figure 11. 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] SEQ ID NO:2

[0067] SEQ ID NO:3

[0068] Each expression sequence contains a T7 promoter and a T7 gene 10 leader ribosome binding site to initiate cell-free mRNA transcription and protein translation, respectively. Double-stranded DNA encoding the minimal expression sequences was digested with BamHI and BglII to generate complementary overhangs and generate DNA minicircles by intramolecular ligation, followed by exonuclease treatment (ExoI, ExoIII) to digest any remaining non-circular DNA. Rolling circle amplification (RCA) was then used to generate high molecular weight hyperbranched concatemers consisting essentially of tandem repeats of each minimal expression sequence. As provided herein, RCA may be performed as disclosed in U.S. Pat. Nos. 10,077,459 and 9,938,568, the disclosures of which are incorporated herein by reference for all purposes. RCA offers the advantage of an easy, economical and robust option to perform in vitro coupled transcription and translation reactions. The RCA reagent, containing water, reaction buffer, 40 μM primers and 20 ng / μL phi29 DNA polymerase, was precleaned of contaminating DNA, followed by the addition of the ligated minicircle template and 400 μM dNTPs. Amplification was performed using a hexamer primer with 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. The RCA product was quantified from a total RCA reaction volume of 100 μL using the Quant-It™ Picogreen® dsDNA Assay Kit (ThermoFisher Inc.) and then directly applied to cell-free protein expression reactions without intermediate purification. To achieve stoichiometric expression of VP1, VP2 and VP3, individual RCA products were mixed in Expressway™ extract (ThermoFisher Inc) at a mass ratio of 1:1:10 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 each RCA DNA to the 50 μL ExpressWay reaction.FluoroTect™ GreenLYS in vitro Translation Label (Promega) was added to all cell-free expression reactions (via anticodon UUU tRNA) to randomly label nascent lysine residues with fluorescent BODIPY-FL label. All cell-free expression reactions were incubated in an Eppendorf ThermoMixer (1200 rpm) for 6 hours at 30° C. and then analyzed by SDS-PAGE to visualize all BODIPY-labeled translation products using a Typhoon Variable Mode Imager (GE Healthcare) by in-gel fluorescence. The data presented in FIG. 1 demonstrates stoichiometric expression of VP1, VP2 and VP3 capsid proteins as generated by a ratio of 1:1:10 of RCA DNA supplemented to cell-free protein expression reactions. In certain embodiments, RCA products encoding assembly activating proteins (AAPs) and expressed in a 1:1:10 ratio (relative to VP1, VP2, VP3) may be used to enhance viral capsid assembly and folding. Alternatively, an RCA mixture encoding VP3 and AAPs 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 immunoglobulin heavy and light chains is demonstrated using two different RCA nucleic acid concatemer products. Minimal expression sequences encoding 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] SEQ ID NO:5

[0072] The expression sequences contained a T7 promoter and an internal ribosome entry site (IRES) from the encephalomyocarditis virus (EMCV) to initiate cell-free mRNA transcription and protein translation, respectively. Double-stranded DNA encoding the minimal expression sequences was digested with BamHI and BglII to generate complementary overhangs and generate DNA minicircles by intramolecular ligation, followed by exonuclease treatment (ExoI, ExoIII) to digest any remaining non-circular DNA. Rolling circle amplification (RCA) was then used to generate high molecular weight hyperbranched concatemers consisting essentially of tandem repeats of each minimal expression sequence. RCA and quantification were performed as disclosed in Example 1. RCA products were quantified from 100 μL total RCA reaction volume using the Quant-It™ Picogreen™ dsDNA Assay Kit (ThermoFisher Inc) and then purified by ethanol precipitation. To achieve stoichiometric expression, individual RCA products encoding light and heavy chains were mixed in 1-Step Human Coupled IVT extracts (ThermoFisher Inc.) at a mass ratio of 1:2, respectively, resulting in a total DNA content of 125 ng per 25 μL reaction. For control purposes, individual light and heavy chains (approximately 26 kD and 52 kD, respectively) were expressed in separate cell-free reactions by adding 125 ng of each 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 (via anticodon UUU tRNA) to randomly label nascent lysine residues with fluorescent BODIPY-FL label. The data presented in Figure 12 demonstrates stoichiometric expression of light and heavy chain polypeptides as defined by the ratio of RCA DNA supplemented to the cell-free protein expression reaction. The cell-free protein expression reaction may be further expanded by increasing the microsomal content of the extract, which promotes signal peptide processing and disulfide bond formation of the final IgG molecule.

[0073] Example 3 Preparation of RCA mixture for generating functional AAV virus Recombinant AAV production typically involves the co-transfection of three different plasmids (Rep / Cap, helper and packaged DNA transgenes) into HEK293T producer cells. Transient transfection procedures are used to produce viral vectors for clinical trials because therapeutic viruses can be generated quickly, in high yields and generally at high titers (without significant pre-optimization) from anchorage-dependent producer cells compared to suspension-adapted cells. However, scale-up of AAV production is limited by the lead time and expense of plasmid DNA production. Here, we demonstrate efficient generation of functional AAV viral vectors by making a mixture of RCA DNA (encoding Rep / Cap, helper and GFP transgenes) in a 1:1:1 ratio 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, performed and quantified as disclosed herein, to generate high molecular weight hyperbranched concatemers containing tandem repeats of each plasmid. To generate AAV particles, individual RCA products were mixed in 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 linear digests of RCA DNA (after ScaI endonuclease cleavage of the concatemers into double-stranded monomers) were transfected into HEK293T cells in a similar ratio of 1:1:1. The DNA mixture was preincubated with DharmaFECT for 10 min at room temperature and then incubated with HEK293T cells for approximately 20 h, after which the transfected cells were fed with fresh medium. Three days after transfection, the cells were harvested and washed, and the AAV crude lysate was harvested by four cycles of freeze-thawing (by a dry ice / ethanol bath at approximately -72°C, followed by warming in a 37°C water bath).Cell debris was removed by centrifugation (10,000g x 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 against appropriate controls for proper gating. The data summarized in Figure 13 demonstrates equivalent AAV production from intact RCA concatemers compared to plasmid DNA. Less virus was produced with RCA DNA mass ratio 1:1:1 compared to RCA DNA molar ratio 1:1:1 using both concatemer and digested forms. Ratiometric comparison revealed that the DNA mass ratio 1:1:1 reduced the relative amount of the longest coding DNA in the DNA mixture, which may be why pHelper DNA is limiting virus production. In subsequent experiments, individual RCA DNAs were mixed with individual plasmids and the molar combination 1:1:1 was evaluated by transfection. In both cases, as shown in FIG. 14, the concatemeric RCA DNA efficiently cooperated with the plasmid DNA, thereby producing equivalent amounts of AAV virus.

[0074] Example 4 Preparation of RCA mixture for functional lentivirus generation Efficient generation of functional lentivirus was demonstrated by making a mixture of RCA DNA (separately encoding VSV-G envelope, Gag / Pol, Rev and GFP mRNA) in stoichiometric ratios and transfecting it into HEK293T cells. 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, performed and quantified as disclosed herein, to generate high molecular weight hyperbranched concatemers containing tandem repeats of each plasmid. Amplification was performed using hexamer primers with the sequence +N+N(atN)(atN)(atN)*N (AT hexamer), and 10 μM alphaS-dATP was optionally included in the pLenti-GFP reaction to thioate the resulting RCA product. To generate lentivirus, the individual RCA products were mixed in 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 ScaI or PvuI endonuclease cleavage) was transfected into HEK293T cells in a similar ratio of 1:1:1:1. The DNA mixture was preincubated with DharmaFECT for 10 min at room temperature and then incubated with HEK293T cells for approximately 20 h and then replaced with fresh medium. Spent medium (containing lentivirus) was harvested 3 days after transfection and filtered through a 0.2 μm syringe filter before transducing naive HEK293T cells in the presence of 5 μg / mL polybrene. Approximately 24 h after viral transduction, the cells were fed with fresh medium and incubated for another 2–3 days. Cells were then harvested (3-4 days post-transduction) and GFP positive cells were quantified by flow cytometry against appropriate gating controls. The data summarized in Figure 15 demonstrate non-optimized lentivirus generation from intact RCA concatemers.The particular stoichiometric mixtures of RCA products or plasmids are examples, and lentiviruses can also be generated using alternative stoichiometric mixtures. For example, high ratio mixtures of RCA DNA with plasmid DNA were tested, specifically the molar combinations 1:3:3:3 and 3:1:1:1 of pLenti-GFP:LP1:LP2:LP3, respectively. In both cases, as shown in FIG. 15, the concatemeric RCA DNA efficiently cooperated with the plasmid DNA, thereby generating comparable amounts of lentivirus.

[0075] Concatamer generation and expression products of concatemers Provided herein are techniques for using concatemers (e.g., concatemer 16) to generate desired expression products, which may include one or more proteins or nucleic acids. The generated expression products may be harvested (e.g., purified, recovered) after expression and used as part of a treatment or therapy protocol for a subject. An embodiment of the present disclosure includes generating one or more expression products from the concatemers provided herein and using the generated expression products to treat a subject. For example, the disclosed techniques may be used to generate a viral vector encoding a desired transgene. Thus, the expression products provided herein may be part of a viral vector that includes a nucleic acid having an expression sequence encoding a desired transgene, and may also be used as part of a gene therapy protocol to treat a subject with the viral vector. Since the transgene is selected based on the need for therapy by the subject, the transgene may be a variable or customizable component of the viral vector production system, while other components of the viral vector production system are constant or less variable. Furthermore, the specific components of the viral vector may be selected based on the desired tropism or patient characteristics. Thus, kits are provided herein that contain prefabricated or pregenerated portions of a viral vector production system that are fixed or selected from a small number of possible options and reagents and precursor molecules, allowing the user to generate customizable portions of the system.

[0076] In another embodiment, the concatemer expression products may be used to generate custom antibodies. In another embodiment, the concatemer expression products may be used to generate CRISPR / Cas9 systems. In another embodiment, the concatemer expression products may be used to generate virus-like particles. Although certain embodiments are disclosed that include expression of products for gene editing, gene therapy, and custom molecules, it should be understood that the disclosed technology may be used to generate expression products suitable for other applications. These include multi-subunit protein complexes (e.g., human DNA polymerase alpha is a complex with 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, illustrations of specific terms should be considered as non-limiting examples. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Approximate language, as used in the present invention throughout this specification and claims, may be applied to modify any quantitative expression that may vary within permissible limits without causing a change in the basic function involved.

[0078] In the present disclosure, nucleic acid amplification techniques may 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 prime a nucleic acid synthesis reaction. A primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. A primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of a primer are determined empirically. The lower limit of the primer length is the minimum length required to form a stable duplex upon hybridization with a target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 nucleotides in length) do not form thermodynamically stable duplexes with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of forming a duplex in a region of the target nucleic acid other than the predetermined nucleic acid sequence. In general, suitable primer lengths range from about 3 nucleotides to about 40 nucleotides in length.

[0079] As used herein, the term "random primer" refers to a mixture of primer sequences that are generated by randomizing the nucleotide at any given position in an oligonucleotide sequence such that the given position can consist of any of the possible nucleotides or analogs thereof (fully randomized). Thus, a random primer is a random mixture of oligonucleotide sequences that consists of all possible combinations of nucleotides in 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 hexamer combinations of the four DNA nucleotides A, C, G, and T, and is represented by the sequence NNNNNN. 6This results in a random mixture containing (4,096) unique hexameric DNA oligonucleotide sequences. Random primers can be used efficiently to prime nucleic acid synthesis reactions when the target nucleic acid sequence is unknown or to perform whole genome amplification reactions. Random primers can also be more effective at priming and generating double-stranded rolling circle amplification (RCA) products than single-stranded RCA products, depending on the primer concentration.

[0080] As used herein, the term "nucleotide analog" refers to a compound that is structurally similar to a naturally occurring nucleotide. A nucleotide analog may have an altered phosphate backbone, sugar moiety, nucleobase, or a combination thereof. A nucleotide analog may be a natural nucleotide, a synthetic nucleotide, a modified nucleotide, or an alternative replacement moiety (e.g., inosine). In general, nucleotide analogs with altered nucleobases confer, among other things, different base pairing and base stacking properties. As used herein, the term "LNA (locked nucleic acid) nucleotide" refers to a nucleotide analog in which the sugar moiety of the nucleotide comprises a bicyclic furanose unit locked in a ribonucleic acid (RNA) mimicking sugar conformation. The structural change from deoxyribonucleotides (or ribonucleotides) to LNA nucleotides, i.e. the introduction of an additional bond between the carbon atoms at the 2' and 4' positions (e.g. 2'-C, 4'-C-oxymethylene bond, see e.g. Singh, SK et al., Chem. Comm., 4, 455-456, 1998 or Koshkin, AA et al., Tetrahedron, 54, 3607-3630, 1998), is restricted from a chemical point of view. The 2' and 4' positions of the furanose units in LNA nucleotides can be linked by O-methylene (e.g. oxy-LNA: 2'-O,4'-C-methylene-β-D-ribofuranosyl nucleotides), S-methylene (thio-LNA) or NH-methylene moieties (amino-LNA), etc. Such bonds restrict the conformational freedom of the furanose ring. LNA oligonucleotides exhibit enhanced hybridization affinity to complementary single-stranded RNA and complementary single- or double-stranded DNA. A-form (RNA-like) double-stranded conformations can be induced by LNA oligonucleotides. Nucleotide analogs with altered phosphate-sugar backbones (e.g., PNA, LNA) often modify, among other things, strand properties, such as secondary structure formation. An asterisk (*) in front of 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 in front of a letter designation means that the nucleotide represented by the letter 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 term "phosphorothioated nucleotide" or "thioated nucleotide" refers to a nucleotide with an altered phosphate backbone, in which the sugar moiety is linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, a phosphorothioate bond contains a sulfur atom instead of a non-bridging oxygen atom. This modification can make the internucleotide 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., a single / double stranded DNA circle) by a rolling circle mechanism. A rolling circle amplification reaction is initiated by hybridization of a primer to a circular, often single stranded nucleic acid template. A nucleic acid polymerase then extends the primer hybridized to the circular nucleic acid template by continuously proceeding around the circular nucleic acid template, replicating the sequence of the nucleic acid template over and over again (rolling circle mechanism). Rolling circle amplification typically produces concatemers that contain tandem repeats of the circular nucleic acid template sequence. Rolling circle amplification can be linear RCA (LRCA), which exhibits linear amplification kinetics (e.g., RCA using a single stranded specific primer), or exponential RCA (ERCA), which exhibits exponential amplification kinetics. Rolling circle amplification can also be performed using multiple primers (multiplex-primed rolling circle amplification or MPRCA) to generate hyperbranched concatemers. For example, in double-primed RCA, one primer can be complementary to the circular nucleic acid template, as in 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 with exponential amplification kinetics that characterize a cascade of multiple hybridizations, primer extensions, and strand displacement events involving both primers and both strands. This often results in the generation of a separate set of concatemeric double-stranded nucleic acid amplification products. RCA can be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase. A suitable polymerase has the ability to synthesize strand-displacement DNA. In certain embodiments, rolling circle amplification can be performed using a random primer mixture that includes nucleotide analogs.

[0082] As used herein, the term "rolling circle amplification (RCA) product" or "RCA-generated DNA" refers to a nucleic acid amplification product, in which a circular nucleic acid template (e.g., a single / double stranded DNA circle) is amplified by a rolling circle amplification reaction mechanism. The template is small in size compared to the RCA product. Rolling circle amplification typically generates concatemers that contain tandem repeats of the circular nucleic acid template sequence. Rolling circle amplification-generated DNA can be generated by linear RCA (LRCA), which exhibits linear amplification kinetics (e.g., RCA using a single stranded specific primer), or by exponential RCA (ERCA), which exhibits exponential amplification kinetics. Rolling circle amplification-generated DNA can also be generated by using multiple primers (multiple prime rolling circle amplification or MPRCA), in which the rolling circle amplification-generated DNA is a hyperbranched concatemer. In double prime RCA, one primer can be complementary to a circular nucleic acid template, as in linear RCA, while the other can be complementary to a tandem repeat unit nucleic acid sequence of RCA-generated DNA, which 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 rolling circle amplification (RCA), can be used to generate large amounts of high-quality DNA starting from a circular nucleic acid template. Rolling circle amplification can generate nucleic acid concatemers that contain tandem repeat units of the corresponding circular nucleic acid template. The nucleic acid concatemers can be linear or branched concatemers.

[0084] As used herein, the term "nucleic acid concatemer" (e.g., concatemer 16 disclosed herein) refers to a nucleic acid molecule having a tandem repeat or tandem repeat unit of a nucleic acid sequence (e.g., expression sequence 12). The terms "concatamer", "nucleic acid concatemer" and "DNA concatemer" may be used interchangeably throughout this disclosure. Concatamers may be single or double stranded. As used herein, the term "double stranded concatemer DNA" refers to a double stranded DNA molecule that contains multiple copies of the same DNA sequence linked in tandem. Concatamers generated by RCA may be larger than 1 kilobase (kb), larger than 10 kb, and larger than 150 kb. In one embodiment, concatemers may range from 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 may vary. Thus, RCA performed on the template solution can generate a pool of concatemers having tandem repeats of the same sequence, but with a variable number of tandem repeats and therefore with variable lengths. In certain embodiments, the nucleic acid concatemers can include a plurality of tandem repeat sequences, where each of the plurality of tandem repeat sequences includes an expressed sequence that encodes 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 is essentially composed of a minimal expression sequence. The resulting concatemer comprises a tandem repeat of the minimal expression sequence derived from the DNA minicircle. In certain embodiments, the minimal expression sequence is essentially composed of a promoter, a cap-independent translation element, and an open reading frame. In certain embodiments, the nucleic acid concatemer comprises modified nucleotides, nucleotide analogs, or a combination thereof.

[0086] As used herein, the term "expression sequence" or "repeated unit of expression sequence" (e.g., expression sequence 12) refers to a DNA sequence capable of expressing RNA and / or protein. Thus, the expression product includes protein, RNA, or a mixture thereof.

[0087] In certain embodiments where expression of a protein is desired, the expression sequence may comprise an expression-competent unit comprising an open reading frame (ORF) and a promoter operably linked to the open reading frame. In one embodiment, the ORF may code for one or more proteins. The encoded proteins may be the same or different. In some embodiments, the expression sequence may comprise one promoter operably linked to two or more ORFs.

[0088] In certain embodiments where expression of RNA is desired, the expression sequence may comprise an RNA expression competent unit comprising at least one promoter and a transcription termination sequence.

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

[0090] Many examples of suitable promoters are known in the art, 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 trunks, species-independent translation leaders (SITS), Kozak consensus sequences, and Shine-Dalgarno sequences. Insulator sequences generally enhance the efficiency of ribosome binding or translation initiation. Numerous examples of suitable insulator sequences exist in the art, including, for example, sequences encoding poly-histidine trunks. In some embodiments, insulator sequences may be empirically determined by inserting spacer sequences 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 may 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 expressed sequence may include codon-optimized sequences, purification tag sequences, amino-terminal peptide fusion sequences derived from an IRES, sequences for protease cleavage or nucleolytic cleavage, or combinations thereof. In some embodiments, the expressed sequence includes both coding and non-coding sequences.

[0093] The codon-optimized sequence of the open reading frame can enhance the translation rate and quality of the RCA product. Codon optimization generally increases protein expression by improving the translation efficiency of the gene of interest. Gene functionality can also be improved by optimizing codon usage in custom-designed genes. In codon-optimized embodiments, low-frequency codons in a species can be replaced by high-frequency codons, for example, the low-frequency codon UUA can be replaced by the high-frequency codon CUG for leucine. Codon optimization can improve mRNA stability, thus modifying the rate of protein translation or protein folding. Furthermore, codon optimization can customize the regulation of transcription and translation, modify ribosome binding sites, or stabilize mRNA degradation sites.

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

[0095] The open reading frame of the expressed sequence may contain an amino terminal peptide fusion sequence derived from an IRES for enhanced ribosome recognition.

[0096] In some embodiments, the expression sequence comprises a coding sequence, where the coding sequence encodes or produces a desired protein expression product in a eukaryotic cell. A coding sequence is a nucleic acid sequence that comprises a particular gene of interest. Generally, a coding sequence comprises a promoter and an open reading frame (ORF). The coding sequence may optionally comprise a cap-independent translation element (CITE). In some embodiments, the coding sequence further comprises a ribosome binding site. The coding sequence may comprise a transcription termination sequence located outside the open reading frame but within the expression sequence.

[0097] In one or more embodiments, each of the plurality of tandem repeat sequences comprises at least one expression sequence. In some embodiments, the at least one expression sequence comprises at least one coding sequence. In such embodiments, the at least one coding sequence of the at least one expression sequence comprises at least one promoter and at least one open reading frame. In some embodiments, each of the plurality of tandem repeat sequences comprises two or more expression sequences. The two or more expression sequences comprising coding sequences may code for the same protein or different proteins. In some embodiments, the expression sequence comprises at least one promoter operably linked to at least one open reading frame. For example, in one aspect, in the expression sequence, one promoter is operably linked to one open reading frame. In another aspect, in the expression sequence, one promoter is operably linked to two different open reading frames. In some embodiments, the expression sequence may comprise two or more promoters operably linked to two or more open reading frames.

[0098] The expression sequence 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 than the other. In this example, a single promoter is operably linked to the two open reading frames via a cap-independent translation element. Each of the open reading frames includes a translation initiation and a translation termination sequence. A translation termination or stop sequence is required in the expression sequence, otherwise an infinite polyprotein may be synthesized, which is undesirable. However, a transcription termination codon may be optional for the first open reading frame, which upon transcription results in the production of a polycistronic mRNA. In such a case, an intervening sequence between the first and second open reading frames may be selected such that upon in vivo protein expression, a single polycistronic mRNA is produced, but which may be translated into two different proteins. After the synthesis of a first protein by translation of a first open reading frame, ribosome slippage to the second translation initiation sequence of the second open reading frame can initiate the synthesis of a second protein from the second open reading frame. This can be achieved by incorporating a "self-cleaving sequence" between the first and second open reading frames. A suitable self-cleaving sequence, for example, a viral P2A motif, facilitates the production of two or more proteins from one single mRNA.

[0099] In some embodiments, the expression sequence includes non-coding sequences, where the non-coding sequences generate the desired RNA expression product. Such expression sequences do not include any coding sequences. Non-coding sequences include promoters and transcription termination sequences. Non-coding sequences generally lack an open reading frame. Expression sequences that include non-coding sequences are also referred to as RNA expression sequences. In some embodiments, the expression sequence consists essentially of non-coding sequences. In some other embodiments, the expression sequence includes both coding and non-coding sequences, where RNA can be generated from the non-coding sequences of the expression sequence. In such embodiments, the desired protein can also be subsequently generated from the coding sequences of the same expression sequence. In some embodiments, the generated RNA can be extracted from the eukaryotic cell for various downstream applications. In one embodiment, the extracted RNA can be subsequently packaged and delivered to another cell by a lentiviral system. Non-coding sequences may include, but are not limited to, sequences for antisense RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), microRNA mimic, transfer RNA (tRNA), ribosomal RNA (rRNA), or combinations thereof. Non-coding sequences may also include CRISPR RNA (tracrRNA, crRNA, sgRNA, or gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Piwi-binding RNA (piRNA), telomerase RNA, spliceosomal RNA, enhancer RNA, retrotransposon, X-inactivation specific transcript (Xist), RNA encoded by RNA polymerase I and RNA polymerase III, or combinations thereof.

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

[0101] The open reading frame includes a translation initiation and a translation termination sequence. In some embodiments, the open reading frame includes a codon-optimized 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 enhancing element (TEE), or a combination thereof.

[0102] The desired protein may be purified by a tag sequence, where the tag sequence may be a fusion tag for affinity purification, a tag for protease cleavage, or a combination thereof. The fusion tag for affinity purification may be used for rapid purification and detection of the expressed protein. Such tags are also called affinity tags. The affinity tag may include polyhistidine tags, glutathione S-transferase tags (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. Although the fusion tag is useful in rapid purification and detection of the desired protein, the tag may not be considered a permanent fixture or domain of the recombinant protein. Thus, removal of the fusion tag is often required for advanced analytical testing of recombinant protein structure and function. The tag for purification may 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 and can include, for example, PreScission™ Protease Tags (GE Healthcare Life Sciences) or Thrombin Protease Tags (GE Healthcare Life Sciences).

[0103] As mentioned 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-specific codon preference, and / or individual codon preference. The codon-optimized sequence of the open reading frame may enhance the translation rate and quality of the RCA product. Codon optimization generally increases protein expression from the coding sequence by improving the translation efficiency of the gene of interest. Gene functionality may also be improved by optimizing codon usage in the custom-designed gene. In codon-optimized embodiments, low-frequency codons in a species may be replaced by high-frequency codons, for example, the low-frequency codon UUA may be replaced by the high-frequency codon CUG for leucine. Codon optimization may improve mRNA stability, thus modifying the rate of protein translation or protein folding. Furthermore, codon optimization may customize the regulation of transcription and translation, modify ribosome binding sites, or stabilize mRNA degradation sites.

[0104] Transcription termination sequences are generally located at the 3' end of genes in DNA templates. Transcription termination sequences provide a signal in newly synthesized mRNA to initiate the process of releasing mRNA from the transcription complex, which may also aid in the efficient translation of desired protein products. Insulator sequences generally enhance the efficiency of ribosome binding or translation initiation. Numerous examples of suitable insulator sequences exist in the art and may be used, including sequences encoding polyhistidine tracts. In some embodiments, insulator sequences may be empirically determined by inserting spacer sequences around the ribosome binding site or optimizing or inserting codons at the N-terminus of expressed proteins.

[0105] In some embodiments, the expression sequence 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 enhanced ribosome recognition, or a combination thereof. The expression sequence further includes a polyA sequence, a transcription termination sequence, an insulator sequence, or a combination thereof.

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

[0107] The double-stranded concatemeric DNA may further comprise 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 sequence used in rolling circle amplification.

[0108] A variety of methods may be used to prepare DNA minicircle templates for use with the disclosed technology. In some embodiments, a linear DNA template may be circularized to generate a DNA minicircle template. In an exemplary embodiment, circularization of the linear DNA template may occur by an enzymatic reaction, for example, by incubation with a ligation enzyme, such as DNA ligase. In some embodiments, both ends of the linear DNA template may be hybridized to a nucleic acid sequence to bring the ends into close proximity. Incubation with a ligation enzyme may then result in circularization of the hybridized linear DNA template to generate a DNA minicircle. Suitable DNA minicircle templates may also be generated by PCR amplification of a portion of a larger DNA (e.g., genomic DNA or DNA from a DNA library) using appropriate PCR primers, followed by circularization of the PCR product. DNA minicircles may also be generated by chemical synthesis of suitable linear oligonucleotides, followed by circularization of the synthesized oligonucleotides. In some embodiments, a synthetic linear oligonucleotide consists essentially of a minimal expression sequence and can be subjected to circularization with DNA ligase to generate a DNA minicircle.

[0109] Provided herein are expression systems (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 CHO, NIH3T3, BHK, HepG2, and HEK293 cell-based expression systems.

[0110] In certain embodiments, "cell-free expression system" refers to an in vitro transcription and translation system. Cell-free expression generally encompasses two forms: (1) mRNA and protein are produced in a single reaction, or (2) mRNA is produced in a first reaction, and the resulting mRNA product is added to a second separate translation reaction. RCA products derived from DNA minicircles may be utilized in either form (1) or (2). For example, in one embodiment, RCA products may be provided for an "in vitro transcription-translation coupled reaction", where RCA-generated DNA is converted to mRNA, and the mRNA expresses the protein simultaneously in one reaction mixture capable of producing both RNA and protein. In another embodiment, RCA products may be provided for a "transcription-translation coupled reaction", where RCA-generated DNA is first converted to mRNA, and the mRNA is added separately to a translation reaction mixture to express the protein. In certain embodiments, concatemers are provided to a cell-free expression system that is unprocessed (or has not undergone any further processing). In one embodiment, the concatemers are added to the cell-free system immediately after amplification such as RCA. The term "further processing" is meant to include the action of restriction digestion, ligation or a combination of these of the concatemers. However, in some embodiments, the RCA products can be separated (e.g., by precipitation) to remove salts or other contaminants, such as primers or small fragmented DNA, from the reaction medium before proceeding to cell-free expression, for example, using eukaryotic cell extracts.

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

[0112] Concatemeric DNA (e.g., double-stranded concatemeric DNA) may 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, a eukaryotic cell is provided that includes an exogenous double-stranded concatemeric DNA that includes a plurality of tandem repeat sequences. In one embodiment, each of the plurality of tandem repeat sequences includes phosphorothioated nucleotides, where the ratio of phosphorothioated nucleotides to total nucleotides is at least 1:1600. The exogenous double-stranded concatemeric DNA that is used for transfection into a eukaryotic cell to generate the cell can be unprocessed or processed RCA-generated DNA. The eukaryotic cell can be a protozoan, a yeast cell, an insect cell, or a mammalian cell.

[0114] The technical effects of the disclosed embodiments include improved expression of desired expression products using nucleic acid concatemers with sizes not typically associated with robust transfection and production of desired expression products. Furthermore, concatemers may contain unknown or variable numbers of tandem repeats of expression sequences, but nevertheless various concatemers may be co-expressed ratiometrically, e.g., based on molar ratios, to generate robust ratios of generated expression products based on evidence. This disclosure demonstrates that concatemers with nucleic acid expression sequences for one or more desired expression products may be used in place of and / or in combination with traditional expression techniques, e.g., plasmids, to generate desired expression products with increased purity and robust co-expression. Furthermore, nucleic acid concatemers may be used to generate more complex structures or assemblies in which multiple co-expression products are associated with each other.

[0115] The above examples are illustrative of some features of the present disclosure, and are selected from among all possible embodiments. Although only certain features are illustrated and described herein, those skilled in the art, having the benefit of this disclosure, may make modifications / alterations to optimize parameters. Thus, the above embodiments are considered to be illustrative in all respects, rather than limiting the embodiments described herein. Where ranges are given, these ranges encompass all subranges therebetween.

[0116] The description uses examples, including the best mode and to enable one of ordinary skill in the art to practice the disclosed embodiments, including making and using the devices or systems, and practicing any incorporated methods. The patentable scope is defined by the claims, and may include other examples that occur to those of ordinary skill 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 literary language of the claims, or if they include equivalent structural elements that have insubstantial differences from the literary language of the claims.

Claims

1. 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; 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. The method includes:

2. 2. The method of claim 1, comprising generating the first nucleic acid concatemer and the second nucleic acid concatemer using a rolling circle amplification method.

3. 3. The method of claim 2, comprising amplifying the circular or plasmid DNA using rolling circle amplification to generate the first nucleic acid concatemer and / or the second nucleic acid concatemer.

4. 4. The method of claim 1, 2 or 3, wherein the first tandem repeat and / or the second tandem repeat is a tandem repeat of at least a portion of a circular or plasmid DNA.

5. 5. The method of claim 1, comprising transfecting a cultured cell with the concatemer mixture to effect co-expression, or wherein the co-expression is effected in a cell-free expression system.

6. 6. The method of any one of claims 1 to 5, wherein the first nucleic acid concatemer, the second nucleic acid concatemer, or both, are greater than 10 kb in length.

7. 7. The method of any one of claims 1 to 6, comprising harvesting the first expression product and the second expression product.

8. 8. The method of claim 1, wherein one or both of the first nucleic acid concatemer or the second nucleic acid concatemer are unprocessed prior to co-expression.

9. 9. The method of any one of claims 1 to 8, wherein a first expression product is a viral envelope or packaging protein and a second expression product comprises a transgene of the virus, and the first expression product and the second expression product form a viral vector for delivering the transgene.

10. 10. The method of 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.

11. The method of claim 10, wherein the first expression product forms a complex with the second expression product.

12. 12. The method of claim 1, wherein the first expression product acts on a third nucleic acid concatemer in the concatemer mixture.

13. Producing a mixture comprising at least two nucleic acid concatemers in a predetermined ratio, each of the nucleic acid concatemers comprising tandem repeats of two or more nucleic acid sequences; co-expressing the mixture of concatemers to produce two or more expression products from each nucleic acid concatemer in the mixture; The method includes:

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

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

16. amplifying at least one template comprising a first nucleic acid sequence using strand displacement rolling circle amplification to generate first concatemers comprising tandem repeats of said first nucleic acid sequence; contacting the first concatemers with second concatemers comprising tandem repeats of a second nucleic acid sequence to form a concatemer mixture in which the first nucleic acid concatemers are present in a predetermined ratio to the second nucleic acid concatemers; 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 a predetermined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture; The method includes:

17. 17. The method of claim 16, comprising a step of allowing a first expression product to form a complex with a second expression product.

18. 18. The method of claim 16 or 17, wherein the first expression product and the second expression product comprise different viral products from the same virus, such as an adenovirus or a lentivirus.

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

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