WO2022098747A1

WO2022098747A1 - Nucleic acid synthesis device and methods of use

Info

Publication number: WO2022098747A1
Application number: PCT/US2021/057882
Authority: WO
Inventors: Solomon SSENYANGE; Javelin CHI
Original assignee: Single Helix Genomics, Inc.
Priority date: 2020-11-03
Filing date: 2021-11-03
Publication date: 2022-05-12

Abstract

The present disclosure provides a device for the in vitro synthesis of nucleic acids, such as RNA. In at least one embodiment, the device includes a solid support comprising (a) a sample inlet chamber in thermal contact with a first thermoelectric device; (b) a reaction chamber in thermal contact with a second thermoelectric device; (c) a bead inlet chamber for introduction of magnetic beads into the device; (d) a bead mixing chamber in thermal contact with the second thermoelectric device; (e) a separation chamber in magnetic contact with a magnet; and (f) a product outlet chamber in thermal contact with a third thermoelectric device. The disclosure also provides associated systems, and methods for nucleic acid synthesis useful with the device.

Description

NUCLEIC ACID SYNTHESIS DEVICE AND METHODS OF USE

FIELD

[0001] The present disclosure relates to devices, systems, and methods useful for the synthesis of nucleic acids, such as the in vitro synthesis of RNA.

BACKGROUND

[0002] The nucleic acid RNA is emerging as an important molecule for use in human therapeutics. RNA-based therapeutics in the areas of vaccines, immunotherapy, and gene therapy are undergoing development and considered among the most promising areas of human medicine. For example, mRNA molecules encoding viral antigens are gaining use as vaccines (see e.g., Fotin-Mleczek et al. 2012. J. Gene Med. 14(6) :428-439). RNA molecules are also undergoing development in potential human enzyme and protein replacement therapies (see e.g., Kariko et al., 2012. Mol. Ther. 20(5) :948-953; Kormann et al., 2012. Nat. Biotechnol. 29(2): 154-157). Additionally, RNA molecules are being developed for use as immunostimulatory therapeutics (see e.g. W02009/095226A2) and in potential therapies based on CRISPR/Cas9 genome editing.

[0003] Processes for the synthesis and manufacture of nucleic acids currently approved by regulatory authorities, however, require many separate manufacturing steps performed by several different devices, and with quality control performed at the DNA level and the RNA level (see e.g., WO2016/180430A1 ). Synthesis of RNA for therapeutics requires a large degree of manual handling in a GMP-regulated laboratory executed by highly-trained technicians, and current established manufacturing processes are time consuming, cost intensive, and require a lot of laboratory space and equipment. A critical step in RNA production is generation of the DNA template, which adds significant cost at the industrial scale. The DNA template, however, is used for only a single in vitro RNA synthesis reaction and then is digested by DNAse and removed during purification to ensure efficacy and safety of the synthesized RNA molecule.

[0004] Accordingly, there remains a need in the art for improved devices, systems, and processes for the efficient, automated in vitro synthesis of nucleic acids, particularly, the rapid synthesis of RNA via in vitro transcription from individual human DNA samples to allow for use in personalized medical treatments.

SUMMARY

[0005] The following paragraphs are intended to introduce the detailed description and not intended to define or limit the subject matter of the present disclosure.

[0006] In at least one embodiment, the present disclosure provides a device for synthesis of a nucleic acid comprising: a solid support comprising: a sample inlet chamber, wherein the inlet chamber comprises an sample inlet access port and is in thermal contact with a first thermoelectric device; a reaction chamber, wherein the reaction chamber is coupled to the sample inlet chamber via a fluidic channel and is in thermal contact with a second thermoelectric device; a bead inlet chamber, wherein the bead inlet chamber comprises an bead inlet access port for introduction of magnetic beads into the device; a bead mixing chamber, wherein the bead mixing chamber is coupled to the reaction chamber via a fluidic channel and to the bead inlet chamber via a fluidic channel, and is in thermal contact with the second thermoelectric device; a separation chamber, wherein the separation chamber is in magnetic contact with a magnet and is coupled to (i) a washing access port via a fluidic channel, (ii) an elution access port via a fluidic channel, and (iii) a waste outlet port via a fluidic channel; and a product outlet chamber, wherein the outlet chamber comprises a product outlet access port and is coupled to the separation chamber via a fluidic channel, and is in thermal contact with a third thermoelectric device.

[0007] In at least one embodiment, the device further comprises a quality control chamber, wherein the quality control chamber comprises a quality control access port and is coupled to the separation chamber via a fluidic channel, and is in thermal contact with a third thermoelectric device.

[0008] In at least one embodiment, the device further comprises a pump unit coupled to the sample inlet access port and/or the product outlet access port, wherein the pump unit is capable of controlling pressure within the device. In at least one embodiment, the pump unit is capable of controlling movement of liquids through the device. In at least one embodiment of the device, the pump unit is capable of increasing the pressure in the reaction chamber of the device to a pressure of between about 5 psi and about 200 psi, thereby accelerating reactions; optionally, a pressure of between about 20 psi and about 150 psi.

[0009] In at least one embodiment of the device, the access ports are sealable; optionally, wherein the access ports when sealed are capable of withstanding a pressure in the device of at least about 5 psi. In at least one embodiment, the access ports comprise sealable caps.

[0010] In at least one embodiment of the device, the first, second, and third thermoelectric devices are independently controllable. In at least one embodiment, the first, second, and/or third thermoelectric device(s) is/are capable of heating and/or cooling the chamber with which it is in thermal contact. In at least one embodiment, the first, second, and/or third thermoelectric device(s) is/are capable of heating and/or cooling the chamber with which it is in thermal contact over a temperature range of from about -90°C to about 100°C; optionally, a temperature range of from about -20°C to about 100°C. In at least one embodiment, the first, second, and/or third thermoelectric device(s) is/are capable of heating and/or cooling the chamber with which it is in thermal contact at a rate of about 1 °C/min to about 3°C/min. In at least one embodiment, the first, second, and/or third thermoelectric device(s) comprise(s) a Peltier device. In at least one embodiment, at least the third thermoelectric device comprises a Peltier device capable of maintaining the outlet chamber at a temperature of between about -90°C and 10°C; optionally, a temperature range of from about -20°C to about 8°C. In at least one embodiment, the first and second thermoelectric devices are capable only of heating the chamber with which each is in thermal contact to a temperature of about room temperature to about 100°C, and the third thermoelectric device is capable only of cooling the outlet chamber to a temperature of between about -90°C and 10°C; optionally, a temperature range of from about -20°C to about 8°C.

[0011] In at least one embodiment of the device, the fluidic channel coupling the separation chamber to the product outlet chamber comprises a macromolecule separation material; optionally, capable of separating single-stranded RNA from DNA template, and/or proteins. [0012] In at least one embodiment of the device, one or more of the access ports and/or fluidic channels comprises a filter that excludes materials of a size greater than the filter size exclusion limit from entering one or more of the chambers; optionally, wherein the filter size exclusion limit is from about 0.10 pm to about 0.50 pm.

[0013] In at least one embodiment of the device, the magnetic field of the magnet is adjustable; optionally, wherein the magnet comprises an electromagnet.

[0014] In at least one embodiment of the device, the solid support comprises a material selected from a polymer, a glass, a ceramic, a metal, or a combination thereof. In at least one embodiment, the solid support comprises a piece prepared by injection molding. In at least one embodiment, the thermoelectric devices are in contact with the underside of the solid support. In at least one embodiment, the thermoelectric devices are integrated in the material of the solid support; optionally, wherein the electrical connections to the thermoelectric devices are integrated in the material of the solid support. In at least one embodiment, the access ports, fluidic channels, and/or chambers are integrated in the material of the solid support. In at least one embodiment, at least one of the chambers comprises an interior surface coated with a metal-based catalyst; optionally, wherein the metal-based catalyst comprises ruthenium or platinum.

[0015] In at least one embodiment of the device, the fluidic channels have a width of about 10 pm to about 3000 pm and a depth of about 10 pm to about 3000 pm.

[0016] In at least one embodiment of the device, the chambers have a volume of from about 50 pL to about 500 pL.

[0017] The present disclosure also provides methods using a device of the present disclosure for the in vitro synthesis of nucleic acids. Accordingly, in at least one embodiment, the present disclosure provides a method for RNA synthesis comprising: (a) introducing a reaction mixture comprising a DNA template, an RNA polymerase, ribonucleotide triphosphates (rNTPs), and a buffer solution through an access port into an inlet chamber in thermal contact with a first thermoelectric device, and allowing the reaction mixture to reach a temperature T1 of about 35°C to about 38°C for a time of at least 1 h;

(b) transferring the reaction mixture through a fluidic channel to a reaction chamber in thermal contact with a second thermoelectric device, and incubating the reaction mixture at a temperature T2 of between about 37°C and about 40°C for a time of at least 1 h, whereby the RNA polymerase catalyzes DNA template-dependent synthesis of RNA;

(c) transferring the reaction mixture through a fluidic channel to a bead mixing chamber in thermal contact with the second thermoelectric device and which contains magnetic beads capable of selectively binding to RNA, whereby the synthesized RNA in the reaction mixture binds to the magnetic beads;

(d) transferring the reaction mixture and RNA-bound magnetic beads through a fluidic channel to a separation chamber in magnetic contact with a magnetic pad, whereby the magnetic beads are immobilized in the separation chamber;

(e) transferring a wash solution from an access port through a fluidic channel into the separation chamber and then out through a waste outlet port through a fluidic channel, whereby RNA polymerase, DNA template, and unreacted rNTPs are removed while the RNA-bound magnetic beads are retained in the separation chamber;

(f) transferring an elution solution from an access port through a fluidic channel to the separation chamber, whereby the bound RNA is eluted from the immobilized beads to form a synthesized RNA solution; and

(g) transferring the synthesized RNA solution through a fluidic channel to an outlet chamber in thermal contact with a third thermoelectric device, and allowing the synthesized RNA solution to reach a temperature T3 of between about -90°C to about 8°C.

[0018] In at least one embodiment of the method, step (b) further comprises increasing the pressure in the reaction chamber to at least 5 psi via application of pressure through a fluidic channel, thereby increasing the rate of the RNA synthesis reaction.

[0019] In at least one embodiment of the method, the transferring of step (g) further comprises passing the synthesized RNA solution through a macromolecule separation material; optionally, wherein the macromolecule separation material is capable of separating singlestranded RNA from DNA template, and/or proteins.

[0020] In at least one embodiment of the method, the transfer through the fluidic channels is driven by positive or negative pressure applied by a pump unit coupled to an access port of the device.

[0021] In at least one embodiment of the method, transfer of macromolecules through at least part of the device is driven by an applied electric potential inducing an electrophoretic force. [0022] In at least one embodiment of the method, the sample mixture has a volume of less than 500 pL; optionally, wherein the sample mixture has a volume of between about 50 pL and about 500 pL.

[0023] In at least one embodiment of the method, the amount of DNA template in the sample mixture is less than 1 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.1 pg and about 50 pg.

[0024] In at least one embodiment of the method, the synthesized RNA solution has a volume of less than 500 pL; optionally, wherein the synthesized RNA solution has a volume of between about 50 pL and about 500 pL.

[0025] In at least one embodiment of the method, the amount of RNA in the synthesized RNA solution is at least 25 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.5 pg and about 500 pg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0027] FIG. 1 depicts a top view of an exemplary in vitro nucleic acid synthesis device of the present disclosure.

[0028] FIG. 2 depicts a side view of an exemplary in vitro nucleic acid synthesis device of the present disclosure.

[0029] FIG. 3 depicts a back view of an exemplary in vitro nucleic acid synthesis device of the present disclosure.

DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the compositions, devices, systems, methods, and processes of the present disclosure are described in greater detail below to illustrate the claimed subject matter. None of the exemplary embodiments described herein are intended to limit the claimed subject matter, which may cover compositions, devices, systems, methods, and processes that differ from those described below. Moreover, the claimed subject matter is not limited to compositions, devices, systems, methods, and processes having all of the features described below, or common to all of the exemplary embodiments described below. Also, the detailed description may include compositions, devices, systems, methods, and processes that are not within the claimed subject matter. Any subject matter disclosed herein and not within the subject matter of the claims of the present disclosure may be within the claimed subject matter of, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[0031] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a fluidic channel” includes more than one fluidic channel, and reference to “a protein molecule” refers to more than one protein molecule. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. For example, a range of 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0032] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range, as will be readily recognized by context. Similarly, other terms of degree such as "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.

[0033] Generally, the nomenclature used herein and the techniques, reagents, procedures, and protocols related to nucleic acid isolation, purification, synthesis (e.g., in v/tra mRNA transcription), amplification (e.g., PGR), and analysis, described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common biochemical and biotechnological techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011 , and now available in journal format online as Current Protocols in Molecular Biology, Vols. 00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0034] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0036] A. Definitions

[0037] “Nucleoside,” as used herein, refers to a molecular moiety that comprises a naturally occurring or a non-naturally occurring nucleobase attached to a sugar moiety (e.g., ribose or deoxyribose), and can include ribonucleosides and deoxyribonucleosides.

[0038] “Nucleotide,” as used herein refers to a nucleoside-5’-phosphate molecule or a structural analog of a nucleoside-5’-phosphate. Exemplary nucleotides include, but are not limited to, ribonucleoside-5’-triphosphates (or “rNTP”) e.g., rATP, rCTP, rGTP, rTTP, and rUTP, deoxyribonucleotide-5’-triphosphates (or “dNTP”) e.g., dATP, dCTP, dGTP, dTTP, and dUTP, and structural analogs of nucleoside-5’-phosphate molecules and that have a modified nucleobase moiety (e.g., a substituted pyrimidine nucleobase such as 5-ethynyl-dU), a modified sugar moiety (e.g., an O-alkylated sugar, or a 2’-4’ “locked” ribose), and/or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thio-phosphate, a methylene, and/or other bridges between phosphates).

[0039] “Nucleic acid,” as used herein, refers to an oligomeric or polymeric molecule of nucleotide or nucleotide analog subunits. Nucleic acid can refer to a naturally occurring or synthetic, oligomer or polymer of ribonucleotides or deoxyribonucleotides, also referred to as an oligonucleotide or polynucleotide, and includes DNA, RNA, in both single and double-stranded form, and hybrids thereof. It is also intended that “nucleic acid” can refer to a oligomeric or polymeric molecule comprising phosphodiester linkages and/or other non-natural linkages (e.g., phosphorothioate, methyl phosphonate, phosphotriester, phosphoramide, boronophosphate) between subunits. [0040] “RNA” as used herein refers to a ribonucleic acid molecule, including oligomers or polymers of the ribonucleotides of the five naturally occurring nucleobases, A, C, G, T, and U, as well as oligomer or polymers of synthetic ribonucleotides. RNA can include the various types of biologically active RNAs, including but not limited to mRNA, tRNA, rRNA, snRNAs, miRNA, siRNA, RNAi, and other non-coding RNAs.

[0041] DNA” as used herein to a deoxyribonucleic acid molecule, including oligomers or polymers of the deoxyribonucleotides of the five canonical nucleic acid bases, A, C, G, T, and U, as well as oligomer or polymers of synthetic deoxyribonucleotides.

[0042] “Polymerase,” as used herein, refers to any natural or non-naturally occurring enzyme or other catalyst that is capable of catalyzing a polymerization reaction, such as the polymerization of nucleotide monomers to form a nucleic acid polymer. The term polymerase encompasses a variety of enzymes including, but not limited to, DNA polymerases, RNA polymerases, and reverse transcriptases. Exemplary polymerases that may be used in the devices and methods of the present disclosure include, but are not limited to, the nucleic acid polymerases, RNA polymerase (e.g., enzyme of class EC 2.7.7.6 or EC 2.7.7.48), DNA polymerase (e.g., enzyme of class EC 2.7.7.7), reverse transcriptase (e.g., enzyme of class EC 2.7.7.49), and DNA ligase (e.g., enzyme of class EC 6.5.1 .1 ).

[0043] “DNA template” as used herein refers to herein to refer to a strand of a nucleic acid molecule that is used by a polymerase (e.g., RNA polymerase) to synthesize a complementary nucleic acid strand, for example, in a transcription reaction.

[0044] “RNA polymerase” as used herein refers to an enzyme that binds to a DNA template and catalyzes the synthesis of a complementary strand of RNA. The naturally occurring process of RNA synthesis catalyzed by an RNA polymerase is commonly referred to as transcription. RNA polymerases useful in the device and methods of the present disclosure include enzymes of class EC 2.7.7.6 or EC 2.7.7.48, such as the enzymes of T7, T3 or SP6 RNA phage polymerase.

[0045] “Transcription” as used herein refers the RNA polymerase catalyzed synthesis of a complementary RNA strand from a DNA template and rNTP monomers, and is intended to include both in vitro transcription. Briefly, the transcription process of begins with the binding of an RNA polymerase to a promoter sequence of a DNA template. The RNA polymerase also has a helicase activity that concurrently unwinds the double helix of the DNA template. The RNA polymerase then progresses along the unwound template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA template sequence also determines where termination of RNA synthesis process occurs.

[0046] B. Integrated Devices for In vitro Synthesis of RNA [0047] The integrated device for in vitro synthesis provided by the present disclosure comprises a solid support (also referred to herein as a “chip,” or “microfluidic chip”), a series of micro vessels of between about 50 pL and about 500 pL volume (also referred to herein as “chambers”) in thermal contact with devices providing independent temperature control, and connected via fluidic channels that are configured for transporting catalysts, reagents, products, and permitting purification, as necessary to carry out an in vitro synthesis reaction and provide a purified synthesis product. The device also includes a number of access ports that allow the introduction of synthesis reagents (e.g., rNTPs, RNA polymerase catalyst, DNA template, primers, buffers), introduction washing and/or elution buffers, and the removal of the desired synthesis product(s). The transport of the fluids containing these various synthesis materials into, through, and out of the device is controlled at least in part by one or more pump units. A pump unit is not incorporated into the solid support but at least some of the access ports are configured and adapted for making a sealable connection to a pump unit, which can thereby control the movement of liquids through the device. Additionally, in at least one embodiment, the solid support is adapted to tolerate increased pressure (e.g., 5 psi - 200 psi) provided by a pump unit, which increased pressure can be used to accelerate the rate of a synthesis and/or binding reaction taking place within a chamber of the device. In at least one embodiment, the integrated device can be used for the enzymatic synthesis of nucleic acids, such as an in vitro transcription for the synthesis of RNA. A more detailed description of \the components and features of the integrated device of the present disclosure are provided with reference to the exemplary embodiment depicted in FIGS. 1-3.

[0048] As shown in FIG. 1 , the integrated device comprises at least the following structures/components incorporated into the structure of the solid support 1 :

(a) a sample inlet chamber 2, wherein the inlet chamber comprises an sample inlet access port and is in thermal contact with a first thermoelectric device 8;

(b) a reaction chamber 3, wherein the reaction chamber is coupled to the sample inlet chamber via a fluidic channel and is in thermal contact with a second thermoelectric device 9;

(c) a bead inlet chamber 12, wherein the bead inlet chamber 12 comprises a bead inlet access port for introduction of magnetic beads into the device;

(d) a bead mixing chamber 4, wherein the bead mixing chamber 4 is coupled to the reaction chamber 3 via a fluidic channel and to the bead inlet chamber 12 via a fluidic channel, and is in thermal contact with the second thermoelectric device 9;

(e) a separation chamber 5, wherein the separation chamber 5 is in magnetic contact with a magnet 11 and is coupled to (i) a washing access port 13 via a fluidic channel, (ii) an elution access port 15 via a fluidic channel, and (iii) a waste outlet port 14 via a fluidic channel; and

(f) a product outlet chamber 6, wherein the product outlet chamber 6 comprises a product outlet access port and is coupled to the separation chamber 5 via a fluidic channel, and is in thermal contact with a third thermoelectric device 10.

[0049] As illustrated by the top view shown in FIG. 1 , the integrated device for synthesis of the present disclosure includes at least a solid support 1 that includes at least five distinct chambers that are connected by fluidic channels. These five chambers can be substantially aligned to provide a substantially linear flow of the reagents and products via fluidic channels from sample inlet chamber 2 to the reaction chamber 3 to bead mixing chamber 4 to separation chamber 5, and finally to the product outlet chamber 6.

[0050] In at least one embodiment, as depicted in FIG. 1 , the integrated device further comprises a sixth chamber, the quality control chamber 7. Like the product outlet chamber 6, the quality control chamber 7 comprises an access port (i.e., the quality control access port) and is coupled to the separation chamber 5 via a fluidic channel. The quality control chamber 7 is incorporated in the solid support of the device like the other five chamber and located in a configuration adjacent to the product outlet chamber 6 and in thermal contact with the third thermoelectric device 10. Due to its direct fluidic connection to the separation chamber 5, the quality control chamber 7 is configured to provide an aliquot of the same final synthesis product equivalent to the product delivered to the product outlet chamber 6. This aliquot provides a sample of the final synthesis product for quality control analysis of the synthesis. The presence of a quality control chamber 7 thus allows the integrated synthesis device to be more easily utilized in accordance with GMP good laboratory management practices that are required for a synthesis product (e.g., an RNA) that is used in a pharmaceutical composition.

[0051] Although FIG. 1 depicts a substantially linear arrangement of the five primary chambers (2, 3, 4, 5, and 6 in FIG. 1 ), the aligned arrangement of the five chambers is not necessary as long as the configuration of the fluidic channels between the chambers maintains the direction and sequence of fluid movements needed to carry out the desired reaction. Thus, it is also contemplated that the arrangement of chambers in the solid support need not be substantially linear, and in some embodiments the five chambers can be arranged in other configurations, such as a zig-zag shaped pattern, a square-wave shaped pattern, an S-shaped pattern, or a circular pattern.

[0052] Generally, the chambers of the solid support are adapted to provide enclosed vessels in which the fluids containing the synthesis reagents and the resulting reaction products can be allowed to react and/or incubate and/or otherwise be manipulated (e.g., heated, cooled, incubated, agitated, diluted, washed, etc.). In addition to the five primary chambers described above, the design integrated device as depicted in FIG. 1 also includes at least one additional chamber 12 configured adjacent to the bead mixing chamber 4 and at least three access ports 13, 14, and 15, configured adjacent to the separation chamber s.

[0053] The bead inlet chamber 12 includes a bead inlet access port and is coupled to the bead mixing chamber 4 via a fluidic channel. The bead inlet chamber 12 is configured to allow magnetic beads to be introduced into the bead mixing chamber 4 where the beads are able to combine with the reaction mixture introduced from the reaction chamber 3. This bead inlet chamber 12 includes an access port adapted to allow the introduction of magnetic beads. The use of magnetic beads in the purification of nucleic acids is well known in the art and described in more detail elsewhere herein. In at least one embodiment, as depicted in FIG. 1 , the bead inlet chamber 12 is not in thermal contact with a thermoelectric device, such as the adjacent second thermoelectric device 9. This permits the use of smaller thermoelectric devices and reduces necessary power input to the device. In some embodiments, however, the bead inlet chamber 12 can be configured in thermal contact with the second thermoelectric device 9, which can facilitate pre-equilibration of the magnetic beads to the temperature of the reaction mixture before they are combined with it in the bead mixing chamber 4.

[0054] As described elsewhere herein, the magnetic beads are typically designed with a coating that selectively binds and thereby immobilizes the desired nucleic acid products of the synthesis reaction. In at least one embodiment, this specific binding (e.g., hybridization) reaction occurs in the bead mixing chamber 4, and the beads and unused reaction materials remaining in the reaction solution are transported to the separation chamber 5. The separation chamber is designed to be in magnetic contact with a magnet 11 embedded in the solid support 1 . A range of magnetic materials and devices are known in the art and can be adapted to incorporation in the solid support of the device to provide the necessary magnetic field in the separation chamber 5 to immobilize the magnetic beads for washing and elution of a synthesis product. In some embodiments, a small permanent magnet can be embedded in the solid support under the separation chamber. In another embodiment, an induction or electromagnet that can be electrically controlled can be incorporated in the solid support. In at least one embodiment, the magnet 11 is an electromagnet; and optionally, an electromagnet that is capable of providing a magnetic field in the separation chamber 5 that is electrically controllable.

[0055] The magnetic field induced in the separation chamber 5 by the embedded magnet 11 immobilizes the magnetic beads. This immobilization of the beads allows any unused reaction materials remaining in solution to be washed out of the chamber through a waste access port 14 while the desired nucleic acid products (e.g., in vitro synthesized mRNA) remains immobilized to the beads. After this washing, the desired nucleic acid synthesis product that is selectively bound to a coating on the magnetic beads can be eluted forming a purified solution of the desired synthesis product that can be transported to the product outlet chamber 6. Accordingly, in at least one embodiment, as depicted in FIG. 1 , the separation chamber 5 is connected via fluidic channels to at least three adjacent access ports 13, 14, and 15 that facilitate the process of washing and eluting that results in a purified synthesis product solution: (i) a washing access port 13 via a fluidic channel, (ii) an elution access port 15 via a fluidic channel, and (iii) a waste outlet port 14 via a fluidic channel. [0056] The washing access port 13 is adapted to introduce a wash solution into the separation chamber 5. This wash solution, which may be introduced as multiple separate aliquots, flows over the immobilized magnetic beads and then through a fluidic channel located the other side of the separation chamber 5 and out of the adjacent waste outlet port 14. The flow of the aliquots of wash solution through the separation chamber 5 effectively dilutes and ultimately completely exchanges with the reaction mixture solution comprising unused reaction materials which is not immobilized by the beads. It is contemplated that additional volume of the wash solution can be retained in a reservoir vessel located external to the solid support but with the reservoir sealably connected to the wash access port 13 such that additional volumes of the wash solution can be accessed on demand. In at least one embodiment, the flow of the wash solution aliquots from the wash access port 13 through the separation chamber and out through the waste access port 14 is driven by positive or negative pressure applied by pump units sealably connected to the washing access port 13 and/or waste outlet port 14. The use of pump units connected to the access ports of the device to move fluids through the device is described elsewhere herein.

[0057] The elution access port 15 is configured in a similar relationship adjacent to the separation chamber 5 and also is adapted to introduce a solution into the separation chamber 5. The elution solution introduced through the elution access port 15, however, is formulated to elute the bound nucleic acid products from the magnetic beads immobilized in the separation chamber 5. Due to its purpose in extracted the desired product, the elution solution is washed through the separation chamber 5 and out the waste outlet access port 14. Rather, the elution solution is introduced into the separation chamber 5 and typically allowed to incubate with the magnetic beads for a time at a particular temperature that facilitates the dissociation of the bound nucleic acid product from the beads. Following incubation, the solution containing eluted product is transported out of the separation chamber 5 through a fluidic channel into the product outlet chamber 6. In some embodiments, additional volume of the elution solution can be introduced through the elution access port 15 into the separation chamber 5 to facilitate the outflow of eluent containing the desired purified nucleic acid product. It is contemplated that additional volume of the desired elution solution can be retained in a reservoir vessel located external to the solid support but with the reservoir sealably connected to the elution access port 15 such that additional volumes of elution solution can be accessed on demand. In at least one embodiment, the flow of the eluting solution from the elution access port 15 through the separation chamber 5 and out through the fluidic channel into the product outlet chamber 6 is driven by positive or negative pressure applied by pump units sealably connected to the elution access port and/or the product outlet chamber access port.

[0058] In at least one embodiment, the access ports, fluidic channels, and/or chambers are integrated in the material of the solid support as depicted in FIGS. 1-3. It is contemplated that the interior surfaces of the chambers can be adapted for the particular purpose of the chamber during the synthesis process. Accordingly, in some embodiments, a chamber can comprise a smooth internal surface, and in some embodiments, a chamber can comprises a textured or roughened interior surface. In at least one embodiment, at least one of the chambers can comprise an interior surface coated with a reagent and/or a catalytic material. In at least one embodiment, at least one of the chambers can comprise an interior surface coated with a metal-based catalyst; optionally, wherein the metal-based catalyst comprises ruthenium or platinum.

[0059] Generally, the chambers in the solid support have a cylindrical shape, e.g., as depicted in FIGS. 1-3, with a diameter of 4 mm or less. The cylinder shape provides for simplified design and fabrication and also supports good fluid flow. However, in some embodiments it may be desirable to include chambers that are not cylindrical, and which have alternative shapes (e.g., triangular, square, hexagonal, oval, teardrop, etc.) to better facilitate desired sample flow characteristics. Generally, the chambers of the integrated device are sized to accommodate a liquid volume of from about 50 pL to about 500 pL. In some embodiments, the volume of each of the chambers of the integrated device can be less than about 500 pL, less than about 400 pL, less than about 300 pL, less than about 250 pL, less than about 200 pL, less than about 150 pL, less than about 100 pL, or less than about 75 pL. In some embodiments, the volume of the chambers of the integrated device are between about 50 pL and about 500 pL, between about 50 pL and about 400 pL, between about 50 pL and about 250 pL, between about 50 pL and about 150 pL, or between about 50 pL and about 100 pL. In some embodiments, each of the chambers may have the same shape, size (e.g., top down view area), and volume. It is also contemplated, that in some embodiments, the size of the individual chambers can vary so as to adapt to the particular purpose of the chamber in the overall use of the integrated device. In some embodiments, the individual chambers may be sized differently, e.g., accommodate larger or smaller volumes than the other chambers. For example, the separation chamber may be sized to accommodate a larger volume due to its use for washing the magnetic beads of unused materials.

[0060] As depicted in FIG. 1 , the solid support provides the primary structure of the device that is capable of accommodating the access ports, fluidic channels, chambers, filters, valves, magnets, thermoelectric devices, and accompanying electrical and pump unit connectors, as described elsewhere herein. Accordingly, the solid support comprises a material or materials that are capable of being fabricated to provide and/or accommodate these substructures and components of the device. For example, the solid support can be fabricated a variety of materials including, but not limited to, glass, quartz, monocrystalline silicon wafers or polymers. Exemplary polymers useful as solid support materials include, but are not limited to, polycarbonate (PC), polydimethylsiloxane(PDMS), polydicyclopentadiene (DCPD), and the like. In at least one embodiment, the solid support comprises a material selected from a polymer, a glass, a ceramic, a metal, or a combination thereof. [0061] Depending on the material(s) selected, the solid support can be fabricated using a variety of fabrication techniques well known in the art including, but not limited to, hot press molding techniques, injection molding, soft lithography, epoxy casting techniques, three dimensional fabrication techniques (e.g., stereolithography), lasers, or other types of micromachining technology. In at least one embodiment, the solid support is prepared by injection molding, and/or comprises a piece prepared by injection molding.

[0062] The overall shape and dimensions of the solid support can be varied based on standard design selection criteria related to the materials used and other factors such as how the integrated device interacts with external devices such as pump units, electrical controllers, and/or instrumentation for product analysis and/or further downstream treatment. In at least one embodiment, as depicted in FIGS. 1-3, the solid support has a thin rectangular box shape. In at least one embodiment, the dimensions of the solid support are about 45 mm x about 20 mm x about 1 mm.

[0063] The device for synthesis of nucleic acids of the present disclosure, as exemplified in the depiction of FIG. 1 , features at least three integrated thermoelectric devices 8, 9, and 10, capable of providing precise temperature control via heating and/or cooling during different stages of the synthesis reaction carried out on the device. Generally, the three thermoelectric devices are embedded in the solid support so as to be in thermal contact with four of the five chambers. The first thermoelectric device 8 is in thermal contact with the sample inlet chamber 2, the second thermoelectric device 9 is in thermal contact with the reaction chamber 3 and the bead mixing chamber 4, and the third thermoelectric device 10 is in thermal contact with the product outlet chamber 6. The configuration of the three independently controllable thermoelectric devices 8, 9, and 10, in thermal contact with the four chambers 2, 3, 4, and 6, as illustrated by the exemplary configuration of FIG. 1 provides the necessary temperature control of the in vitro synthesis reaction with efficiency of using fewer devices (and associated connectors) thereby allowing for a smaller, more easily fabricated, and/or less costly device. [0064] As described herein, the three thermoelectric devices are each independently controllable and capable of heating and/or cooling the chambers in which they are thermal contact over specific temperature ranges and for specific time periods. In at least one embodiment, the first, second, and/or third thermoelectric device(s) is/are capable of heating and/or cooling the chamber with which it is in thermal contact over a temperature range of from about -30°C to about 100°C. The rate of heating and/or cooling can also be controlled independently and can be important in carrying out certain in vitro synthesis reactions.

Accordingly, in at least one embodiment, the first, second, and/or third thermoelectric device(s) is/are capable of heating and/or cooling the chamber with which it is in thermal contact at a rate of about 1 °C/min to about 3°C/min.

[0065] Generally, each of the first, second, and third thermoelectric devices are capable of heating and/or cooling the chamber with which it is in thermal contact. It is contemplated, however, that in some embodiments it may be necessary for certain of the thermoelectric devices to heat the chamber with which it is in contact or only cool the chamber. In a particular use of the device where only heating is required of certain chambers it is contemplated that the device will only incorporate a thermoelectric device capable of heating in thermal contact with those certain chambers. Similarly, where certain chambers only require cooling, the device can be designed with a thermoelectric device capable of cooling in thermal contact with that chamber. A range of controllable thermoelectric heating elements capable of heating a chamber with which it is in thermal contact are well known in the art. Thermoelectric devices capable of cooling a chamber with which it is thermal contact include a Peltier device. It is contemplated that in order to be able to both heat and cool a chamber, all three of the thermoelectric devices can incorporate both a heating element and Peltier device. However, it is also contemplated that in some embodiments the three independently controllable thermoelectric devices incorporated in the solid support of the device may include only a heating element or only a Peltier device for cooling. For example, the device may comprise a solid support wherein the first and second thermoelectric devices are capable only of heating the chamber with which each is in thermal contact (e.g., heating from about room temperature to about 100°C), and the third thermoelectric device is capable only of cooling the product outlet chamber (e.g., cooling to a temperature of between about -90°C and 10°C). The ability to maintain the product outlet chamber at a low temperature (e.g., below 10°C) is particularly desirable in applications where the device is used for the synthesis of RNA, as RNA is particularly susceptible to rapid degradation at higher temperatures. For example, where the RNA synthesis product is intended for use as a vaccine, the product outlet chamber may be immediately cooled to a temperature of about -90°C for storage until use.

[0066] Various options are available in the art for the selection of thermoelectric devices that can be adapted for use in the device of the present disclosure. Generally, the thermoelectric device should have a surface in thermal contact with the solid support that allows sufficient heat transfer to and/or from the chamber with which it is associated. In at least one embodiment, as depicted in the bottom view of FIG. 2 of the device of FIG. 1 , the three thermoelectric devices 8, 9, and 10, are in contact with the solid support material under the four chambers 2, 3, 4, and 6. It is contemplated that the solid support forms the base or floor of the chamber and has a conductivity (based on material and/or thickness) that permits the thermoelectric device in contact with it to control the temperature of any liquid present in the chamber with sufficient precision for the particular synthesis application.

[0067] In at least one embodiment, as depicted in the side view of FIG. 3, the three thermoelectric devices 8, 9, and 10, are embedded in the material of the solid support under the chambers 2, 3, 4, and 6. For example, the solid support can be fabricated via injection molding of a polymer to accommodate the three thermoelectric heating and/or cooling devices under specific portions of the device, such as in the configuration depicted in FIG. 1 . The side view of FIG. 3 shows the three thermo electric devices 8, 9, and 10 embedded in the solid support in thermal contact with the four chambers under which they are embedded.

[0068] The thermoelectric devices can be controlled via electronic signals to heat and/or cool over specific temperature ranges, and/or for specific time periods. The electronic signals are provided via electrical connections to the thermoelectric devices that are also integrated into the material of the solid support. FIG. 2 (which is a back view of FIG. 1) depicts one illustrative embodiment of the configuration of the electrical connections 16 to the thermoelectric devices. [0069] The design of the integrated nucleic acid synthesis device, as exemplified by the embodiment of FIG. 1 , includes chambers adapted for the inclusion and use of magnetic beads during a synthesis process. Magnetic beads (or magnetic particles) are well known and widely used for isolation, separation, and purification of nucleic acids in a variety of in vitro processes including synthesis, amplification, and sequencing of nucleic acids. In a typical application, the magnetic beads include a surface coating adapted for binding of a desired nucleic acid that is present in a reaction mixture. For example, magnetic beads can be modified with a surface coating that includes a poly-T oligonucleotide sequence that will specifically bind to a complementary poly-A sequence that is present on mRNA molecules synthesized an in vitro transcription reaction mixture. The poly-T modified magnetic beads are then combined with in vitro reaction mixture and allowed to incubate under conditions of buffer and temperature and for a period of time that allows binding via specific hybridization of the poly-A sequences of the mRNA to the poly-T sequences attached to the beads. This specific binding of the mRNA effectively immobilizes the desired in vitro transcription product to the beads. A magnetic field can then be applied to the mixture to immobilize the beads themselves while they are washed with solutions that remove any unused portions of the reaction mixture (e.g., DNA template strands, unused rNTP substrate, RNA polymerase, and primers). Once sufficiently washed, the magnetic beads can be further treated with a solution that selectively elutes the mRNA from the immobilized beads. Thus, as described, magnetic beads of providing a solid phase affinity purification of in vitro synthesized nucleic acids.

[0070] The solid support, in addition to the various chambers described herein, comprises a number of access ports and fluidic channels that are configured to allow fluidic materials involved in the synthesis reaction to be introduced into, transported through, and removed from the integrated synthesis device. Generally, the access ports allow access for introducing and removing fluids to/from the device. For example, the sample inlet chamber comprises a sample inlet access port which allows the introduction of a sample (e.g., patient’s DNA) and reagents (e.g., RNA polymerase and rNTPs) into the sample inlet chamber (1 ) of the device. A variety of access port designs can be used depending on the desired method for introducing the liquid. In at least one embodiment, the access port is configured to accept the introduction of a liquid through a disposable micropipette tip. Alternatively, it is contemplated that the access port is configured to accept the introduction of a liquid through a syringe tip, or the needle of syringe; optionally, wherein the access port is fitted with septum through which the needle is inserted, the insertion and/or removal of a liquid material, e.g., the starting materials and/or synthesis product.

[0071] As described elsewhere herein, in at least one embodiment, the transport of fluids through the fluidic channels and chambers of the device are driven by either negative or positive pressure applied by a pump unit external to the solid support. Accordingly, it is further contemplated that any of the access ports of the device are configured to be covered with a sealable cap. As depicted in FIG. 3, in one exemplary embodiment, sealable caps 17 are included on the sample inlet chamber 2, the bead mixing chamber 4, the separation chamber 5, and the product outlet chamber 6. The sealable cap 17 on the sample inlet chamber 2 is depicted in an open position. The sealable cap 17 on the product outlet chamber 6 is further adapted with a pump unit connection 18. A range of sealable cap designs are known in the art that can be used with the access ports of the device as configured in the solid support. In at least one embodiment, the caps are adapted to withstand negative or positive pressure over a range of pressures (e.g., from about 5 psi to about 200 psi) when applied by an external pump unit attached to another access port of the device.

[0072] Biological macromolecules such as nucleic acid polymers and proteins comprise many charged chemical moieties that result in the macromolecules typically having a large overall positive or negative charge that can correlate with overall size. In the case of nucleic acids, the phosphodiester backbone of the polymer results in large overall negative charge that correlates with the polymer length. Due to their large overall charge, these macromolecules can be transported through fluids and gels via the electrophoretic force provided by an electric potential placed across solution of these macromolecules. Electrophoresis is commonly used to separate in a gel matrix charged macromolecules such as nucleic acids and proteins based on their overall charge. It is contemplated that an electrophoretic force applied via an electric potential applied across the integrated device of the present disclosure can be used to transport and/or otherwise control the movement of nucleic acid and/or protein components in the reaction mixtures at least through a portion of the device. In such an embodiment, the device further comprises at least two electrodes capable of applied an electric potential across at least a portion of the device resulting in electrophoretic movement of charged molecules through the device. In at least one embodiment, the electrodes of opposite charge can be configured at opposite ends of the overall fluid flow path through device, for example, a cathode at the sample inlet access port and an anode at the product outlet access port. The electrodes could also be configured closer together to facilitate the use of an electrophoretic force to control movement of macromolecules through just a portion of the overall flow path through the device. For example, a cathode configured at the separation chamber or in the fluidic channel out of the separation chamber and an anode in the final product outlet chamber. In such an embodiment, the electrophoretic force could be used to transport the synthesized nucleic acid to the final product outlet chamber and also provide further separation of potential contaminants.

[0073] In another related embodiment, it is contemplated that the fluidic channel coupling the separation chamber 5 to the sample outlet chamber 6 can further comprise a macromolecule separation material, such as electrophoretic gel material, or size exclusion gel or affinity gel, that can provide further purification of the reaction mixture after the separation chamber. A wide range of macromolecule separation materials (e.g., electrophoretic gels, ion exchange gels, size exclusion gels, affinity gels) are well known in the art and could be incorporated in the fluidic channel(s) of the device. For example, in one embodiment, the fluidic channel coupling the separation chamber 5 to the sample outlet chamber 6 further comprises a macromolecule separation material capable of separating single-stranded RNA from DNA template, and/or proteins.

[0074] Generally, the fluidic channels are configured to allow the fluid compositions introduced into the device to transported to and from the different access ports and chambers. At least one exemplary embodiment of a configuration of the fluidic channels between the access ports and chambers is depicted in FIG. 1. Like the access ports and chambers, the fluidic channels are integrated into the material of the solid support. The fluidic channels are grooves in the solid support material having a square or rectangular cross-section. A square or rectangular cross-section provides for simplified design and fabrication and also can facilitate good fluid flow. It is also contemplated that the fluidic channels can have a rounded, cylindrical crosssection. Other fluidic channel designs may be desirable to better facilitate desired sample flow characteristics, for example grooves having a triangular, hexagonal, or oval-shaped cross- sectional shape. Generally, the dimensions of the fluidic channels are designed according to well-known microfluidic principles to accommodate the desired volume and flow-rate through the device, and in view of the method of driving the flow (e.g., pump unit pressure). In at least one embodiment, the fluidic channels have a width of about 10 pm to about 3000 pm and a depth of about 10 pm to about 3000 pm. Generally, the fluidic channels can be fabricated in a solid support using the same methods and techniques for fabricating the integrated chambers and access ports of the solid support described elsewhere herein. Such techniques are well known in the art and can be selected depending on materials and other design features of the device.

[0075] The integrated synthesis device of the present disclosure can also include one or more filters that exclude materials above a certain size from entering a chamber or other portions of the device. For example, the device can include a filter that excludes small particles that sometimes contaminate biological samples, such as aggregated protein, from entering the sample inlet chamber when the sample is introduced into the device. Semi-permeable materials with known exclusion sizes (or molecular weight cutoffs) that can be incorporated as a filter in the device are well-known in the art. In at least one embodiment, one or more of the access ports and/or fluidic channels comprises a filter that excludes materials of a size greater than a filter size exclusion limit from entering one or more of the chambers. The size exclusion limit of the filter can be selected based on the types of samples (e.g., reagents, nucleic acids), and the desired methods of sample preparation used before the sample is inserted in the device. For example, in at least one embodiment, the filter size exclusion limit is from about 0.10 pm to about 0.50 pm, which excludes most contaminant particles that can clog the device during a typical in vitro nucleic acid synthesis reaction.

[0076] In addition to excluding particulate contaminants in the sample from entering and clogging the device, it is also contemplated that a filter can be incorporated in the device to retain the magnetic beads in the separation chamber s. Although the magnet 11 is intended to provide sufficient magnetic field to retain the beads during washing and/or elution, it is contemplated that a filter may also be incorporated at the exits of the separation chamber 5 as a back-up to prevent the beads from entering the product outlet chamber 6 or the quality control chamber 7. Accordingly, in at least one embodiment, the device comprises a filter between the separation chamber 5 and the product outlet chamber 6 that excludes magnetic beads.

[0077] C. Methods of Use of Integrated Devices

[0078] The present disclosure also provides methods for in vitro nucleic acid synthesis using an integrated device as described herein. It is contemplated that the device can be adapted to range of standard in vitro nucleic acid synthesis techniques and protocols that incorporate template-dependent polymerase catalyzed synthesis of nucleic acid molecules.

[0079] In at least one embodiment, the integrated device of the present disclosure can be used in a method for RNA synthesis (e.g., in vitro mRNA transcription), wherein the method comprises:

(a) introducing a reaction mixture comprising a DNA template, an RNA polymerase, ribonucleotide triphosphates (rNTPs), and a buffer solution through an access port into an inlet chamber in thermal contact with a first thermoelectric device, and allowing the reaction mixture to reach a first temperature T1 of about 35°C to about 38°C for a time of at least 1 h;

(b) transferring the reaction mixture through a fluidic channel to a reaction chamber in thermal contact with a second thermoelectric device, and incubating the reaction mixture at a second temperature T2 of between about 37°C and about 40°C for a time of at least 1 h, whereby the RNA polymerase catalyzes DNA template-dependent synthesis of RNA;

(c) transferring the reaction mixture through a fluidic channel to a bead mixing chamber in thermal contact with the second thermoelectric device and which contains magnetic beads capable of selectively binding to RNA, whereby the synthesized RNA in the reaction mixture binds to the magnetic beads; (d) transferring the reaction mixture and RNA-bound magnetic beads through a fluidic channel to a separation chamber in magnetic contact with a magnetic pad, whereby the magnetic beads are immobilized in the separation chamber;

(g) transferring the synthesized RNA solution through a fluidic channel to an outlet chamber in thermal contact with a third thermoelectric device, and allowing the synthesized RNA solution to reach a third temperature T3 of between about -90°C to about 8°C.

[0080] It is contemplated that the above method for using the integrated device to synthesize mRNA can be used to synthesize mRNA with any of the structural/sequence features produced by the in vivo mRNA transcription and maturation process. mRNA when it is synthesized by in vivo transcription in an eukaryotic organism undergoes a variety of post-transcriptional modifications such as splicing, 5’-capping, 5’-UTR, 3’-UTR, and 3’ polyadenylation. 5’-capping modifies the 5’ end of the mRNA with a cap structure consisting of a 5' 7-methyl guanosine which protects the mRNA from nuclease digestion and promotes the translation process within the organism. Polyadenylation is catalyzed by a template independent polymerase and results in 3’ poly-A structure (or tail) on the transcribed mRNA molecule. The poly-A tail contributes to mRNA stability and translational regulation in the cell. In some cases, in vivo transcribed mRNA also includes 5’-UTR and/or 3’-UTR sequences located upstream and downstream of the coding region. These untranslated region sequences are involved in regulating the in vivo translation of the mRNA transcript.

[0081] In order to mimic the structure and stability of in vivo transcribed mRNA, in vitro synthesized RNA can also include enzymes and reagents for synthesis of the cap structure (e.g., using mRNA capping enzymes from vaccinia virus), and modification with a poly-A tail (e.g., using an E. coli poly(A) polymerase). Alternatively, cap structure analogs, such as (m7G(5')ppp(5')G), may be used to co-transcriptionally cap RNA transcripts during in vitro synthesis. As described elsewhere herein, the capping of in vitro synthesized mRNA modified with a poly-A tail allows for facile separation and purification of the mRNA using poly-T modified magnetic beads. Additionally, the DNA template can be designed to include 5’-UTR and/or 3’- UTR regions where it is desirable to synthesize an mRNA transcript that includes these features upstream and/or downstream of the translated coding region. The design of modified mRNA molecules with increased stability for use in medicinal formulations (e.g., vaccines) are known in the art (see e.g., WG2002/098443A2, WO2013/143699A1 , WG2013/143700A2, WO2013/143698A1 , WO2015/024667A1 , each of which is hereby incorporated by reference herein for all purposes). Any of the mRNA designs known in the art for use in medicinal formulation may be implemented in the synthesis methods using the integrated device of the present disclosure.

[0082] Generally, an in vitro mRNA synthesis reaction is initiated by combining in a reaction mix of rNTP substrate (e.g., rATP, rCTP, rGTP, and rUTP) in buffer, an RNA polymerase mix (e.g., T7 phage polymerase), and the desired DNA template. The in vitro transcription of the DNA template to produce the RNA product can be catalyzed by an RNA polymerase, such as T7, T3 or SP6 RNA phage polymerase, in the presence of a set of rNTPs. Generally, the RNA polymerase processes the DNA template strand in a 3’ to 5’ direction using complementary base pairing to synthesize the complementary RNA strand in the 5’ to 3’ direction. The DNA template base thymine (T) is replaced with the ribonucleotide uracil (U) in the synthesized RNA strand. Where it is desired to cap and add poly(A) tails to the mRNA product, the reaction mix is further supplemented with an mRNA capping enzyme (e.g., from vaccinia virus) and a poly-A polymerase (e.g., from E. coli).

[0083] The enzymes, reagents, and protocols useful for in vitro mRNA transcription and capping are well known in the art and commercially available (see e.g., New England Biolabs, Ipswich, MA, USA; ThermoFisher Scientific, Waltham, MA, USA). The synthesis device of the present disclosure is capable of accommodating and being used with commercially available enzymes and reagents for in vitro mRNA transcription and/or other polymerase-based nucleic acid synthesis reactions.

[0084] The desired DNA template sequence that is transcribed to form the RNA product can be isolated from a biological sample (e.g., from an organism, including a human patient), using standard procedures for DNA isolation and amplification. Alternatively, a DNA template can be synthesized de novo based on a known sequence of interest. In some embodiments, for example when using plasmid DNA as the DNA template, the DNA is linearized by treatment with a restriction enzyme. PGR amplification products and synthetic oligonucleotides can also be used as DNA templates for transcription reactions and typically do not require treatment to linearize.

[0085] In at least one embodiment of the method of RNA synthesis using the integrated device of the present disclosure, step (b) of the method further comprises increasing the pressure in the reaction chamber to at least 5 psi via application of pressure through a fluidic channel, thereby increasing the rate of the RNA synthesis reaction; optionally, wherein the pressure in the reaction chamber is increased to at least 10 psi, at least 15 psi, or at least 20 psi.

[0086] In at least one embodiment of the method for RNA synthesis using the integrated device of the present disclosure, the transferring of step (g) further comprises passing the synthesized RNA solution through a macromolecule separation material; optionally, wherein the macromolecule separation material is capable of separating single-stranded RNA from DNA template, and/or proteins.

[0087] In at least one embodiment of the method of RNA synthesis using the integrated device of the present disclosure, the transfer through the fluidic channels is driven by positive or negative pressure applied by a pump unit coupled to an access port of the device.

[0088] In at least one embodiment of the method of RNA synthesis using the integrated device of the present disclosure, the transfer of macromolecules through at least part of the device is driven by an applied electric potential inducing an electrophoretic force.

[0089] In at least one embodiment of the method of RNA synthesis using the integrated device of the present disclosure, the sample mixture has a volume of less than 500 pL; optionally, wherein the sample mixture has a volume of between about 50 pL and about 500 pL. In some embodiments, the amount of DNA template in the sample mixture is less than 1 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.1 pg and about 50 pg.

[0090] In at least one embodiment of the method of RNA synthesis using the integrated device of the present disclosure, the synthesized RNA solution has a volume of less than 500 pL; optionally, wherein the synthesized RNA solution has a volume of between about 50 pL and about 500 pL. In some embodiments, the amount of RNA in the synthesized RNA solution is at least 25 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.5 pg and about 500 pg.

[0091] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0092] Additional embodiments of the invention are set forth in the following claims.

[0093] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMS What is claimed is:

1 . A device for synthesis of RNA comprising: a solid support comprising: a sample inlet chamber, wherein the inlet chamber comprises an sample inlet access port and is in thermal contact with a first thermoelectric device; a reaction chamber, wherein the reaction chamber is coupled to the sample inlet chamber via a fluidic channel and is in thermal contact with a second thermoelectric device; a bead inlet chamber, wherein the bead inlet chamber comprises an bead inlet access port for introduction of magnetic beads into the device; a bead mixing chamber, wherein the bead mixing chamber is coupled to the reaction chamber via a fluidic channel and to the bead inlet chamber via a fluidic channel, and is in thermal contact with the second thermoelectric device; a separation chamber, wherein the separation chamber is in magnetic contact with a magnet and is coupled to (i) a washing access port via a fluidic channel, (ii) an elution access port via a fluidic channel, and (iii) a waste outlet port via a fluidic channel; and a product outlet chamber, wherein the outlet chamber comprises a product outlet access port and is coupled to the separation chamber via a fluidic channel, and is in thermal contact with a third thermoelectric device.

2. The device of claim 1 , wherein:

(a) the device further comprises a quality control chamber, wherein the quality control chamber comprises a quality control access port and is coupled to the separation chamber via a fluidic channel, and is in thermal contact with a third thermoelectric device;

(b) the device further comprises a pump unit coupled to the sample inlet access port and/or the product outlet access port, wherein the pump unit is capable of controlling pressure within the device;

(c) the device further comprises electrodes capable of capable of controlling electrophoretic movement of charged molecules through the device;

(d) the access ports are sealable; optionally, wherein the access ports when sealed are capable of withstanding a pressure in the device of at least about 5 psi;

(e) the first, second, and third thermoelectric devices are independently controllable and capable of heating and/or cooling the chamber with which it is in thermal contact;

(f) the fluidic channel coupling the separation chamber to the product outlet chamber comprises a macromolecule separation material;

-23- (g) an access port and/or fluidic channel comprises a filter that excludes materials of a size greater than the filter size exclusion limit from entering one or more of the chambers; and/or

(h) the magnetic field of the magnet is adjustable. The device of any one of claims 1 -2, wherein the device comprises a pump unit capable of:

(a) controlling movement of liquids through the device;

(b) increasing the pressure in the reaction chamber of the device to a pressure of between about 5 psi and about 200 psi, thereby accelerating reactions; optionally, a pressure of between about 20 psi and about 150 psi. The device of any one of claims 1 -2, wherein the access ports comprise sealable caps. The device of any one of claims 1 -2, wherein the first, second, and/or third thermoelectric device is capable of heating and/or cooling the chamber with which it is in thermal contact:

(a) over a temperature range of from about -30°C to about 100°C; and/or

(b) at a rate of about 1 °C/min to about 3°C/min. The device of any one of claims 1 -2, wherein the first, second, and/or third thermoelectric device comprises a Peltier device; optionally, wherein at least the third thermoelectric device comprises a Peltier device capable of maintaining the outlet chamber at a temperature of between about -20°C and 10°C. The device of claim 6, wherein the first and second thermoelectric devices are capable only of heating the chamber with which each is in thermal contact to a temperature of about room temperature to about 100°C, and the third thermoelectric device is capable only of cooling the outlet chamber to a temperature of between about -20°C and 10°C. The device of any one of claims 1 -2, wherein the fluidic channel coupling the separation chamber to the product outlet chamber comprises a macromolecule separation material capable of separating single-stranded RNA from DNA template, and/or proteins. The device of any one of claims 1 -2, wherein one or more of the access ports and/or fluidic channels comprises a filter that excludes materials, wherein the filter size exclusion limit is from about 0.10 pm to about 0.50 pm. The device of any one of claims 1 -2, wherein the magnetic field of the magnet is adjustable; optionally, wherein the magnet comprises an electromagnet. The device of any one of claims 1 -2, wherein the solid support comprises:

(a) a material selected from a polymer, a glass, a ceramic, a metal, or a combination thereof; and/or

(b) a piece prepared by injection molding. The device of any one of claims 1 -2, wherein the thermoelectric devices are:

(a) in contact with the underside of the solid support; and/or

(b) integrated in the material of the solid support; optionally, wherein the electrical connections to the thermoelectric devices are integrated in the material of the solid support. The device of any one of claims 1 -2, wherein the access ports, fluidic channels, and/or chambers are integrated in the material of the solid support. The device of any one of claims 1 -2, wherein at least one of the chambers comprises an interior surface coated with a metal-based catalyst; optionally, wherein the metal-based catalyst comprises ruthenium or platinum. The device of any one of claims 1 -2, wherein the fluidic channels have a width of about 10 pm to about 3000 pm and a depth of about 10 pm to about 3000 pm. The device of any one of claims 1 -2, wherein the chambers have a volume of from about 50 pL to about 500 pL. A method for RNA synthesis comprising :

(a) introducing a reaction mixture comprising a DNA template, an RNA polymerase, ribonucleotide triphosphates (rNTPs), and buffer solution through an access port into an inlet chamber in thermal contact with a first thermoelectric device, and allowing the reaction mixture to reach a temperature T1 of about 32°C to about 38°C for a time of at least 1 h;

(b) transferring the reaction mixture through a fluidic channel to a reaction chamber in thermal contact with a second thermoelectric device, and incubating the reaction mixture at a temperature T2 of between about 37°C and about 42°C for a time of at least 1 h, whereby the RNA polymerase catalyzes DNA template-dependent synthesis of RNA;

(g) transferring the synthesized RNA solution through a fluidic channel to an outlet chamber in thermal contact with a third thermoelectric device, and allowing the synthesized RNA solution to reach a temperature T3 of between about -90°C to about 8°C. The method of claim 17, wherein step (b) further comprises increasing the pressure in the reaction chamber to at least 5 psi via application of pressure through a fluidic channel, thereby increasing the rate of the RNA synthesis reaction. The method of any one of claims 17-18, wherein the transferring of step (g) further comprises passing the synthesized RNA solution through a macromolecule separation material; optionally, wherein the macromolecule separation material is capable of separating single-stranded RNA from DNA template, and/or proteins. The method of any one of claims 17-19, wherein transfer through the fluidic channels is driven by positive or negative pressure applied by a pump unit coupled to an access port of the device. The method of any one of claims 17-20, wherein transfer of macromolecules through at least part of the device is driven by an applied electric potential inducing an electrophoretic force. The method of any one of claims 17-21 , wherein the sample mixture has a volume of less than 500 pL; optionally, wherein the sample mixture has a volume of between about 50 pL and about 500 pL. The method of any one of claims 17-22, wherein the amount of DNA template in the sample mixture is less than 1 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.1 pg and about 50 pg.

-26- The method of any one of claims 17-23, wherein the synthesized RNA solution has a volume of less than 500 pL; optionally, wherein the synthesized RNA solution has a volume of between about 50 pL and about 500 pL. The method of any one of claims 17-24, wherein the amount of RNA in the synthesized RNA solution is at least 25 pg; optionally, wherein the amount of DNA template in the sample mixture is between about 0.5 pg and about 500 pg.

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