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US-12624378-B2 - Cell-free protein synthesis

US12624378B2US 12624378 B2US12624378 B2US 12624378B2US-12624378-B2

Abstract

Provided herein are cell free protein synthesis (CFPS) systems comprising a plurality of ribosomes attached to or encapsulated within a structure, or a plurality of structures, and, optionally, a solid support. Also provided are related kits and uses of the CFPS systems. Methods of producing a protein and methods of treating a disease are provided herein.

Inventors

  • Nicholas N. Watkins
  • Neil Reginald Beer
  • Kenneth W. Turteltaub

Assignees

  • LAWRENCE LIVERMORE NATIONAL SECURITY, LLC

Dates

Publication Date
20260512
Application Date
20240214

Claims (20)

  1. 1 . A method for continuous synthesis of a protein at a rate of 1.4×10 −2 mg/hr/μL-2.5 mg/hr/μL, comprising: adding a solution comprising one or more in vitro translation reagents through a fluidic inlet of a reusable cell free protein system (CFPS), wherein the CFPS comprises a plurality of ribosomes attached to a structure or a plurality of structures, housed, contained, or attached to a solid support at a density of at least 10 3 ribosomes per cm 2 of the structure or plurality of structures, wherein the solid support comprises a chamber, the fluidic inlet, a fluidic outlet, and the solution for containing synthesized protein, and wherein the plurality of ribosomes are positioned between the fluidic inlet and the fluidic outlet; and collecting the solution after passing through the fluidic outlet, wherein the solution after passing through the fluidic outlet comprises the synthesized protein but does not comprise the plurality of ribosomes.
  2. 2 . The method of claim 1 , wherein the one or more in vitro translation reagents comprise a messenger RNA (mRNA) encoding the protein, a set of transfer RNAs (tRNAs), an enzymatic cofactor, a set of amino acids, and an energy source.
  3. 3 . The method of claim 1 , wherein the method further comprises recycling the solution collected after passing through the fluidic outlet and adding it back through the fluidic inlet or outlet of the CFPS.
  4. 4 . The method of claim 3 , wherein the CFPS further comprises a filter capable of filtering reagents but not the synthesized protein.
  5. 5 . The method of claim 4 , wherein the filter is positioned near the outlet.
  6. 6 . The method of claim 1 , wherein the CFPS further comprises a filter to retain the plurality of ribosomes in the solid support.
  7. 7 . The method of claim 1 , further comprising washing the CFPS to allow for further production of a new protein.
  8. 8 . The method of claim 1 , wherein the solid support is attached to the structure or the plurality of structures.
  9. 9 . The method of claim 8 , wherein the structure is a micropillar, a nanopillar, a nanotube, an aerogel, a bead, a lattice, a foam, or a sponge.
  10. 10 . The method of claim 9 , wherein the nanotube is a carbon nanotube.
  11. 11 . The method of claim 1 , wherein the plurality of ribosomes are attached to the structure or a plurality of structures at a density of at least 10 6 to 6×10 14 ribosomes per cm 2 of structure.
  12. 12 . The method of claim 1 , wherein the plurality of ribosomes are attached to the structure through a linker.
  13. 13 . The method of claim 12 , wherein each linker attached to the structure is of approximately the same length.
  14. 14 . The method of claim 12 , wherein each linker attached to the structure is of different length, optionally, by at least 50% different length.
  15. 15 . The method of claim 12 , wherein the linker comprises a polymer, optionally, a polyethylene glycol.
  16. 16 . The method of claim 12 , wherein the linker comprises a functional group at one end of the linker or comprises a functional group at each end.
  17. 17 . The method of claim 1 , wherein the solid support is a chip or a bead.
  18. 18 . The method of claim 1 , wherein each structure is attached to the solid support under a controlled condition.
  19. 19 . The method of claim 18 , wherein the controlled condition comprises electrostatic or magnetic forces.
  20. 20 . The method of claim 19 , wherein each structure is attached only when a magnetic field is applied to the CFPS.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62/796,427, filed Jan. 24, 2019, the entire contents of which is incorporated herein by reference. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 53495B_SeqListing.xml; Size: 3,361 bytes; Created: Feb. 12, 2024. BACKGROUND In the healthcare sector, there is a crucial need for the rapid and portable manufacture of proteins, e.g., therapeutic proteins. The ability to quickly produce targeted proteins for therapeutics and vaccines to stop or slow infection before it propagates would greatly reduce the susceptibility to future pandemics. The rapid production of a personalized medicine would improve a patient's prognosis by reducing the time between the onset of symptoms and therapy. A point-of-care system would reduce new infections by minimizing the relocation of infected patients, and also would transfer the capability of custom therapeutics from centralized factories to flexible and nimble platforms in hospitals, clinics, and even the bedside. Pharmaceutical companies desire a small-quantity, quick turnaround protein manufacturing capability during their precision medicine patient trials, which currently suffer delays of 4 to 6 months in the creation of the personalized drugs (personal communication). The current industry standard for mass protein synthesis consists of large, labor-intensive facilities where genetically-modified cells are cultured to create desired proteins. This in vivo protein production model—which takes on the order of months to produce a new drug lot—cannot be scaled down into reactors that would generate practical amounts of protein for point-of-care therapeutics or low-latency patient trials. Other drawbacks include decreased production and yield from unwanted genetic mutations during culturing and a limited product scope since some proteins may be unstable and/or toxic to the cells that are creating them [Spirin et al., Synthesis (Stuttg) 242(4882): 1162-1164 (1988)]. Cell-free protein synthesis (CFPS) methods have streamlined and simplified the process by replacing cells with only the necessary components to translate proteins from messenger RNA (mRNA), creating a larger variety of purified protein products that could not otherwise be made in a cellular system [Shimizu et al., Nat. Biotechnol, 19(8): 751-755 (2001); Rosenblum and Cooperman, FEBS Letters 588(2): 261-268 (2014)]. CFPS systems can be scaled down to more portable formats, as some have shown [Siuti et al., Anal. Chem. 84(2): 1092-1097 (2012); Timm et al., Small 6:810-817 (2015)]. However, even the most advanced miniaturized technology to date produces only single doses of up to 2 mg in 8 hours [Timm et al., 2015, supra]. This is still impractical for point-of-care therapeutic systems and biopharmaceutical applications that may need to produce on the order of grams of various proteins per day. SUMMARY Provided herein for the first time are data which demonstrate the design and manufacture of a variety cell free protein synthesis (CFPS) systems comprising localized ribosomes on structures, wherein the ribosomes may be repeatedly used in in vitro translation (IVT) reactions to produce proteins. Without being bound to any particular theory, the CFPS systems described herein are capable of synthesizing proteins at rates per reaction chamber volume of several orders of magnitude higher than those described in the art, including cell-based methods and cell-free methods. Advantageously, the CFPS systems provided herein are characterized by a large surface-to-volume ratio which maximizes the surface area available for biomolecular interactions between the components of the IVT reactions, e.g., ribosomes, translation mixture, and mRNA. Also, the CFPS systems provided herein may be run in a continual fashion, such that the IVT reaction products (e.g., synthesized proteins) and inhibitory reaction byproducts are continuously collected or washed away, optionally, for downstream processing, and replaced by fresh consumables so that the IVT reactions may be repeated with the same high density array of ribosomes at high reaction rates. Accordingly, the present disclosure provides a CFPS system comprising a plurality of ribosomes attached to or encapsulated within a structure, or a plurality of structures. In exemplary embodiments, the CFPS system comprises a solid support, a plurality of structures, and a plural