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KR-20260062985-A - Method and composition for delivery of non-modified mRNA constructs using mesenchymal stem cells

KR20260062985AKR 20260062985 AKR20260062985 AKR 20260062985AKR-20260062985-A

Abstract

The present disclosure provides an approach for enhancing the expression of an unmodified mRNA construct encoding a secreted protein in mesenchymal stem cells. A composition for delivering an unmodified mRNA construct using mesenchymal stem cells is also provided.

Inventors

  • 로흐너 에두아르드
  • 솔메르 예스페르
  • 위트먼 네빈

Assignees

  • 스마트셀라 솔루션즈 아베

Dates

Publication Date
20260507
Application Date
20241011
Priority Date
20231013

Claims (20)

  1. A mesenchymal stem cell comprising a substantially single-stranded composition of an mRNA construct introduced into a cell by a non-endosome delivery pathway, wherein the mRNA construct comprises a coding sequence, the nucleosides within the mRNA construct are chemically unmodified, and optionally, the codons of the mRNA construct are selected to reduce the uridine content.
  2. Mesenchymal stem cells comprising a substantially single-stranded composition of an mRNA construct introduced into a cell by a non-endosome delivery pathway, wherein the mRNA construct comprises a coding sequence, the codons of the mRNA construct are selected to reduce the uridine content.
  3. In paragraph 2, a mesenchymal stem cell in which all nucleosides in the mRNA construct are chemically unmodified.
  4. In any one of paragraphs 1 to 3, the mRNA construct is a mesenchymal stem cell encoding a protein, for example, a secreted protein.
  5. In any one of claims 1 to 4, the mRNA construct is a mesenchymal stem cell encoding a cytokine, enzyme, chemokine, antibody or fragment thereof, immunomodulator or growth factor.
  6. Mesenchymal stem cells according to any one of claims 1 to 5, wherein the method of synthesizing the mRNA construct is selected from the group consisting of an enzymatic (IVT) method, a solid-phase method, a liquid-phase method, a combined synthesis method, a small region synthesis method, and a ligation method.
  7. Mesenchymal stem cells according to any one of claims 1 to 6, wherein the non-endosome delivery route is selected from the group consisting of biolistic particle delivery, ultrasonic spraying, soniphyllation, buffer-mediated delivery, delivery using phagocytosis, pinocytosis, clathrin-dependent delivery and/or clathrin-independent delivery by viral particles and/or polymers, microinjection, streptolysin-O permeation, permeation using anionic peptides, and electroporation.
  8. Mesenchymal stem cells according to any one of claims 1 to 7, wherein 1 ng of mRNA to 1 mg of mRNA, for example 10 ng of mRNA to 100 μg of mRNA, for example 0.5 μg of mRNA to 50 μg of mRNA, for example 1 μg of mRNA to 25 μg of mRNA, for example 5 μg of mRNA to 20 μg of mRNA is introduced into 1 x 10⁶ mesenchymal stem cells.
  9. Mesenchymal stem cells according to any one of claims 1 to 8, wherein at least 5X10⁹ , for example at least 5X10¹², for example at least 5X10¹⁵ , of mRNA molecules having a length of up to 5000 nucleotides are introduced into 1X10⁶ mesenchymal stem cells.
  10. Mesenchymal stem cells according to any one of claims 1 to 9, wherein 1X10⁶ cells comprise the mRNA composition in an amount of 1 ng to 1 mg, for example 10 ng to 100 μg, for example 0.5 μg to 50 μg, for example 1 μg to 25 μg, for example 5 μg to 20 μg.
  11. In any one of claims 1 to 10, the 1X106 cells are mesenchymal stem cells comprising at least 5X109 , for example at least 5X1012 , for example at least 5X1015 mRNA molecules of the mRNA composition having a length of up to 5000 nucleotides.
  12. A mesenchymal stem cell according to any one of claims 1 to 11, wherein up to 10% of all RNA in the mRNA composition is double-stranded RNA, for example, up to 8%, for example, up to 6%, for example, up to 5%, for example, up to 3%, for example, up to 1%.
  13. In any one of claims 1 to 12, the method for removing dsRNA of an mRNA composition is selected from the group consisting of cellulose chromatography, high-performance liquid chromatography, silica-based chromatography, and poly-A affinity annealing, for mesenchymal stem cells.
  14. In any one of claims 1 to 13, the codons of the coding sequence are selected to reduce the uridine content by a method, said method comprising the following steps, for mesenchymal stem cells: a. Codon optimization; b. Reduce uridine content by selecting a low uridine content or uridine-free codon.
  15. In paragraph 14, the codons are optimized for rebalancing codon usage, reduction of sequence complexity, avoidance of rare codons, minimization of secondary structure and/or reduction of complexity, in mesenchymal stem cells.
  16. Mesenchymal stem cells according to any one of claims 1 to 15, wherein the absolute uridine content of the mRNA construct in the mRNA composition is reduced by at least 0.5%, for example, at least 1%, for example, at least 2.5%, for example, at least 5%, for example, at least 10%, for example, at least 15% compared to the uridine content of naturally occurring mRNA.
  17. In any one of claims 1 to 16, the cell is a mesenchymal stem cell derived from a tissue selected from the group consisting of bone marrow, adipose tissue, peripheral blood, umbilical cord, amniotic fluid, cord blood, placenta, and Wharton's jelly.
  18. In any one of paragraphs 1 to 17, the cell is a mesenchymal stem cell that differentiates from a stem cell.
  19. In any one of claims 1 to 18, the cells are mesenchymal stem cells that are cryopreserved and/or thawed before administration to a subject.
  20. An mRNA composition comprising a substantially single-stranded mRNA construct encoding a protein, wherein the codons of the mRNA construct are selected to reduce the uridine content.

Description

Method and composition for delivery of non-modified mRNA constructs using mesenchymal stem cells The success of COVID-19 mRNA vaccines has demonstrated the feasibility of mRNA formulations for human use, thereby opening up new biotechnological platforms for a wide range of preventive and therapeutic purposes. Current mRNA vaccines utilize modified mRNA (mmRNA) formulations in which chemically modified nucleosides (e.g., modified uracil residues) are introduced into the mRNA structure. Chemically modified nucleosides have been used to enhance the stability of mRNA formulations or to evade detection by the innate immune system. To successfully deliver mmRNA formulations in vivo, mmRNA has been encapsulated in lipid nanoparticles (LNPs). While this has proven effective, the current approach has potential limitations. The inherent instability of mRNA necessitates a delivery system that protects it from degradation by nucleases and enables intracellular uptake upon in vivo administration. Current approaches using LNPs were first clinically used to allow for the in vivo delivery of siRNA (Coelho et al. (2013) New Eng. J. Med. 369:819-829; Adams et al. (2018) New Eng. J. Med. 379:11-21). LNPs protect RNA cargo and are uptaken via the endosome pathway, where a portion of the RNA cargo is released from the endosome for final translation. Early versions of lipid nanoparticles containing ionizable aminolipids used to encapsulate siRNA are described, for example, in Jayarama et al. (2012) Angew Chem. Int. Ed. Engl. 51:8529-8533. The original version of LNP allowed absorption in the liver and eventually led to the approval of the first siRNA therapeutic, but it was associated with a significant level of side effects (Coelho et al. (2013) New Eng. J. Med. 369:819-829; Adams et al. (2018) New Eng. J. Med. 379:11-21). However, a new generation of ionizable LNPs has been designed that induces greater release of RNA cargo and brings about significant improvements in safety and efficacy (Cheng et al. (2020) Nature Nanotech. 15:313-320). These latest versions have enabled large-scale administration with relatively rare severe side effects and are being utilized to address the wave of new therapeutic candidates. Despite some improvements to LNP technology, significant potential limitations still exist, particularly regarding the repeated administration often required in the treatment of chronic diseases using mRNA agents. Even with the chemical modification of mRNA and packaging into more advanced lipid nanoparticles, protein expression levels decrease upon chronic repeated administration. This severely limits the opportunities to use mRNA technology in continuous therapy, such as continuous treatment to deliver therapeutic proteins of interest encoded by mRNA. Therefore, despite various advancements in the use of mRNA preparations in humans, there is still a need in the field of technology for additional methods and approaches, particularly for methods and approaches to deliver mRNA preparations in vivo using alternative means other than LNP. The present state of the art involves using mRNA containing chemically modified nucleosides to evade innate immune system receptors such as RNAse and toll-like receptors. Examples of chemically modified nucleosides include pseudouridines or N1-methyl-pseudouridines. However, the production of mRNA containing chemically modified nucleosides is costly and labor-intensive. The present disclosure relates to a method and composition for delivering a secreted protein in vivo using mesenchymal stem cells (MSCs) containing substantially single-stranded mRNA without the need for chemically modified nucleosides, achieving a delivery efficiency more than 200 times higher than that observed with LNP delivery mRNA constructs containing chemically modified nucleosides. This remarkable effect is achieved by using a combination of a cell-based system for the in vivo delivery of mRNA constructs, codon optimization of the mRNA to reduce uridine content, and purification of the produced mRNA. Therefore, the mRNA does not need to contain any chemically modified nucleosides. This is a surprising effect because conventional techniques (e.g., Liu et al., 2022) teach that chemical modification in mRNA is pivotal. However, this combination of cell-based delivery with substantially single-stranded mRNA makes chemically modified nucleosides unnecessary. Since the mRNA has been purified to remove dsRNA, the “substantially single-stranded mRNA” does not exclude the possibility that a small portion of the RNA may be double-stranded. The present disclosure provides a method and a composition for delivering an mRNA construct in vivo by loading the mRNA construct into a cell, wherein the cell subsequently acts as a delivery vehicle for the mRNA construct. In addition, the compositions and methods of the present disclosure have the advantage of utilizing unmodified mRNA constructs, such as mRNA constructs in which all uri