MAKING AND USING CARDIOPOIETIC CELLS

Abstract

This document relates to methods and materials for making and using cardiopoetic stem cells. For example, methods and materials for delivering one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors to a stem cell to generate a cardiopoetic stem cell are provided.

Claims

1. A method for generating a cardiopoetic stem cell, said method comprising delivering a messenger RNA (mRNA) encoding an early mesodermal transcription factor to a stem cell under conditions wherein said mRNA expresses said early mesodermal transcription factor.

2. The method of claim 1, wherein said stem cell is an adult stem.

3. (canceled)

4. The method of claim 1, wherein said stem cell is an adipose-derived mesenchymal stem cell (AD-MSC).

5-6. (canceled)

7. The method of claim 1, wherein said early mesodermal transcription factor is brachyury (T).

8. The method of claim 1, said method further comprising delivering a mRNA encoding a cardiac transcription factor to said stem cell under conditions wherein said mRNA expresses said cardiac transcription factor.

9. (canceled)

10. The method of claim 1, said method further comprising delivering a glycogen synthase kinase (GSK) inhibitor to said stem cell.

11. (canceled)

12. The method of claim 1, said method further comprising delivering a wingless/integrated (Wnt) inhibitor to said stem cell.

13. (canceled)

14. The method of claim 1, wherein said cardiopoetic stem cell is generated within 5 days of delivering said mRNA encoding an early mesodermal transcription factor to said stem cell.

15. An engineered cardiopoetic stem cell generated using the method of claim 1.

16. A method for treating a mammal with a cardiac event, wherein said method comprises: (a) administering an engineered cardiopoetic stem cell of claim 15 to said mammal, or (b) generating an engineered cardiopoetic stem cell in said mammal by delivering a messenger RNA (mRNA) encoding an early mesodermal transcription factor to a stem cell within said mammal under conditions wherein said mRNA expresses said transcription factor, wherein severity of said cardiac event is reduced following step (a) or step (b).

17. The method of claim 16, wherein said mammal is a human.

18. The method of claim 16, wherein said cardiac event is selected from the group consisting of myocardial infarction, heart failure, recurrent myocardial infarction, repeat hospitalization for cardiac-related events, and ischemic heart disease.

19. The method of claim 16, wherein said method comprises step (a).

20. The method of claim 19, wherein said stem cell is an adult stem.

21. (canceled)

22. The method of claim 21, wherein said stem cell is an adipose-derived mesenchymal stem cell (AD-MSC).

23. The method of claim 22, wherein said AD-MSC is a human AD-MSC.

24. (canceled)

25. The method of claim 16, wherein said method comprises step (b).

26. The method of claim 25, wherein said stem cell is an adult stem.

27. (canceled)

28. The method of claim 27, wherein said stem cell is an adipose-derived mesenchymal stem cell (AD-MSC).

29. (canceled)

30. The method of claim 16, wherein said method comprises steps (a) and (b).

Description

DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A-1B show that adipose-derived MSCs exhibit characteristic CD markers. FIG. 1A contains representative fluorescent images of AD-MSCs stained with CD90 (BV421), CD73 (PE), and CD45 (APC). MSCs do not express CD45. Scale bar=50 μm.

[0013] FIG. 1B contains representative flow cytometry scatter plots of AD-MSCs stained with CD90 (BV421), CD73 (PE), and CD45 (APC).

[0014] FIGS. 2A-2B show that transfection of AD-MSCs with M.sup.3RNA encoding brachyury (T) increases the expression of T in a rapid and transient fashion compared to non-transfection control. FIG. 2A contains representative ICC images showing expression of T at indicated time points post-transfection. FIG. 2B contains a quantitation of nuclear fluorescence intensity using ImageJ (arbitrary fluorescence intensity with Mean±SEM in at least three experiments and at least six images/experiment).

[0015] FIGS. 3A-3B show that transfection of AD-MSCs with M.sup.3RNA encoding T increases the expression of Nkx2.5, a cardiac transcription factor downstream of T. FIG. 3A contains representative ICC images showing expression of Nkx2.5 at indicated time points post-transfection. FIG. 3B contains a quantitation of nuclear fluorescence intensity using ImageJ (arbitrary fluorescence intensity with Mean±SEM in at least three experiments and at least six images/experiment).

[0016] FIGS. 4A-4B show that a western blot of nuclear fraction of AD-MSCs transfected with T shows increased levels of cardiopoetic markers Nkx2.5 and Mef2c compared to non-transfection control. FIG. 4A contains a representative blot using Li-Cor. FIG. 4B contains a quantitation of protein levels based on fluorescence intensity after normalization to total protein.

DETAILED DESCRIPTION

[0017] This document provides engineered CP cells and methods for making and using engineered CP cells. For example, one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors can be delivered to a stem cell to generate a CP cell (e.g., an engineered CP cell). In some cases, one or more RNAs encoding one or more early mesodermal transcription factors can be delivered to tissue (e.g., heart tissue) within a mammal where the RNA is expressed to generate a functional polypeptide. In some cases, a microencapsulated-modified-messenger RNA (M.sup.3RNA) platform can be used to deliver one or more mRNAs encoding one or more early mesodermal transcription factors to a stem cell to generate an engineered CP cell.

[0018] In some cases, generating CP cells as described herein (e.g., by delivering one or more RNAs encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, to a stem cell such as a human AD-MSC) can be performed in vitro. In cases where cardiopoetic cells are generated in vitro, one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors can be delivered to a stem cell in culture.

[0019] In some cases, generating CP cells as described herein (e.g., by delivering one or more RNAs encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, to a stem cell such as a human AD-MSC) can be performed in vivo. In cases where cardiopoetic cells are generated in vivo, one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors can be delivered to a stem cell in vivo (e.g., administered locally or systemically to a mammal).

[0020] In some cases, CP cells can be generated as described herein (e.g., by delivering one or more RNAs encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, to a stem cell such as a human AD-MSC) in as few as 5 days.

[0021] A nucleic acid encoding one or more early mesodermal transcription factors can be any appropriate type of nucleic acid. Examples of nucleic acids that can encode one or more early mesodermal transcription factors include, without limitation, DNA, RNA (e.g., mRNA), and combinations thereof. In some cases, a nucleic acid encoding one or more early mesodermal transcription factors can be a mRNA.

[0022] A nucleic acid encoding one or more early mesodermal transcription factors can encode any appropriate early mesodermal transcription factor. A non-limiting example of an early mesodermal transcription factor is T. In some cases, a M.sup.3RNA can include mRNA encoding T. For example, one or more nucleic acids (e.g., one or more RNAs) encoding T can be delivered to a stem cell to generate a CP cell.

[0023] In some cases, one or more nucleic acids (e.g., one or more RNAs) encoding one or more cardiac transcription factors (e.g., polypeptides useful for regenerating cardiac function and/or tissue) also can be delivered to a stem cell to generate a CP cell. Examples of cardiac transcription factors include, without limitation, POU homeodomain proteins (e.g., Oct-4), NK2 homeobox proteins (e.g., NKX2 proteins such as Nkx2.5), myocyte enhancing factors (e.g., MEF2 proteins such as Mef2c), GATA binding proteins (e.g., GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6), T-box transcription factors (e.g., TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22), mesoderm posterior proteins (e.g., MESP1 and MESP2), neutrophil-activating proteins (e.g., NAP-2 and NAP-3), transforming growth factors (e.g., TGF-α and TGF-β), erythroblastic leukemia viral oncogene-3 (ErBb3), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF-2), platelet-derived growth factors (e.g., PDGFA, PDGFB, PDGFC, and PDGFD), Interleukin-2 (IL-2), CD19, CD20, and CD80/86. For example, one or more nucleic acids (e.g., one or more RNAs) encoding one or more Oct4, Nkx2.5, and Mef2c can be delivered to a stem cell to generate a CP cell. For example, one or more nucleic acids (e.g., one or more RNAs) encoding T and Oct4 can be delivered to a stem cell to generate a CP cell.

[0024] In some cases, one or more glycogen synthase kinase (GSK) inhibitors also can be delivered to a stem cell to generate a CP cell. In some cases, a GSK inhibitor can be a small molecule inhibitor. A non-limiting example of a GSK inhibitor is CHIR99021. For example, CHIR99021 can be delivered to a stem cell to generate a CP cell. For example, one or more nucleic acids (e.g., one or more RNAs) encoding T and CHIR99021 can be delivered to a stem cell to generate a CP cell.

[0025] In some cases, one or more wingless/integrated (Wnt) inhibitors also can be delivered to a stem cell to generate a CP cell. In some cases, a Wnt inhibitor can be a small molecule inhibitor. A non-limiting example of a Wnt inhibitor is IWP-2. For example, IWP-2 can be delivered to a stem cell to generate a CP cell. For example, one or more nucleic acids (e.g., one or more RNAs) encoding T and IWP-2 can be delivered to a stem cell to generate a CP cell.

[0026] In some cases, one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors (and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors) can be modified. For example, a nucleic acid can be modified for sustained gene expression. For example, nucleotides can be modified for increased stability. In some cases, one or more uracil residues of an RNA can be replace with a modified uracil residue. Examples of modified uracil residues include, without limitation, pseudouridine (Ψ), dihydrouridine (D), and dideoxyuracil.

[0027] One or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, can be delivered to a stem cell using any appropriate method. In some cases, one or more nucleic acids can be encapsulated within a delivery vehicle (e.g., a particle). For example, when one or more nucleic acids are encapsulated within a particle, particle-mediated delivery methods can be used to deliver the encapsulated nucleic acid to a stem cell. In some cases, one or more nucleic acids can be delivered to a stem cell using a M.sup.3RNA platform. In some cases, one or more nucleic acids can be delivered to a stem cell using delivery methods as described elsewhere (see, e.g., WO 2018/098312).

[0028] One or more RNAs encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, can be delivered to any appropriate type of stem cell. A stem cell can be an adult stem cell. A stem cell can be from any appropriate tissue type and/or cell type (e.g., a mesenchymal stem cell (MSC) such as an adipose-derived stem cell (AD-MSC), a bone marrow-derived stem cell (BMSC), an endothelial stem cell, and a dental pulp stem cell). A stem cell can be from any appropriate type of mammal (e.g., humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats). For example, a stem cell can be an AD-MSC such as a human AD-MSC.

[0029] This document also provides methods for improving cardiac function within a mammal. For example, CP cells provided herein (e.g., CP cells engineered by delivering one or more RNAs encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, to a stem cell such as a human AD-MSC) can be used to improve cardiac function within a mammal. In some cases, CP cells provided herein can be delivered to and/or generated within heart tissue to improve cardiac function. For example, one or more CP cells can be delivered to and/or generated within heart tissue within a mammal to improve the mammal's cardiac function.

[0030] In cases where CP cells are generated in vitro, one or more CP cells can be delivered to a mammal in need thereof (e.g., a mammal in need of cardiac tissue regeneration and/or improved cardiac function). Any appropriate method can be used to deliver CP cells to heart tissue within a mammal.

[0031] In cases where CP cells are generated in vivo, one or more nucleic acids (e.g., one or more RNAs) encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, can be delivered to a mammal in need thereof (e.g., a mammal in need of cardiac tissue regeneration and/or improved cardiac function). Any appropriate method can be used to deliver one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, to a mammal. For example, one or more nucleic acids encoding one or more early mesodermal transcription factors, and, optionally, one or more nucleic acids encoding one or more cardiac transcription factors, one or more GSK inhibitors, and/or one or more Wnt inhibitors, can be delivered to a mammal using a particle-mediated delivery method. Particle-mediated delivery methods can be as described elsewhere (see, e.g., WO 2018/098312).

[0032] CP cells provided herein can be delivered to and/or generated within any type of mammal. Examples of mammals that CP cells provided herein can be delivered to and/or generated within include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. In some cases, CP cells provided herein can be delivered to and/or generated within a human.

[0033] When CP cells provided herein are delivered to and/or generated within mammal, the mammal can be experiencing, or can be at risk of experiencing, any adverse cardiac event (e.g., a major adverse cardiac event). Examples of major adverse cardiac events include, without limitation, myocardial infarction (e.g., acute myocardial infarction), heart failure, recurrent myocardial infarction, repeat hospitalization for cardiac-related events, and ischemic heart disease. In some cases, CP cells provided herein can be delivered to and/or generated within a mammal experiencing myocardial infarction, such as acute myocardial infarction.

[0034] In some cases, the methods and materials described herein can be used to improve cardiac function. Examples of improved cardiac function include, without limitation, increased survivorship, reduced hospitalization, symptom-free tolerance of physical activity, improved global physical fitness, improved cardiac ejection fraction, improved cardiac output, improved stroke volume, improved cardiac mass index, and reduced scar size.

[0035] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Induction of Cardiopoesis from Human Adipose-Derived Mesenchymal Stem Cells Using Microencapsulated-Modified-Messenger RNA

[0036] The following demonstrates the successful use of microencapsulated-modified-messenger RNA (M.sup.3-RNA) in conjunction with small molecule inhibitors to create an engineered cardiopoetic stem cell lineage from human adipose-derived mesenchymal stem cells (AD-MSCs).

[0037] Human AD-MSCs were transfected with mRNA encoding brachyury (T) and/or Oct4 for up to 48 hours prior to the addition of GSK inhibitor (CHIR99021) and/or Wnt inhibitor (IWP-2) for another 24-48 hours. The cells were probed for cardiac mesoderm marker Mesp1 and cardiopoetic markers Nkx2.5 and Mef2c expression using immunocytochemistry.

[0038] Transfection of human AD-MSCs with Oct4 mRNA induced nuclear Oct4 expression 4 hours post-transfection that peaked at 24 hours. Co-transfection with Oct4 and T mRNA induced Mesp1 expression. A combined or sequential transfection of AD-MSCs with either Oct4 and/or T, along with the addition of CHIR99021 and/or IWP-2 was then employed to assess cardiopoetic markers Nkx2.5 and Mef2c expression. While many permutations yielded comparable results, transfection with T alone for 48 hours followed by CHIR99021 for 48 hours was the most effective approach in inducing maximal Nkx2.5 and Mef2c expression.

[0039] These results demonstrate that transfection of human AD-MSCs with M.sup.3-RNA encoding cardiogenic transcription factors with small molecular inhibitors induces an engineered cardiopoetic stem cell lineage.

[0040] This example demonstrates a novel approach to engineer cardiopoetic stem cells using Oct4 and T mRNA with small molecule inhibitors in vitro using human AD-MSCs in as few as 5 days. This approach circumvents many limitations of current cocktail-based cardiopoesis protocols and serves as a highly translatable platform to ease the cost burden in inducing cardiopoesis for human clinical applications.

Example 2: Engineering Cardiopoesis Using Microencapsulated-Modified-Messenger RNA M.SUP.3.RNA

[0041] Current strategies of management of heart failure patients focus on preserving or delaying deterioration, but are unable to restore cardiac function to pre-injury state. Cell therapy, such as CP cells, has shown to be a promising approach to address this issue. Early and advanced clinical trials using CP cells have shown signals of efficacy in improving cardiac function. The CP platform has been developed over the past decade, incorporating a lineage-specifying approach termed cardiopoesis to increase the regenerative potency of mesenchymal stem cells (MSCs). However, the manufacturing cost, duration of induction, and complexity of cell manufacturing impair its potential for broad-based use in practice. To address this, the novel microencapsulated-modified-messenger RNA (M3RNA) platform was developed as a cost-effective and time-saving approach to induce cardiopoesis.

[0042] Flow cytometry analyses showed that AD-MSCs exhibit characteristic CP markers CD90.sup.+/CD73.sup.+/CD45.sup.−. Fluorescent images of AD-MSCs stained with CD90 (BV421), CD73 (PE) and CD45 (APC), but did not stain with CD45 (FIG. 1A). Flow cytometry scatter plots of AD-MSCs also revealed staining with CD90 (BV421), CD73 (PE) and CD45 (APC) (FIG. 1B).

[0043] Human AD-MSCs were transfected with M.sup.3RNA encoding T and/or cardiac transcription factors Nkx2.5 and/or Mef2c for 48 hours followed by incubation in media for 24 hours. Changes in cardiopoetic markers were probed using immunocytochemistry (ICC) and western blots of nuclear fractions.

[0044] Rapid and transient expression of T polypeptides was observed in AD-MSCs transfected with M.sup.3RNA encoding T (FIGS. 2A-2B). T polypeptide expression peaked at 24 hours post-transfection.

[0045] Increased polypeptide levels of Nkx2.5, a cardiac transcription factor downstream of T, were observed in AD-MSCs transfected with M.sup.3RNA encoding T (FIGS. 3A-3B).

[0046] Increased levels of cardiopoetic markers Nkx2.5 and Mef2c were observed in AD-MSCs transfected with M.sup.3RNA encoding T (FIGS. 4A-4B). Expression of cardiopoetic markers Nkx2.5 and Mef2c peaked at 72 hours post-transfection.

[0047] These results demonstrate that transfection of (AD-MSCs) with M.sup.3RNA encoding early mesodermal and/or cardiac transcription factor(s) induces an engineered cell phenotype comparable to CP cells.

Other Embodiments

[0048] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.