METHOD OF ENHANCING RNA EXPRESSION IN A CELL

Abstract

The present invention describes a virus-derived factor which when provided to cells, e.g., by transfecting the cells with RNA encoding the virus-derived factor, enhances expression of RNA encoding a peptide or protein in the cells. In particular, the virus-derived factor enhances survival of cells, in particular when transfected repetitively with RNA, and reduces an IFN response of cells to transfected RNA. Accordingly, the present invention provides methods and means for enhancing expression of RNA in cells. The cells are preferably transfected with the RNA.

Claims

1. A method for expressing a peptide or protein in a cell comprising the steps of (i) introducing RNA encoding the peptide or protein into the cell and (ii) providing a virus-derived factor comprising Toscana virus NSs protein or a functional variant of Toscana virus NSs protein to the cell.

2. The method of claim 1, wherein the virus-derived factor is Toscana virus NSs protein.

3. The method of claim 1, wherein the RNA is introduced into the cell repetitively.

4. The method of claim 1, wherein the RNA is introduced into the cell by electroporation or lipofection.

5. The method of claim 1, wherein the RNA is in vitro transcribed RNA.

6. The method of claim 1, wherein providing the virus-derived factor to the cell comprises introducing RNA encoding the virus-derived factor into the cell.

7. The method of claim 1, further comprising providing vaccinia virus B18R and/or vaccinia virus E3 to the cell.

8. The method of claim 7, wherein providing vaccinia virus B18R and/or vaccinia virus E3 to the cell comprises introducing RNA encoding vaccinia virus B18R and/or RNA encoding vaccinia virus E3 into the cell.

9. The method of claim 1, wherein providing the virus-derived factor to the cell enhances stability and/or expression of the RNA in the cell.

10. The method of claim 9, wherein the enhancement of expression of the RNA in the cell comprises an increase in the level of expression and/or an increase in the duration of expression of the RNA in the cell.

11. The method of claim 1, wherein providing the virus-derived factor to the cell enhances cell viability.

12. The method of claim 1, wherein the cell is a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a T cell, an antigen presenting cell, or a human cell.

13. (canceled)

14. (canceled)

15. (canceled)

16. A method for providing cells having stem cell characteristics comprising the steps of (i) providing a cell population comprising somatic cells, (ii) providing a virus-derived factor comprising Toscana virus NSs protein or a functional variant of Toscana virus NSs protein to the somatic cells, (iii) introducing RNA encoding one or more reprogramming factors into the somatic cells, and (iv) allowing the development of cells having stem cell characteristics.

17. The method of claim 16, wherein the virus-derived factor comprises Toscana virus NSs protein.

18. The method of claim 16, wherein providing the virus-derived factor to the somatic cells comprises introducing RNA encoding the virus-derived factor into the cell.

19. The method of claim 16, which further comprises introducing miRNA enhancing reprogramming of the somatic cells to cells having stem cell characteristics into the somatic cells.

20. The method of claim 16, wherein the one or more reprogramming factors comprise: (i) OCT4 and SOX2; (ii) OCT4, SOX2, and KLF4; (iii) OCT4, SOX2, and c-MYC; (iv) OCT4, SOX2, KLF4, and c-MYC; (v) OCT4, SOX2, and NANOG; (vi) OCT4, SOX2, and LIN28; (vii) OCT4, SOX2, NANOG, and LIN28; (viii) OCT4, SOX2, KLF4, and NANOG; (ix) OCT4, SOX2, KLF4, and LIN28; (x) OCT4, SOX2, KLF4, NANOG, and LIN28; (xi) OCT4, SOX2, c-MYC, and NANOG; (xii) OCT4, SOX2, c-MYC, and LIN28; (xiii) OCT4, SOX2, c-MYC, NANOG, and LIN28; (xiv) OCT4, SOX2, KLF4, c-MYC, and NANOG; (xv) OCT4, SOX2, KLF4, c-MYC, and LIN28; or (xvi) OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. The method of claim 16, further comprising the step of culturing the somatic cells in the presence of at least one histone deacetylase inhibitor.

27. The method of claim 26, wherein the at least one histone deacetylase inhibitor comprises valproic acid.

28. The method of claim 16, wherein the step of allowing the development of cells having stem cell characteristics comprises culturing the somatic cells under embryonic stem cell culture conditions.

29. The method of claim 16, wherein the stem cell characteristics comprise an embryonic stem cell morphology.

30. The method of claim 16, wherein the cells having stem cell characteristics: (i) have normal karyotypes, express telomerase activity, express cell surface markers that are characteristic for embryonic stem cells and/or express genes that are characteristic for embryonic stem cells; (ii) exhibit a pluripotent state; and/or (iii) have the developmental potential to differentiate into advanced derivatives of all three primary germ layers.

31. (canceled)

32. (canceled)

33. The method of claim 16, wherein the somatic cells are selected from the group consisting of lung fibroblasts, foreskin fibroblasts, dermal fibroblasts, keratinocytes and endothelial progenitor cells.

34. (canceled)

35. (canceled)

36. A method for providing differentiated cell types comprising the steps of (i) providing cells having stem cell characteristics using the method of claim 16, and (ii) culturing the cells having stem cell characteristics under conditions that induce or direct partial or complete differentiation to a differentiated cell type.

Description

FIGURES

[0307] FIG. 1: NSs can replace EKB for inhibition of IFN-response to synthetic mRNA in human fibroblasts

[0308] FIG. 2: NSs enables RNA-based Reprogramming

[0309] FIG. 3: NSs inhibits IFN-response to SFV-self-replicating RNA and prevents protein kinase R activation

[0310] FIG. 4: NSs increases transfection rates with VEEV self-replicating RNA, increases translation and blocks IFN-response

[0311] FIG. 5: NSs improves the expression of VEEV trans-replicons and inhibits IFN-response

[0312] FIG. 6: NSs improves transreplicon expression in immune cells

[0313] FIG. 7: NSs improves expression of VEEV trans-replicating and self-replicating RNA in immune cells and prevents cell death

EXAMPLES

Example 1: NSs can Replace EKB for Inhibition of IFN-Response to Synthetic mRNA in Human Fibroblasts

[0314] The generation of induced pluripotent stem (iPS) cells holds great promises to liberate stem cells research from ethical concerns associated with the use of human embryonic stem (hES) cells. Continuous expression of exogenous pluripotency-associated transcription factors (TF) until the pluripotency is maintained by the endogenous pluripotency network is thereby the key to achieve reprogramming of somatic cells. In regard to clinical translation of the iPS-technology it is crucial to circumvent any risk of genomic integration in the targeted cells genome. Synthetic mRNA is therefore probably the most suitable vector since synthetic mRNA is strictly transient and complex RNA mixtures can be easily transfected into somatic cells. Nevertheless synthetic mRNA is recognized by cellular receptors that can distinguish between specific molecular patterns distinct from cellular RNAs. This leads to defense mechanisms of the cell resulting in apoptosis, cytoskeletal rearrangements, RNA degradation and a shutdown of translation which hinders successful reprogramming of cells. We were able to establish an non-modified RNA-based reprogramming approach in which the viral escape proteins E3, K3 and B18R (EKB) are used to counteract the IFN-response of the cell allowing successful reprogramming of human fibroblasts and blood-outgrowth endothelial progenitor cells (EPCs) (Hum Gene Ther. 2015 November; 26(11):751-66). The viral escape protein NSs from Toscana virus was shown to be a potent inhibitor of IFN-response. Here we investigate the effect of NSs on the IFN-response of human fibroblasts. In a first step NSs effect on reporter expression (GFP), reduction of IFN-response genes (OAS1/IFN) and cell survival after daily lipofections was analyzed in comparison to the gold standard EKB.

[0315] Primary human foreskin fibroblasts (HFFs, System Bioscience) were plated into 6 wells (100,000 cells/well) and lipofected the next day using 6p1 RNAiMAX (Invitrogen) and 1.4 g non-modified synthetic mRNA (in vitro transcribed). The synthetic mRNA mixtures were thereby composed of 0.8 g green florescent protein (GFP) together with different mixtures of IFN-escape proteins E3 (E), K3 (K), B18R (B) and/or NSs (N) as indicated in FIG. 1A. 0.2 g synthetic mRNA of each IFN-escape protein was used and synthetic mRNA encoding Luciferase (Luc) was utilized to sum up the mixtures to 1.4 g. Lipofections were performed according to the manufacturers instructions. Cells were harvested 24 h post transfection and half of the cells were used for analysis of GFP expression by FACS. It became obvious that all samples were lipofected equally with a transfection efficiency of 91-96% (FIG. 1A, left panel). We observed a 3-fold increase of GFP mRNA translation when co-transfected with EKB. This increase was slightly lower when N was used alone and slightly higher when co-transfected with EN. Additional IFN-escape proteins (K and B) did not further increase translation of GFP mRNA.

[0316] The rest of cells from the previous experiment was pelleted, total RNA was isolated and mRNA-expression of IFN and OAS1 was quantified by qRT-PCR (FIG. 1B). As expected, EKB is able to downregulate induction of IFN and OAS1 expression by synthetic mRNA when co-transfected. This could also be achieved with N alone, even better in case of IFN induction. N in combination with other viral escape proteins (EKB) reduced IFN-response of the cell to synthetic mRNA to a minimum.

[0317] HFFs were plated into 6 wells (100,000 cells/well) and lipofected the next three consecutive days using 6p1 RNAiMAX (Invitrogen) and 1.4 g synthetic mRNA. The synthetic mRNA mixtures were thereby composed of 0.8 g GFP together with different mixtures of IFN-escape proteins E3 (E), K3 (K), B18R (B) and/or NSs (N) as indicated in FIG. 1C. 0.2 g synthetic mRNA of each IFN-escape protein was used and synthetic mRNA encoding Luciferase (Luc) was utilized to sum up the mixtures to 1.4 g. Lipofections were performed according to the manufacturers instructions. 24 h after the last lipofection, cell viability was assayed using the Cell Proliferation Kit II (Roche) with normalization to mock transfected cells. As expected cells die when they are repetitive transfected with synthetic mRNA. This can be rescued by co-transfection with EKB. Unexpectedly, N alone was also able do enhance survival of cells and could therefore replace EKB. If combined with E3 there was also a slight proliferative effect observed.

[0318] Cells from the previous experiment were pelleted 24 h after the last transfection, total RNA was isolated and mRNA-expression of IFN and OAS1 was quantified by qRT-PCR (FIG. 1D). In accordance to the survival analysis in FIG. 1C, EKB reduces induction of IFN and OAS1 by synthetic mRNA significantly. This could also be achieved when EKB is replaced by N alone. If N is combined with other viral escape proteins, the IFN-response of the cells is nearly extinguished.

[0319] The data shows that NSs can replace EKB in inhibition of IFN-response to synthetic mRNA in human fibroblasts. NSs alone is able to counteract a downregulation of translation, it inhibits the upregulation of IFN-response genes and prevents loss of cell viability.

Example 2: NSs Enables RNA-Based Reprogramming

[0320] As shown in Example 1, NSs can replace EKB in inhibition of IFN-response to synthetic mRNA in human fibroblasts. As stated before, we previously developed an RNA-based reprogramming approach in which the viral escape proteins E3, K3 and B18R (EKB) are used to counteract the IFN-response of the cell allowing successful reprogramming of human fibroblasts and blood-outgrowth endothelial progenitor cells (EPCs) into human induced pluripotent stem (iPS) cells (Hum Gene Ther. 2015 November; 26(11):751-66). Here we investigate whether NSs alone is also able to facilitate RNA-based reprogramming of human fibroblasts.

[0321] FIG. 2A shows the timeline for the reprogramming of primary human dermal fibroblasts (HDFs, Innoprot). 40,000 cells were plated into a 12-well-plate and after 4 hours of cultivation lipofected on four consecutive days with synthetic mRNA mixtures that were composed of 0.33 g non-modified synthetic mRNA encoding the reprogramming transcription factors (TF) OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (03SKMNL) (3:1:1:1:1:1) with 0.08 g of each B18R, E3 and K3 (EKB) or 0.24 g of NSs (N) alone and in both cases with 0.17 g of a miRNA mixture composed of miRNAs 302a-d and 367 (1:1:1:1:1). Cells were cultivated in human embryonic stem (hES) cell medium (NutriStem media, Stemgent) and lipofections using 2.5 L RNAiMAX (Life Technologies) per 12 well were performed according to the manufacturers instructions. From day 9 on, colony formation was observed and analysis of colonies were performed on d10.

[0322] Established colonies were stained for alkaline phosphatase (AP) on day 10 using an AP Staining Kit (SBI) and following the manufacturers instructions. For an overview, representative stainings are shown FIG. 2B. It became obvious that formation of AP positive colonies (red/dark) is quite similar when EKB or N alone is used.

[0323] AP positive colonies from two independent experiments were counted and are shown in FIG. 2C as mean colony countSD (n=2). The data confirms the results from the overview: RNA-based reprogramming of human fibroblasts into iPS cells can be performed with EKB or NSs alone with similar reprogramming efficiencies.

[0324] Colony morphology of resulting iPS-cell colonies when using NSs alone instead of EKB were similar hES cell-like with tightly packed small cells in distinct colonies and well-defined borders (FIG. 2D). These colonies could as well be stained positive for AP (FIG. 2E) (red/dark) and the hES cell surface marker TRA-1-81 (FIG. 2F) (green/white). TRA-1-81 live staining was performed with the Stain-Alive TRA-1-81 antibody (Stemgent) according to the manufacturers instructions. Representative pictures of colonies are shown.

[0325] To further assess pluripotency of colonies, cells were pelleted, total RNA isolated and mRNA-expression of the hES-markers OCT4 (endogenous), NANOG (endogenous), LIN28 (endogenous), TERT and REX1 was quantified by qRT-PCR. mRNA expression was normalized to that of HPRT and is shown as mean fold inductionSD (n=2) compared to the transcript levels of input cells. All analyzed markers were highly expressed compared to input cells indicating pluripotency of reprogrammed cells. Again there was no significant difference observed when NSs alone instead of EKB was used to facilitate RNA-based reprogramming of human fibroblasts (FIG. 2G).

[0326] NSs is able to reduce the IFN-response of human fibroblasts to synthetic mRNA to a similar degree then the use of the three viral escape proteins E3, K3 and B18R. This allows multiple transfections of an RNA-based reprogramming cocktail leading to the successful reprogramming of human fibroblasts into human iPS cells. Successful reprogramming of cells using NSs alone instead of EKB was thereby confirmed by hES-cell like morphology, AP-activity and the expression of hES-cell surface and endogenous markers of resulting iPS-cell colonies. Reprogramming efficiency and quality of reprogramming using NSs was shown to be as good as EKB enabled RNA-based reprogramming.

Example 3: NSs Inhibits IFN-Response to SFV-Self-Replicating RNA and Prevents Protein Kinase R Activation

[0327] Human foreskin fibroblasts were plated into 6-well-plates and transfected the next day. To this aim 2.5 g GFP encoding self-replicating RNA (viral origin Semliki Forest Virus), or 1.25 g self-replicating RNA together with 1.25 g mRNA encoding either Vaccinia virus E3 or Toscana virus NSs were complexed with MessengerMax and added to the cells. Cells were harvested 17 h after transfection and used to determine interferon response by qPCR (upregulation of IFN and OAS1) and PKR-degradation and activation by immunoblotting against total PKR and autophosphorylated PKR (p-PKR). NSs resulted in a more pronounced reduction of IFN response than E3 (FIG. 3A). NSs overexpression reduced PKR expression compared to untransfected (untr.) cells, cells transfected with replicon alone, or replicon+E3. However, it does not prevent activation of residual PKR (FIG. 3B). FIG. 3C shows a densitometric quantification of the band intensities of the PKR blot.

Example 4: NSs Increases Transfection Rates with VEEV Self-Replicating RNA, Increases Translation and Blocks IFN-Response

[0328] Human foreskin fibroblasts were plated into 6-well-plates and transfected the next day. To this aim 1.1 g GFP encoding self-replicating RNA (viral origin Venezuelan equine encephalitis virus) and 1.4 g mRNA encoding either infrared fluorescent protein (iRFP) or Vaccinia virus E3 or Toscana virus NSs were complexed with MessengerMax and added to the cells. 24 h later, cells were harvested to determine transfection rates by flow cytometry and interferon response by qPCR (upregulation of IFNb and OAS1). Flow cytometric analysis shows an increased transfection rate upon cotransfection of E3 and NSs (untr.=untransfected cells) (FIG. 4A,B). Cotransfer of NSs resulted in a more pronounced reduction of IFN response than cotransfer of E3 (FIG. 4C).

Example 5: NSs Improves the Expression of VEEV Trans-Replicons and Inhibits IFN-Response

[0329] Human foreskin fibroblasts were electroporated with 1.4 g mRNA encoding VEEV replicase, 0.3 g transreplicating RNA encoding GFP and 0.3 g transreplicating RNA encoding luciferase. To inhibit interferon response the samples were supplemented with 2 g NSs mRNA, E3 mRNA, or E3 and B18 mRNA (1 g each). 24 h later cells were harvested to analyze GFP expression by flow cytometry (FIG. 5A), and interferon response by qPCR (upregulation of IFNb and OAS1) (FIG. 5B). Furthermore, luciferase expression was measured for 5 days at the time points indicated in FIG. 5C.

[0330] NSs improves expression as good as the combination of E3 and B18, and inhibits IFN response comparably.

Example 6: NSs Improves Transreplicon Expression in Immune Cells

[0331] Resting peripheral CD8 positive human T cells were electroporated with 5 g mRNA encoding VEEV replicase, and 5 g transreplicating RNA encoding luciferase. To inhibit interferon response the samples were supplemented with 5 g NSs mRNA or E3 mRNA. Luciferase expression was assessed at the time points indicated in FIG. 6A.

[0332] Human peripheral immature dendritic cells were electroporated with 1.4 g mRNA encoding VEEV replicase, 0.3 g transreplicating RNA encoding luciferase and 0.3 g transreplicating RNA encoding GFP. To inhibit interferon response the samples were supplemented with 2 g NSs mRNA or E3 mRNA. Luciferase expression was assessed at the time points indicated in FIG. 6B.

Example 7: NSs Improves Expression of VEEV Trans-Replicating and Self-Replicating RNA in Immune Cells and Prevents Cell Death

[0333] (A) Human peripheral immature dendritic cells were lipofected with MessengerMax (lnvitrogen) in 96-well-plates with 25 ng total RNA per well. 3 ng transreplicating RNA encoding luciferase were co-transfected or not with 12 ng mRNA encoding VEEV replicase (+Rep), and 10 ng mRNA encoding GFP (+GFP) or the inhibitor NSs (+NSs) as indicated. Luciferase expression was assessed at the indicated time points (BrightGlo Assay; Promega). (B) Viability of the transfected cells was also assessed (CellTiterGlo assay; Promega) and plotted against viability of untransfected cells. (C, D) In parallel, cells were also transfected with 10 ng VEEV-replicon encoding luciferase, and either GFP or NSs. Luciferase expression (C) and viability (D) were followed for 72 h after lipofection.

[0334] In dendritic cells, NSs prolongs expression of self- and trans-replicating RNA and inhibits cell death.