Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

Abstract

In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.

Figure 1.

In vitro-transcribed RNA is immunogenic and contains dsRNA contaminants. (A) 200 ng of in vitro transcripts encoding mEPO and containing the indicated modified nucleosides were blotted and analyzed with K1 and J2 dsRNA-specific mAbs. The dsRNA positive control contained a 328 bp long dsRNA (25 ng). (B) DCs were treated with Lipofectin-complexed Renilla luciferase (T7TSRenA₃₀), firefly and Metridia luciferases (T7TSLucA₃₀, T7TSMetlucA₃₀), and mEPO (TEVmEPOA₅₁) mRNAs. TNF-α levels were measured in the supernatants at 24 h. (C) DCs were treated with TransIT-complexed in vitro transcripts encoding Renilla and firefly luciferases (T7TSRenA₃₀, T7TSLucA₃₀), eGFP (TEVeGFPA₅₁) and mEPO (TEVmEPOA₅₁). IFN-α levels were measured in the supernatants at 24 h. Error bars are standard error of the mean. Data shown is from one experiment that is representative of 3–6.

Figure 2.

HPLC purification of RNA identifies contaminants eluting before and after the expected product. Chromatogram of Ψ-modified TEVeGFPA_n mRNA. RNA was applied to the HPLC column and eluted using a linear gradient of Buffer B (0.1 M TEAA, pH 7.0, 25% acetonitrile) in Buffer A (0.1 M TEAA, pH 7.0). The gradient spanned 38–55% Buffer B over 22 min (red line). Absorbance at 260 nm was analyzed (black line), which demonstrated the expected sized RNA as well as smaller and larger RNA species. Data shown are from one experiment that is representative of over 200.

Figure 3.

HPLC purification of in vitro-transcribed nucleoside modified mRNA removes dsRNA contaminants and eliminates immunogenicity. (A) 200 ng of RNA encoding the indicated protein and containing the indicated modified nucleosides with or without HPLC purification were blotted and analyzed with the J2 dsRNA-specific mAb. (B) 200 ng of RNA encoding the indicated protein and containing Ψ-modifications with or without HPLC purification were blotted and analyzed with the J2 dsRNA-specific mAb. Blots were reprobed with a ³²P-labeled probe for the 3′-UTR of the RNAs to control for amount of RNA analyzed. (C) DCs were treated with TEVRenA₅₁ RNA containing the indicated nucleoside modifications with or without HPLC purification and complexed to Lipofectin. TNF-α levels were measured in the supernatants at 24 h. Differences in the effect of nucleoside modification on immunogenicity of Renilla-encoding mRNA compared to Figure 1B is likely due to donor variation and differences in UTRs of the RNAs. (D) DCs were treated with TEVLucA₅₁ RNA containing the indicated nucleoside modifications with or without HPLC purification and complexed to TransIT. IFN-α levels were measured in the supernatants at 24 h. Error bars are standard error of the mean. Data shown is from one experiment that is representative of 3 or more.

Figure 4.

HPLC purification of in vitro-transcribed nucleoside-modified mRNA eliminates activation of genes associated with RNA sensor activation. (A) Heatmap representing changes in expression of genes activated by RNA sensors were derived from microarray analyses of DCs treated for 6 hr with TransIT alone or transit-complexed TEVRenA₅₁ RNA with the indicated modifications with or without HPLC purification. RNA from medium-treated cells was used as the baseline for comparison. (B) Northern blot of RNA from DCs treated with medium or TransIT alone or TransIT-complexed TEVRenA₅₁ RNA with the indicated modifications with or without HPLC purification and probed for IFN-α, IFN-β, TNF-α and GAPDH mRNAs.

Figure 5.

HPLC purification of in vitro transcribed mRNA enhances translation. 293T (A) and human DCs (B and C) were transfected with TransIT- (A and C) or Lipofectin- (B) complexed TEVRenA₅₁ or TEVmEPOA₅₁ mRNA with the indicated modifications with or without HPLC purification and analyzed for Renilla luciferase activity or levels of supernatant-associated mEPO protein at 24 h. (D) Human DCs were transfected with Ψ-modified TEVeGFPA_n mRNA with or without HPLC purification (0.1 µg/well) complexed with Lipofectin or TransIT and analyzed 24 h later. Error bars are standard error of the mean. Data shown is from one experiment that is representative of three or more.

Figure 6.

Analysis of RNA contaminants removed by HPLC purification. (A) One hundred microgram of Ψ-modified T7TSLucA₃₀ RNA was applied to the HPLC column and 3 fractions were collected, all RNAs eluting before the main transcription product (I), the expected RNA (II), and all RNAs eluting after the main transcription product (III). The gradient began at 38% Buffer B and increased to 43% Buffer B over 2.5 min and then spanned 43–65% Buffer B over 22 min. Unmodified and m5C/Ψ-modified T7TSLucA₃₀ RNA had similar fractions obtained. (B) The RNAs from each fraction were complexed to TransIT and added to DCs and IFN-α in the supernatant was measured 24 h later. Error bars are standard error of the mean. (C) 200 ng of RNA from the 3 fractions and the starting unpurified RNA were blotted and analyzed with the J2 dsRNA-specific mAb.

Figure 7.

Daily transfection with HPLC-purified m5C/Ψ-modified mRNA does not reduce cell proliferation. Primary keratinocytes were transfected daily with TransIT alone or m5C/Ψ-modified RNA-encoding Renilla luciferase with or without HPLC purification complexed with TransIT. Every 2–3 days, cultures were split and equal numbers of cells for each condition were plated. Total cell numbers for each condition were divided by the total cell number in untreated cells to calculate the percent of control proliferation.