2019

Beissert T, Perkovic M, Vogel A, Erbar S, Walzer KC, Hempel T, Brill S, Haefner E, Becker R, Türeci Ö, Sahin U. (2019) A trans-amplifying RNA vaccine strategy for induction of potent protective immunity. Mol Ther. pii: S1525-0016(19)30412-5.

DOI; PMID

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Here we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10-100 fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- over saRNA-encoded replicase to drive expression of the transreplicon is most likely attributable to its higher translational efficiency and lack of interference with cellular translation. Testing the novel taRNA system in mice, we observed that doses of influenza hemagglutinin antigen-encoding RNA as low as 50 ng were sufficient to induce neutralizing antibodies and mount a protective immune response against live virus challenge. These findings, together with a favorable safety profile, a simpler production process and the universal applicability associated with this bipartite vector system, warrant further exploration of taRNA. 

Sorn P, Holtsträter C, Löwer M, Sahin U, Weber D.  (2019) ArtiFuse – Computational validation of fusion gene detection tools without relying on simulated reads. Bioinformatics.  pii: btz613.

DOI; PMID

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Gene fusions are an important class of transcriptional variants that can influence cancer development and can be predicted from RNA sequencing (RNA-seq) data by multiple existing tools. However, real world performance of these tools is unclear due to the lack of known positive and negative events, especially with regard to fusion genes in individual samples. Often simulated reads are used, but these cannot account for all technical biases in RNA-seq data generated from real samples. Here we present ArtiFuse, a novel approach that simulates fusion genes by sequence modification to the genomic reference, and therefore can be applied to any RNA-seq dataset without the need for any simulated reads. We demonstrate our approach on eight RNA-seq datasets for three fusion gene prediction tools: Average recall values peak for all three tools between 0.4 and 0.56 for high quality and high coverage datasets. As ArtiFuse affords total control over involved genes and breakpoint position, we also assessed performance with regard to gene-related properties, showing a drop in recall value for low expressed genes in high coverage samples and genes with co-expressed paralogues. Overall tool performance assessed from ArtiFusions is lower compared to previously reported estimates on simulated reads. Due to the use of real RNA-seq datasets we believe that ArtiFuse provides a more realistic benchmark that can be used to develop more accurate fusion gene prediction tools for application in clinical settings.

ArtiFuse is implemented in Python. The source code and documentation is available at https://github.com/TRON-Bioinformatics/ArtiFusion. 

Grunwitz C, Salomon N, Vascotto F, Selmi A, Bukur T, Diken M, Kreiter S, Türeci Ö, Sahin U. (2019) HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memoryOncoimmunology. 8:e1629259.

DOI; PMID

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HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers. 

Vascotto F, Petschenka J, Walzer KC, Vormehr M, Brkic M, Strobl S, Rösemann R, Diken M, Kreiter S, Türeci Ö, Sahin U. (2019) Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell responseOncoimmunology. 8:e1601480.

DOIPMID

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TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8+ T cells and CD11b+ conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8+ T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound.

 

Orlandini von Niessen AG, Poleganov MA, Rechner C, Plaschke A, Kranz LM, Fesser S, Diken M, Löwer M, Vallazza B, Beissert T, Bukur V, Kuhn AN, Türeci Ö, Sahin U. (2019) Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3′ UTRs Identified by Cellular Library Screening. Mol Ther. 27:824-836.

DOI; PMID

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Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3′ UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3′ UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) the delivery of vaccine antigens in order to mount T cell immune responses and (2) the introduction of reprogramming factors into differentiated cells in order to induce pluripotency, that mRNAs tagged with the 3′ UTR elements discovered in this study outperform those with commonly used 3′ UTRs. This approach further leverages the utility of mRNA as a gene therapy drug format.

 

Vormehr M, Türeci Ö, Sahin U. (2019) Harnessing Tumor Mutations for Truly Individualized Cancer Vaccines. Annu Rev Med. 70:395-407.

DOI; PMID

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T cells are key effectors of anticancer immunity. They are capable of distinguishing tumor cells from normal ones by recognizing major histocompatibility complex-bound cancer-specific peptides. Accumulating evidence suggests that peptides associated with T cell-mediated tumor rejection arise predominantly from somatically mutated proteins and are unique to every patient’s tumor. Knowledge of an individual’s cancer mutanome (the entirety of cancer mutations) allows harnessing this enormous tumor cell-specific repertoire of highly immunogenic antigens for individualized cancer vaccines. This review outlines the preclinical and clinical state of individualized cancer vaccine development and the challenges ahead.