Viral and Synthetic RNA Vector Technologies and Applications Juliane W Schott, Michael Morgan, Melanie Galla, Axel Schambach  Molecular Therapy  Volume 24, Issue 9, Pages 1513-1527 (September 2016) DOI: 10.1038/mt.2016.143 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Dendrogram of RNA technologies and overview of delivery particle/RNA composition. Current RNA technologies can be classified with respect to RNA origin (viral/nonviral), replication profile (replicating/nonreplicating), delivery mode (viral/nonviral) and RNA polarity (plus-sense (+)/minus-sense (−)). (a–f) For simplicity, only major structural components and RNA elements are shown; accessory proteins and some structural and enzymatic components are not depicted. RNA orientation is indicated by the 3′ and 5′ end marks. GOI, gene of interest; (A)n, poly(A)-tail. (b,c) Env, envelope glycoprotein; MA, matrix; CA, capsid; nucleocapsid (NC) proteins are not shown; R, redundant region; U5, unique 5; aPBS, artificial primer binding site; U3, unique 3. (d) Nonsegmented negative-strand (NSNS) RNA vector technologies. RNA design is exemplarily shown for first-generation transmission-competent vector genomes. H, hemagglutinin; HN, hemagglutinin-neuraminidase; F, fusion protein; M, matrix; N, nucleoprotein; P, phosphoprotein; L, major polymerase subunit. Arrows indicate possible sites for GOI insertion. (e) Recombinant Alphavirus (upper part) and Flavivirus (lower part) vector particles. RNA design is exemplarily shown for nontransmissible vector genomes. Of note, options for GOI insertion other than those depicted are also possible. E1/E2, envelope glycoproteins; C, capsid; M + E, membrane protein + envelope protein; nsP1 to nsP4, Alphavirus nonstructural proteins 1–4; 26S P, subgenomic 26S promoter; NS1–NS5, Flavivirus nonstructural proteins 1–5. (f) Alphavirus (upper part) and Flavivirus (lower part) naked RNA replicons. Molecular Therapy 2016 24, 1513-1527DOI: (10.1038/mt.2016.143) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Packaging of recombinant SeVV particles. Plasmids encoding a SeV or SeVV antigenomic RNA and the nucleocapsid proteins N, P, and L are cotransfected into packaging cells. These plasmids harbor a bacteriophage promoter (T7 P) and—in case of the SeV/SeVV plasmid—also a T7 terminator (T7 T) and a ribozyme (Rbz) (derived from Hepatitis delta virus) to create precise termini of the (+)-sense RNA transcript, a prerequisite for later RNA replication. A helper virus (commonly a Vaccinia Virus recombinant)193 is used to provide the bacteriophage RNA polymerase required for plasmid transcription. The three proteins and the antigenomic RNA self-assemble into nucleocapsids, which are ribonucleoprotein complexes (RNP) with RNA-dependent RNA polymerase function. This complex transcribes the RNA of both viral structural and nonstructural proteins from the replicated (−)-RNP. Accumulation of viral proteins within the packaging cell causes a switch from a transcription to a replication mode, leading to amplification of the viral genome. The GOI is also transcribed and coexpressed within packaging cells (not shown). The structural proteins M, F, and HN and the (−)-RNP genomes then assemble into particles budding from the plasma membrane. For nontransmissible SeVV particles, one or more structural genes are deleted from the vector genome and must be provided in trans during packaging, either through cotransfection of expression plasmids or via specific packaging cell lines that stably or inducibly express the respective gene(s). While the lack of structural genes precludes progeny production by nontransmissible SeVV, the retention of nucleocapsid genes endows competence for self-amplification of SeVV RNA. Of note, recovered vector particles are commonly amplified through two rounds of packaging cell transduction followed by particle concentration. SeV, Sendai virus; SeVV, SeV vector(s); H, hemagglutinin; HN, hemagglutinin-neuraminidase; F, fusion protein; M, matrix; N, nucleoprotein; P, phosphoprotein; L, major polymerase subunit. Molecular Therapy 2016 24, 1513-1527DOI: (10.1038/mt.2016.143) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Design of AV vectors and packaging constructs. All templates and elements are shown in the respective DNA (plasmid) composition. Except for the DNA-layered vector system (c), templates usually harbour bacteriophage promoters (T7/SP6) for RNA generation through IVT (a,b,d). DNA-layered templates are equipped with a polymerase II promoter (e.g., CMV) for RNA expression within target cells. For RNA replication, all vectors encompass the AV nonstructural genes nsP1-4, and the gene of interest (GOI) is expressed under control of the subgenomic 26S promoter. (a) In case of transmission-competent particles, the GOI expression cassette using a second 26S promoter is added to the full-length AV genome either up- or downstream of the AV structural genes. (b–d) In all other cases, the GOI replaces the structural genes and is inserted downstream of the native 26S promoter. (b) For nontransmissible particle production, the structural genes are provided in trans by helper constructs, either using a monopartite helper design or a bipartite helper system194 with higher safety profile, expressing capsid and spike proteins from two independent RNA molecules (this diminishes potential generation of replication-competent viruses by RNA recombination). These commonly also carry 26S promoters to amplify the helper RNA during packaging. However, promoter-less helper RNAs have been reported to be effective for virus replicon particle (VRP) production as well.195 (c,d) DNA-layered and naked RNA replicon systems are devoid of all structural genes. P, promoter; p(A), polyadenylation signal; AV, Alphavirus; CMV, Cytomegalovirus; IVT, in vitro transcription. Molecular Therapy 2016 24, 1513-1527DOI: (10.1038/mt.2016.143) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Production of AV vectors and particles. (a,b) Vector and helper RNAs generated through IVT are introduced into packaging cells. The nonstructural proteins nsP1-4 encoded on the (+)-sense RNA are directly translated by the host cell machinery. The nsP1–4 complex then mediates replication of the vector RNA. (a) With vector RNA for transmission-competent particles, nsP1–4 under control of the 26S promoter mediate transcription of (+)-sense subgenomic (SG) RNA from the (–)-sense RNA. The SG RNA encodes all structural genes, which are translated by the host cell machinery. (b) For production of nontransmissible particles, nsP1–4 also replicate the helper RNAs by trans-complementation. Directed by the 26S promoters, nsP1–4 transcribe mRNA encoding the structural genes from the (−)-sense helper RNAs, which are then translated by the host cell machinery. Alternatively, stable packaging cell lines are available for induced structural protein expression.196 (a,b) The GOI, which is also placed downstream of a 26S promoter (see Figure 3), is coexpressed within packaging cells in both systems (not shown). Structural proteins and (+)-sense vector RNA assemble into recombinant viral particles competent for target cell transduction. Here, nsP1–4 expression again launches RNA replication and transcription as well as GOI expression. While nontransmissible viral replicon particles (VRP) lack structural gene information on the replicon RNA, transmission-competent recombinant AV particles (rAV) are capable of virus production within transduced cells. Vector and helper functions can also be expressed from DNA-based plasmids.197 (c,d) DNA-layered vectors are plasmid-encoded AV replicons. Naked RNA replicon vectors are synthesized through IVT. Both are directly transfected into target cells, circumventing the need for packaging cell-mediated particle production. Translation of nsP1–4 then launches RNA replication and transcription followed by GOI expression. AV, Alphavirus; IVT, in vitro transcription. Molecular Therapy 2016 24, 1513-1527DOI: (10.1038/mt.2016.143) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions