Next generation self-replicating RNA vectors for vaccines and immunotherapies.

RNA technology has recently come to the forefront of innovative medicines and is being explored for a wide range of therapies, including prophylactic and therapeutic vaccines, biotherapeutic protein expression and gene therapy. In addition to conventional mRNA platforms now approved for prophylactic SARS-CoV2 vaccines, synthetic self-replicating RNA vaccines are currently being evaluated in the clinic for infectious disease and oncology. The prototypical srRNA vectors in clinical development are derived from alphaviruses, specifically Venezuelan Equine Encephalitis Virus (VEEV). While non-VEEV alphaviral strains have been explored as single cycle viral particles, their use as synthetic vectors largely remains under-utilized in clinical applications. Here we describe the potential commonalities and differences in synthetic alphaviral srRNA vectors in host cell interactions, immunogenicity, cellular delivery, and cargo expression. Thus, unlike the current thinking that VEEV-based srRNA is a one-size-fits-all platform, we argue that a new drug development approach leveraging panels of customizable, synthetic srRNA vectors will be required for clinical success.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

Metabolism goes viral.

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication.

A protein-polymer hybrid mediated by DNA.

Protein-polymer hybrids (PPHs) represent an important and rapidly expanding class of biomaterials. Typically in these hybrids the linkage between the protein and the polymer is covalent. Here we describe a straightforward approach to a noncovalent PPH that is mediated by DNA. Although noncovalent, the DNA-mediated approach affords the highly specific pairing and assembly properties of DNA. To obtain the protein-DNA conjugate for assembly of the PPH, we report here the first direct copper catalyzed azide-alkyne cycloaddition-based protein-DNA conjugation. This significantly simplifies access to protein-DNA conjugates. The protein-DNA conjugate and partner polymer-DNA conjugate are readily assembled through annealing of the cDNA strands to obtain the PPH, the assembly of which was confirmed via dynamic light scattering and fluorescence spectroscopy.

Genetically encoded initiator for polymer growth from proteins.

Despite the importance of protein-polymer bioconjugates, there is no general method for producing homogeneous recombinant protein that contains polymer initiators at defined sites. To address this deficiency, we designed the amino acid 4-(2'-bromoisobutyramido)phenylalanine (1) as an initiator in atom-transfer radical polymerization (ATRP) that would provide a stable linkage between the protein and growing polymer. We synthesized 1 and evolved a Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA(CUA) pair to genetically encode this initiator in response to an amber codon. To demonstrate the utility of this initiator, we produced green fluorescent protein (GFP) with 1 site-specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with a monomer, oligo(ethylene oxide) monomethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate where the polymer is connected at our selected site on GFP.

Generating permissive site-specific unnatural aminoacyl-tRNA synthetases.

Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. Using these techniques, researchers can precisely control the position and number of novel chemical moieties in a protein, via introducing the novel R group of UAAs, that are genetically encoded in the protein's primary structure. The substrate recognition properties of a natural aminoacyl-tRNA synthetase (aaRS) must be modified in order to incorporate UAAs into proteins. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibility of this important technology. At present, engineered unnatural aminoacyl-tRNA synthetases (UaaRS) are evaluated on their translational efficiency (the extent to which they allow for incorporation of UAAs into protein) and fidelity (the extent to which they prevent incorporation of natural amino acids). We propose that a third parameter of substrate recognition, permissivity, is equally important. Permissive UaaRSs, whose relaxed substrate recognition properties allow them to incorporate multiple unnatural amino acids (but not natural amino acids), would eliminate the need to generate new UaaRSs for many new UAAs. Here, we outline methods for quickly and easily assessing the permissivity of existing UaaRSs and for generating permissive UaaRSs. In proof of principle experiments, we determined the degree of permissivity of two UaaRSs for a family of structurally related fluorinated UAAs ((19)F-UAAs). We then increased the permissivity of the initial UaaRSs to allow for incorporation of the family of (19)F-UAAs. Finally, we validated the utility of these new (19)F-UAAs as probes for fluorine NMR studies of protein structure and dynamics. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins.

Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity.

Many unnatural amino acid synthetases have been evolved to enable the site-specific in vivo incorporation of many useful functionalities into proteins. While these unnatural amino acid-tRNA synthetase-tRNA(CUA) pairs do not incorporate endogenous amino acids, their substrate specificity has not been assessed for other unnatural amino acids. Here we demonstrate that the unnatural synthetases can be permissive to many unnatural amino acid substrates. The utility of unnatural synthetases can be further expanded by manipulating the synthetase active sites by mutagenesis. Here we have also shown that an l-2-naphthylalanine synthetase can be converted into a permissive l-4-benzoylphenylalanine synthetase with a single mutation without compromising fidelity. Permissive unnatural amino acid synthetases should significantly expand the tool set available for manipulation of proteins.

Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan.

The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.