Introduction to RNA Vaccines Post COVID-19.

Available prophylactic vaccines help prevent many infectious diseases that burden humanity. Future vaccinology will likely extend these benefits by more effectively countering newly emerging pathogens, fighting currently intractable infections, or even generating novel treatment modalities for non-infectious diseases. Instead of applying protein antigen directly, RNA vaccines contain short-lived genetic information that guides the expression of protein antigen in the vaccinee, like infection with a recombinant viral vector. Upon decades of research, messenger RNA-lipid nanoparticle (mRNA-LNP) vaccines have proven clinical value in addressing the COVID-19 pandemic as they combine benefits of killed subunit vaccines and live-attenuated vectors, including flexible production, self-adjuvanting effects, and stimulation of humoral and cellular immunity. RNA vaccines remain subject to continued development raising high hopes for broader future application. Their mechanistic versatility promises to make them a key tool of vaccinology and immunotherapy going forward. Here, I briefly review key developments in RNA vaccines and outline the contents of this volume of Methods in Molecular Biology.

Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes.

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding. However, the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science. Here, we report an efficient, non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus (TSWV), an RNA virus with a host range of over 1000 plant species. We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts. The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors. In systemically infected plant tissues, the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency. Plants with heritable, bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection. Moreover, we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100% of regenerated plants and further augmented the recovery of heritable mutations. Because many plants are recalcitrant to stable transformation, the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

Biodistribution and environmental safety of a live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

New platforms are needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as a vector for several licensed vaccines and platform for novel candidates. On the basis of YF17D, we developed an exceptionally potent COVID-19 vaccine candidate called YF-S0. However, use of such live RNA viruses raises safety concerns, such as adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic disease [YEL-AND] and yellow fever vaccine-associated viscerotropic disease [YEL-AVD]). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in signal transducer and activator of transcription 2 (STAT2) signaling as a new preclinical model of YEL-AND/AVD. Compared with YF17D, YF-S0 showed improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering that yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence compared with readily transmitting YF-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other vaccines based on the same YF17D platform.

Carotenoid fortification of zucchini fruits using a viral RNA vector.

BACKGROUND: Carotenoids are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. RESULTS: Here, we demonstrate an alternative biotechnological strategy for carotenoid fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated to zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and ß-carotene (provitamin A), lutein and phytoene, in both rind and flesh. CONCLUSION: Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Development of a Honey Bee RNA Virus Vector Based on the Genome of a Deformed Wing Virus.

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs.

Cytoplasmic RNA virus-derived vectors have emerged as attractive vehicles for microRNA (miRNA) delivery as they possess no potential risk of chromosomal insertion. However, their relatively short-term expression limits their use in biological applications that require long-term miRNA manipulation, such as somatic cell reprogramming. Here, we show that a cytoplasmic RNA virus vector based on a replication-defective and persistent Sendai virus (SeVdp) serves as an effective platform for long-term production of miRNAs capable of inducing sequence-specific target suppression. The SeVdp vector was able to simultaneously deliver embryonic stem cell-enriched miRNAs, as well as multiple transcription factors, into fibroblasts, resulting in effective reprogramming into induced pluripotent stem cells. Furthermore, we report that the murine miR-367 hairpin produced elevated levels of mature miRNA when it was incorporated into the SeVdp vector and served as an effective backbone for production of artificial miRNAs. These SeVdp vector-derived artificial miRNAs efficiently inhibited expression of target genes. Our findings provide novel insights into a powerful tool for long-term and targeted gene silencing in areas such as regenerative medicine, gene therapy, and cell therapy.

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.

Introduction to RNA Vaccines.

RNA vaccines are attractive, because they exhibit characteristics of subunit vaccines and live-attenuated vectors, including flexible production and induction of both humoral and cellular immunity. While human proof-of-concept for RNA vaccines is still pending, the nascent field of RNA therapeutics has already attracted substantial industry and government funding as well as record investments of private venture capital. Most recently, the WHO acknowledged messenger RNA (mRNA) as a new therapeutic class. In this chapter, we briefly review key developments in RNA vaccines and outline the contents of this volume of Methods in Molecular Biology.

RNA-based viral vectors.

The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (-) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of negative strand RNA virus genomes rapidly followed as more sophisticated approaches to provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA templates were developed. These advances have driven an explosion of RNA virus vaccine vector development. That is, development of approaches to exploit the basic replication and expression strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing.