Transgenic Tomatoes for Delivery of RNA Therapeutics
RNA Interference (RNAi) is a gene-silencing process in which double-stranded RNAs trigger the destruction of corresponding RNAs in a sequence-specific manner via the production of small interfering RNAs (siRNAs). RNA interference has been used to suppress viral infections by administration or expression of 21-25 nucleotides siRNAs targeting essential components of various viruses. The rapid mutation and recombination rates of viruses such as hepatitis C virus (HCV), avian influenza (H5N1) and the human immunodeficiency virus (HIV) make monotherapeutics involving single targets ineffective in the long term. Thus we are focusing on the use of combinatorial RNAi to circumvent virus evasiveness. Combinatorial RNAi molecules can inhibit a virus's life cycle by targeting multiple genes. To undertake the economic challenges of deployment of RNAi for antiviral therapeutics, as an unconventional approach, we are critically examining the possibility of using transgenic tomato as a delivery tool for combinatorial RNAi (coRNAi) triggers in mammals. An edible fruit like tomato can serve as an economic and sustainable oral delivery tool of the RNAi triggers produced in planta. RNAi by ingestion is used routinely in the model system C. elegans. The RNAi signal can then persist to the next generation after the initial feeding to silence expression of the targeted gene. Although the mammalian digestive system is very different from that of nematodes, the potential benefit from such a simple delivery route for RNAi triggers warrants its examination.
As opposed to animal cells, plant cells tolerate long strand dsRNAs of up to several kilobases (kbs) and thus can produce a large repertoire of siRNAs from a single transcription unit. Previously, we have demonstrated that siRNAs targeting a 400 nucleotide conserved region of the influenza NS1 protein can be constitutively produced in transgenic tobacco and these plant-derived siRNAs are effective in virus suppression in transfection studies involving mammalian cells (Zhou et al. FEBS lett., 2004). This strategy can be easily scaled up to express several kb of sequences that would contain multiple viral genome targets for combinatorial silencing. Thus plants are excellent natural "factories" to produce complex RNAi triggers economically on an agricultural scale.
To examine the question of whether tomato-derived RNAs can in fact survive the mammalian digestive system and make it to the bloodstream and organs, we have performed feeding studies with rabbits that have been fed a simple tomato diet before blood and tissues are drawn and analyzed for the presence of tomato-specific transcripts. Our preliminary data showed the survival of food-ingested RNA and its entry into blood and tissues of the animal. Thus indicating that there should be mechanisms for some of the food-ingested RNA to gain entry into blood stream and different organs.
In this project, our objective is to demonstrate whether antiviral RNAi triggers produced in transgenic tomato fruits can accumulate in mammals after their ingestion. For this purpose, we have created hairpin RNA expression plasmid constructs using: a) a 500 bp fragment from the conserved NP gene (nucleoprotein) of the H1N1 influenza virus; b) a 450 bp fragment of the core protein sequence from the HCV genome; and c) a 300 bp fragment of the Tat-encoding sequence of HIV. These constructs were then used to create transgenic tomato lines using the cultivar Moneymaker as the genetic background. After about 2 years of work by the group of Prof. Ralph Bock at the Max Planck Institute at Golm, Germany, we have now obtained homozygous tomato lines for each of these three constructs. The plasmid construct introduced into tomato plants has a nptII gene that confers kanamycin resistance and a GUS gene that serves as a reporter gene to help screen the transformed tomato plants. All the transgenic plants were screened for the presence of GUS gene by histochemical GUS staining of leaf tissues and for the presence dsRNA construct using genomic PCR. Based on the GUS assay, three high GUS expressing and one low GUS expressing plants were selected from each of the three groups of transgenic plants, HIVi, NPi and HCVi. The expression levels of GUS protein in leaves and fruits were also quantified by performing fluorimetric GUS assay. The expression of siRNAs corresponding to viral dsRNA constructs in the transgenic fruits was detected by Northern blot analysis and quantified using TaqMan-based real-time PCR method. We are in the process of feeding wild type and transgenic tomatoes to the rabbits. We will then compare animals that have been fed rabbit feed (Control) versus those that have been fed a tomato-based diet with either wild-type or transgenic tomatoes. As before, blood and tissue samples will be isolated from these animals and total RNA prepared using protocols that would preserve small RNAs. TaqMan-based real-time PCR will be carried out to detect tomato-specific transcripts and expressed viral siRNAs in blood and tissues of rabbits.