Edible Transgenic Plants

About

Invention Summary  Rutgers scientists have developed compositions and methods for the production of edible plants expressing small RNAs (sRNAs), such as small interfering RNAs (siRNAs), which are important for down-regulation of therapeutic targets.  Specifically, Rutgers scientists have constructed several binary vectors using:  a 500 base pair (bp) fragment from the conserved nucleoprotein (NP) gene of the influenza virus H1N1 (IFV);  a 498 bp fragment of the core protein sequence from the hepatitis C virus (HCV) genome; and  a 306 bp fragment of the Tat-encoding sequence of human immunodeficiency virus (HIV). The constructs were used to create transgenic tomato lines using the commercial cultivar Moneymaker as the genetic background. Each plasmid construct introduced into tomato plants has a nptII marker gene that confers kanamycin resistance and a bacterial β-glucuronidase (GUS) gene as a reporter gene to help screen the transformed tomato plants. All tomato plants were tested for transgene activity by rapid leaf tissue histochemical staining and PCR genotyping, and four plants from each of the three groups of transgenic plants, designated as HIV interfering (HIVi), IFV interfering (NPi), and HCV interfering (HCVi), with different levels of GUS expression were selected and asexually propagated. Studies with purified sRNAs transfected into mammalian cells showed that tomato-expressed siRNAs down-modulate the targeted viral sequences in the cells with a high degree of specificity and efficacy  Parallel studies with rabbits fed with the transgenic tomato fruits showed that sRNAs in fruits can be efficiently taken up into the circulatory system of mammals after ingestion, making this approach a viable oral delivery method  Further time course studies af ter termination of tomato feeding indicated that the plant siRNAs can remain stable in rabbits for more than 2 weeks  Additional studies are currently being performed to demonstrate efficacy for viral resistance in small animal models as well as to determine dosage dependence.   Application  Agricultural Biotechnology; Pharmaceutical; Anti-viral; Small RNA Delivery for Therapeutics; Foods; Vegetables, etc.   Advantages  Exceptional stability of sRNAs in plant extracts suggests potential means of product packaging, storage and delivery.  High degree of specificity compared to previous work on synthetic siRNA or small hairpin RNA designed for a specific site on target genes, which did not lead to effective gene silencing.  Ability to target multiple genes or virus of interest via gene stacking in the vector design for siRNA production in transgenic plants.  Alternative cheaper approach compared with current methods, as the transgenic plants can be incorporated into the diet.  The proposed method also avoids the need for repeated injections, which are often required in RNA treatments and are both inconvenient and painful.