Allele Will Receive Its 3rd US Patent On RNAi

On December 1st, 2009 Allele Biotech will be granted its 3rd US patent on using RNA polymerase III (Pol III) for creating RNAi inside mammalian cells. Previously, US patent 7,294,504 was granted to Allele Biotech that covers commercial kits with DNA template components designed for expression of shRNA, miRNA, or siRNA; US patent 7,422,896 further granted claims covering broader designs of using a Pol III promoter such as a U6 promoter for RNAi, including the use of a constitutive or inducible enhancer. The current US patent 7,625,750, protects the use of above technologies in conjunction with arrays, particularly addressable, high density DNA arrays. The RNAi encoding DNA molecules, anchored to the surface through a special peptide, can be transduced via transduction peptide into target cells grown on the array surface. The DNA will then be released from the surface after the completion of DNA transfer by a membrane protease. "This array format for RNAi using the Pol III technology should have higher efficiency and controllability than soft agar embedded siRNAs for transfecting cells, with great potential in large-scale RNAi functional screening and validation. Combined with Allele Biotech’s existing lentiviral vector-based shRNA platform, the addressable RNAi arrays provide us with the best methods available for RNAi screening”, said Dr. Jiwu Wang, CEO of Allele Biotech and the inventor of the patent.



Allele Biotech provides reagent kits and custom services from using its patented technologies in the field of RNAi. The 3 patents issued to Allele Biotech within the past 2 years are so far the only US patents on the methods and compositions of using Pol III promoter for expressing dsRNA-mediated gene silencing. Allele Biotech aims to strengthen its market position by providing superior products and services while actively protecting its intellectual properties. The current strategy includes noting providers and users of existing products that apparently fall under Allele’s patent protection in order to provide reasonable sub-licensing or co-development options.



Allele Biotech is expanding its RNAi capabilities by incorporating the Pol III promoter-driven shRNA cassettes into its popular Phoenix retroviral system and the recently added lentiviral vector system. The RNAi service will also be integrated with Allele’s viral packaging service which offers the best value in terms of per viral particle cost in the market today. High throughput and high content screenings to be conducted at Allele could be further aided by the use of low mutation oligo annealing and wall-less cell array technologies by collaborating with partner companies. In addition, RNAi target design and selection are carried out with an advanced algorism and the most effective empirical rules through Allele’s RNAi services.



Allele Biotech's RNAi technologies were developed with help from the National Institutes of Health (NIH) through several grants. The research team at Allele is currently applying for another NIH project to use these technologies in synthetic lethal screening for cancer therapy. Dr. Wang said that "It is now our goal as well as responsibility to make the RNAi technologies helpful to as many researchers as possible in their pursuit of the best results from gene function studies”.

Difference between hESCs and NSC-Derived Integration-Free iPSCs

By introducing Oct4 and Nanog into human fetal neural progenitor cells, the Muotri lab at UCSD, in collaboration with colleagues from the Yeo and Gage labs of UCSD and Salk, was able to create induced pluripotent stem cells (iPS cells) using the oriP/EBNA1 episomal system to introduce the aforementioned Oct4 and Nanog. While it has been shown by others that with less differentiated cells, such as progenitor cells, it is relatively easy to reprogram using two, or sometimes even one, of the commonly used iPSC generating factors (Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin28) as reported in the AlleleNews article “iPSC generated by using a single reprogramming factor” on August 3rd, 2009, the new publication in PLoS ONE analyzed in detail the differences between embryonic stem cells (ES cells) and iPS cells.



There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:

(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.



Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076



Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.