RNAi Litigation

[This Declaration is dated July 21, 2009]
[Case 1:11-cv-10484-PBS Document 85-2 Filed 07/09/12]
[Case 1:09-cv-11116-PBS Document 51 Filed 07/21/09]

Some of the observations in Dr. Tuschl’s description of his Tuschl I work may be used to show that the Tuschl I inventors knowledge of 21-23 nt literally correct structure preceded Dr. Bass’ disclosure of the siRNA structure.

9. Prior to developing the Tuschl II composition and method for RNAi, I worked with Dr. Bartel, Dr. Sharp, and Dr. Zamore on the Tuschl I invention, which also relates to RNAi. The Tuschl I collaboration led to two main developments: (1) the creation of a system, referred to as the “Drosophila lysate system,” for studying the process of RNAi, and (2) the intermediary discovery that introducing long dsRNA into the Drosophila lysate system leads to the creation of short RNA fragments that are involved in RNAi.

The “Drosophila lysate System”

10. In mid-1999, Dr. Sharp, Dr. Bartel, Dr. Zamore, and I decided to pursue an in vitro system for studying the recently-discovered phenomenon ofRNAi. “In vitro” refers to a system that exists in the controlled environment of a test tube. At the time, Dr. Zamore and I were working as post-doctoral fellows in Dr. Bartel’s laboratory at the Whitehead Institute for Biomedical Research (”Whitehead”). The system we developed is referred to as the “Drosophila lysate system.” In very general terms, the Drosophila lysate system is comprised of the lysate extracted from the inside of certain cells of the fruit fly, Drosophila.

The lysate contains the contents originally found in the cells, including the cells’ naturally occurring DNA, RNA, enzymes, and debris. In this system, we introduced long strands of dsRNA, which were previously made separately, into test tubes containing the Drosophila lysate. We observed that the long dsRNAs initiated RNAi. The other Tuschl I co-inventors and I published the results of this research in Tuschl et al., “Targeted mRNA degradation by double-stranded RNA in vitro,” Genes & Development 13:3191-3197 (December 15, 1999), …

11. Using the Drosophila lysate system, we were able to show that the introduction of long dsRNA into test tubes containing the Drosophila cellular lysate could cause silencing of genes. Though we were able to observe that the introduction of long dsRNAs was causing the silencing of genes, we did not know how it was happening. At the time of publication, we acknowledged that “[t]he molecular mechanisms by which dsRNA generates the RNAi effect are unknown.” That is, we did not know what components in the lysate or what characteristics of the long dsRNA molecules were responsible for triggering the RNAi response.

21-23 Nucleotide Length.

12. In or about January, 2000, the other Tuschl I co-inventors and I determined that in the Drosophila lysate system, there was some process occurring in the lysate that was causing cleavage of the long input dsRNA into fragments of about 21-23 nucleotides. It was further determined that if we removed RNA fragments that we believed to be about 21-23-nucleotides in length from the Drosophila lysate system in which they were created, and added them to a brand new system to which a long dsRNA precursor had never been added, the roughly 21-23 nucleotide fragments alone could result in the silencing of genes.

13. Based on these findings, the first Tuschl I provisional patent application, USSN 601193,594 (”the ‘594 application”), was filed in the USPTO on March 30, 2000 naming Dr. Sharp, Dr. Bartel, Dr. Zamore and me as inventors. … The following day, March 31,2000, the Tuschl I inventors published the data in Zamore et al., “RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals,” Cell 101:25-33 (March 31, 2000).

14. All of the information and figures included in the ‘594 application can be traced back to the Tuschl et al. (1999) Genes & Development and Zamore et al. (2000) Cell papers. Specifically, figures 1-5 of the ‘594 application were also published as Figures 1-5 in the Tuschl et al. (1999) Genes & Development paper. Figures 6-9 and 11 of the ‘594 application were also published as Figures 1,2,4,5, and 7 of the Zamore et at. (2000) Cell paper. Figure 12 (left sidebar) of the ‘594 application was described as unpublished data in the Zamore et al. (2000) Cell paper on page 30, column 2.

15. In a contemporaneous publication by Bass, “Double-Stranded RNA as a Template for Gene Silencing,” Cell 101 :226-238 (April 28, 2000), the research described in the Zamore et al. (2000) Cell paper was recognized as an in vitro Drosophila lysate system that produces 21-23 nucleotide RNA fragments that cause RNAi. However, the author recognized that “[t]he in vitro [i.e., test tube] observations of Zamore et al. (2000) have not yet been directly connected to RNAi in vivo [i.e., in a living organism].” The term in vivo refers to experiments that are performed in living organisms as opposed to experiments that are performed in vitro, or in the controlled environment of a test tube. In other words, our research generated no evidence that our Tuschl I invention could silence genes in living fruit fly (i.e., Drosophila) cells that were the source of the lysate for the artificial system, much less in humans–or for that matter in any mammalian species.

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