Patenting Interfering RNA

1. Patenting Interfering RNA John LeGuyader – SPE Art Unit 1635 (571) 272-0760 John.leguyader@uspto.gov

2. Interfering RNAGlossary of Terms • RNAi – RNA interference • dsRNA – double stranded RNA • Dicer – RNase endonuclease • siRNA – small interfering RNA, double stranded, 21-23 nucleotides • shRNA – short hairpin RNA (doubled stranded by virtue of a ssRNA folding back on itself) • ssRNA – single stranded RNA • RISC – RNA-induced silencing complex • Helicase – unwinds siRNA and RNase endonuclease

3. RNAi - Mechanism of Action • Introducing long double stranded RNA leads to sequence-specific degradation of homologous gene transcripts • Long double stranded RNA metabolized to small 21-23-nucleotide siRNAs by endogenous RNase Dicer • siRNAs bind to protein complex RISC with dual function helicase – unwinding and RNase activity • Unwinds siRNA allowing antisense strand to bind to target • Hydrolyses of target by endonuclease activity of helicase

4. ssDNA - 5 deoxynucleotides 8-30 mer Endonuclease - ribonuclease Antisense binds homologous RNA target RNAse H endonuclease attacks DNA/RNA duplex Sequence-dependent degradation of cognate mRNA siRNA: ds- or ssRNA  21 mer Endonuclease - ribonuclease Antisense binds homologous RNA target Helicase unwinds/endonuclease attacks RNA/RNA duplex Sequence-dependent degradation of cognate mRNA RNAi Antisense

5. RNAi Patentability issues • Consider a broad claim to: An siRNA that inhibits expression of a nucleic acid encoding protein X. • Can also be claimed more broadly as short interfering nucleic acid.

6. RNAi Patentability Issues35 U.S.C. 101 – Utility • Credible/Specific/Substantial/Well Established • Used to routinely investigate gene function in a high throughput fashion or to modulate gene expression in human diseases (see Chi et al. (PNAS) 100(11):6343-6346, 2003) • Some knowledge of gene function required • Enough to warrant target inhibition

7. RNAi Patentability Issues35 U.S.C. 101 – Utility • If no function for target nucleic acid (protein or regulatory) is shown or was known: • RNAi would likely lack utility • also raises possible enablement (how to use) and/or written description issues • probe function alone for target not sufficient to provide utility for RNAi

8. RNAi Patentability Issues35 U.S.C. 112, first paragraph, Enablement • high in vivo unpredictability due to general lack of knowledge regarding efficacy and in vivo target site determination, and delivery issues • to date only one human antisense with FDA approval

9. RNAi Predictability • Highly dependent on target position • Screening required to identify accessible target regions • Antisense targets can also be RNAi targets but with exceptions, e.g. intron targets not active for RNAi • Small mismatches (1-2 nts) can be tolerated • Long dsRNAs cause severe sequence-non-specific effects • induces apoptosis from shut down of translation • RNAi of about 21nts required to evade effects • Little in vivo efficacy to date

10. RNAi Patentability Issues35 U.S.C. 112, first paragraph, Written Description • adequate description for broad/generic claims relates to what is known and/or disclosed regarding target site accessibility for the gene at issue • the example claim defines the invention in purely functional terms and lacks structural correlates • RNAi described only by function may lack written description

11. RNAi Patentability Issues35 U.S.C. 112, first paragraph, Written Description • The Analysis: • identify all disclosed relevant distinguishing characteristics as they relate to the scope and content of the claims • identify any disclosed structure/function relationships • what target regions are accessible for RNAi and provide for inhibition • showing of antisense targets across mRNA may be sufficient, but not all antisense targets are open to siRNA • intron targets may not be active for siRNA but may be for antisense • identify all elements claimed and their support in the description • identify species explicitly or implicitly disclosed • reconcile with the level of skill in the art

12. RNAi Patentability Issues35 U.S.C. 112, first paragraph, Written Description • A broad genus of siRNAs inhibiting expression of a nucleic acid encoding a protein may not be described by the sequence of a gene alone since the genus reads on targeting many different nucleic acids. • Description of genus sufficient to comply with written description requires description of a representative number of species • In the case of a small genus covering a limited defined target, one species may be respresentative

13. RNAi Patentability Issues35 U.S.C. 112, first paragraph, Written Description • Written Description Conclusions: • Broad claims to siRNAs inhibiting expression of a nucleic acid encoding a protein may lack an adequate written description since the claim reads on targeting many different nucleic acids. • Analysis turns on what is shown in the specification and what was known about the various versions of the gene at the time of filing. • Provide evidence RNAi targets shown functionally correlate with targeting other versions of the gene. • The more you show and/or is known, the more you can possibly claim.

14. Gene Walk

15. Gene Walk Conclusions • Probability of finding an individual functional RNAi is high • A broad claim to “An isolated RNAi that inhibits the expression of human gene X.” may be enabled by providing the sequence for gene X and gene walk data (no magic number) • Predictability of any single RNAi being effective is low • claims to specific RNAi sequence may require evidence of function • The current state of predictability for RNAi may support that breadth/scope claimed is enabled • but this may also raise prior art issues depending on what was known at the time of filing

16. RNAi Patentability Issues35 U.S.C. 102 – Novelty/Anticipation • Anticipation of specific RNAi • must be explicitly taught in the prior art for anticipation to be applicable.

17. RNAi Patentability Issues35 U.S.C. 103 - Obviousness • Expect an obviousness rejection against broad RNAi claims to known genes if the prior art suggested inhibiting the gene by nucleic acid-based methods or other means and the gene sequence was known. • The current knowledge and level of skill in the art is high such that one of ordinary skill in the art would expect at least an RNAi against a known gene, absent evidence to the contrary. • Narrow claims to specific RNAi sequences may be free of the art, where there would be no motivation to modify the prior art to achieve the specific RNAi sequence claimed.

18. RNAi Patentability Issues35 U.S.C. 103 - Obviousness • Motivation • used to routinely investigate gene function in a high throughput fashion or to modulate gene expression in human diseases (see Chi et al. (PNAS) 100(11):6343-6346, 2003) • target identified in the prior art as desirable for silencing (disease gene, virus), and which has been inhibited in cells • neither necessarily identifies any specific RNAi sequence

19. RNAi Patentability Issues35 U.S.C. 103 - Obviousness • Expectation of Success • expectation of RNAi gene silencing highly likely for target sites identified as accessible to antisense inhibition (see Vickers et al. (J. Biol. Chem.) 278: 7108-7118, 2003) • low expectation of success for in vivo applications • low expectation for specific target sequences which are not identified • identification of some sequence as appropriate target should be provided, e.g. known antisense target

20. Case Law • Antisense-specific: • Enzo Biochem Inc. v. Calgene Inc., 52 USPQ2d 1129 (CAFC 1999) • enablement - antisense highly unpredictable • the decision is based on patents with effective filing dates of at least 1989 and the technology at that time • decision does not necessarily determine the outcome for examination of antisense patent applications recently filed because current knowledge and level of skill in the art is high (antisense has progressed as a technology since 1989)

21. Recommendations • Claim functional RNAi by specific sequence. • List results of “gene walk” • showing activity of each RNAi • “gene walk” data may provide representative number of species for broad scope of a generic claim, but there is no magic number

22. Recommendations • Provide objectiveevidence that in vitro results are representative of in vivo applicability. • Respond to examiner-cited unpredictable factors with objective evidence to the contrary. • Expert opinions are more favorably viewed when supported using objective evidence. • Provide objective evidence that a particular animal model is generally accepted as representative of disease or methods of treating, particularly for humans. • Objective evidence includes arguments, case law, journal articles, and experimental data and comparisons commensurate with the disclosure as filed.

23. RNAi Questions?