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`Filed on behalf of: Sarepta Therapeutics, Inc.
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`UNITED STATES PATENT AND TRADEMARK OFFICE
`______________________
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`______________________
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`SAREPTA THERAPEUTICS, INC.
`Petitioner
`v.
`NIPPON SHINYAKU CO., LTD.
`&
`NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY
`Patent Owners
`______________________
`
`Case No. IPR2021-01134
`Patent No. 9,708,361
`______________________
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`DECLARATION OF DR. DAVID R. COREY
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`U.S. Patent No. 9,708,361
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`TABLE OF CONTENTS
`INTRODUCTION ................................................................................ 1
`I.
`QUALIFICATIONS ............................................................................. 1
`II.
`III. MATERIALS CONSIDERED ............................................................. 3
`IV. SUMMARY OF MY OPINIONS ........................................................ 4
`V.
`BACKGROUND AND THE STATE OF THE ART .......................... 8
`A.
`From DNA to Protein ................................................................. 8
`B.
`Duchenne Muscular Dystrophy (DMD) ................................... 13
`C.
`Exon Skipping Antisense Oligomers (AOs) as DMD
`Therapies .................................................................................. 15
`1.
`Exon Skipping AOs ....................................................... 15
`2.
`Natural Precedents for Exon Skipping AOs in DMD .... 16
`3.
`Exon Skipping AOs in DMD ......................................... 17
`4.
`Exon 53 Targeting AOs ................................................. 25
`5.
`Known Techniques for Evaluating Exon Skipping
`AOs ................................................................................ 32
`The Asserted Prior Art References........................................... 33
`1.
`Popplewell ...................................................................... 33
`2.
`Sazani ............................................................................. 37
`VI. THE ’361 PATENT ............................................................................ 39
`A. Overview .................................................................................. 39
`B.
`The Claims of the ’361 Patent .................................................. 40
`C.
`The Disclosures of the ’361 Patent .......................................... 42
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`D.
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`2.
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`3.
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`4.
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`5.
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`AOs Disclosed in the Japanese Application .................. 42
`1.
`Additional AOs Disclosed in the ’361 Patent ................ 43
`2.
`The Disclosures of the ’361 Patent ................................ 43
`3.
`VII. LEVEL OF ORDINARY SKILL IN THE ART ................................ 44
`VIII. CLAIM 1 IS OBVIOUS OVER POPPLEWELL AND SAZANI ..... 45
`A. A POSA Would Have Been Motivated to Make and Test
`AOs of Claim 1 ......................................................................... 45
`1.
`A POSA Would Have Been Motivated to Make Exon
`53 Targeting AOs ........................................................... 45
`A POSA Would Have Been Motivated to Make and
`Test AOs Targeting the (+30+65) Hotspot Target
`Region of Exon 53 ......................................................... 47
`A POSA Would Have Been Motivated to Make and
`Test 25-mer AOs Targeting the (+36+60) Sequence of
`Exon 53 as Claimed in the ’361 Patent .......................... 50
`A POSA Would Have Been Motivated to Make and
`Test a PMO and a 2’-OMePS Consisting of SEQ ID
`NO: 57 (+36+60) ............................................................ 53
`Popplewell Does Not Discourage AOs that Are 25
`Bases in Length .............................................................. 56
`A POSA Would Have Had a Reasonable Expectation of
`Making and Testing a PMO and 2’-OMePS Targeting the
`(+36+60) Sequence of Exon 53 ................................................ 57
`A POSA Would Have Had a Reasonable Expectation that a
`PMO or a 2’-OMePS Consisting of SEQ ID NO: 57 Would
`Cause Exon 53 Skipping .......................................................... 58
`IX. CLAIMS 2–7 ARE OBVIOUS OVER POPPLEWELL AND
`SAZANI .............................................................................................. 61
`A. Analysis of Claim 2 .................................................................. 61
`ii
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`B.
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`C.
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`X.
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`Analysis of Claims 3–5 ............................................................ 62
`B.
`Analysis of Claim 6 .................................................................. 62
`C.
`D. Analysis of Claim 7 .................................................................. 64
`THE ’361 PATENT DOES NOT EVIDENCE SUPERIORITY OF
`A CLAIMED AO OVER POPPLEWELL ......................................... 67
`XI. OTHER RESEARCHERS’ NEAR-SIMULTANEOUS ARRIVAL
`AT THE (+36+60) TARGET REGION OF EXON 53 ...................... 78
`XII. CONCLUSION ................................................................................... 80
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`I.
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`U.S. Patent No. 9,708,361
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`INTRODUCTION
`I, David R. Corey, have been retained by Sarepta Therapeutics, Inc.
`1.
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`(“Sarepta”) as an independent expert in the design and evaluation of antisense
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`oligomers for therapeutic purposes.
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`2.
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`I am being compensated for the time I spend on this matter, but my
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`compensation is not contingent upon my opinions or the outcome of this or any
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`other proceeding.
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`II. QUALIFICATIONS
`I am a Professor of Pharmacology at the University of Texas
`3.
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`Southwestern Medical Center (UTSWMC). I also hold the Rusty Kelley
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`Professorship in Medical Science. For over 30 years, I have investigated various
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`technologies for altering genetic materials and their therapeutic potentials as
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`treatments of genetic diseases. My current research focuses on designing and
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`testing antisense oligomers, peptide nucleic acids, antigene oligomers, and double
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`stranded RNAs/single-stranded silencing RNAs (RNA interference) for treating
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`rare genetic diseases such as Duchenne Muscular Dystrophy, Huntington’s
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`Disease, Machado Joseph Disease, Dentatorubral-pallidoluysian atrophy,
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`Friedreich’s ataxia, and Fuchs corneal endothelial dystrophy.
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`4.
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`I received my B.A. in Chemistry in 1985 from Harvard University. I
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`received my Ph.D. in Chemistry in 1990 from the University of California,
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`Berkeley. My dissertation research focused on methods of using oligomer
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`U.S. Patent No. 9,708,361
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`conjugates for altering genetic materials such as DNA and RNA in a sequence-
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`specific manner.
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`5.
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`From 1990 to 1992, I was a post-doctoral fellow at the University of
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`California, San Francisco. In 1992, I joined the UTSWMC as an Assistant
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`Professor in the Department of Pharmacology. I received a second appointment
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`from the Department of Biochemistry in 1997. I was promoted to an Associate
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`Professor in 1998 and a Professor in 2003. In 2014, I received the Rusty Kelley
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`Professorship in Medical Science.
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`6.
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`I have been on the Board of Directors of the Oligonucleotide
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`Therapeutics Society since 2009. In this role, I have helped to organize several
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`Annual Oligonucleotide Therapeutics Society Meetings. I was a Chair of the 10th
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`Annual Oligonucleotide Therapeutics Society Meeting held in 2014. Currently, I
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`am President Elect of the Oligonucleotide Therapeutics Society.
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`7.
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`I have been on editorial boards for several journals including the
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`Journal of RNAi and Gene Silencing (2005–2015), Artificial DNA (2009–2016),
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`Methods (2012–2019), and Scientific Reports (2016). Currently, I am Executive
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`Editor of Nucleic Acids Research, a prominent journal in the field of nucleic acids
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`and related technologies. I am also a Senior Editor of Nucleic Acid Therapeutics.
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`I have also recently served as a reviewer for numerous journals including Nature,
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`Science, Proceedings of the National Academy of Sciences USA, RNA,
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`U.S. Patent No. 9,708,361
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`Biochemistry, Journal of the American Chemical Society, Nature Neuroscience,
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`Nature Medicine, and Cell Reports. I have reviewed grant applications for various
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`institutes and foundations including the National Institutes of Health, the National
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`Cancer Institute, the National Institute of General Medical Sciences, the National
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`Science Foundation, the Wellcome Trust, and Muscular Dystrophy Campaign.
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`8.
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`I have authored over 170 peer-reviewed publications related to nucleic
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`acids and related technologies such as antisense oligomers. I have also published
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`several book chapters on designing and making antisense oligomers, including
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`those that are chemically modified. I am regularly invited to present my work at
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`conferences, and I have given more than 50 invited seminars since 2014.
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`9. My professional qualifications are described in further detail in my
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`curriculum vitae, which is attached as Appendix A.
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`III. MATERIALS CONSIDERED
`In forming my opinions, I have considered the materials cited in this
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`report, as well as those listed in Appendix B. In addition to these materials, I may
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`consider additional documents and information in forming any supplemental
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`opinions. To the extent I am provided with additional documents or information,
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`including any expert declarations in this proceeding, I may offer further opinions.
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`IV. SUMMARY OF MY OPINIONS
`11. The claims of the ’361 patent are directed to antisense oligomers
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`(“AOs”) that induce skipping of exon 53 of the human dystrophin gene and consist
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`of the sequence of SEQ ID NO: 57, which targets and is perfectly complementary
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`to the (+36+60) sequence of exon 53. The claimed AOs would have been obvious
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`to a person of ordinary skill in the art (“POSA”) as of August 31, 2011, the date
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`that NS filed its PCT application which contained for the first time SEQ ID NO:
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`57. As described herein, my opinions set forth in this declaration would not
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`change if the September 1, 2010 filing date of NS’s original Japanese application is
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`used as the date for determining prior art.
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`12. By August 31, 2011, exon skipping AOs were recognized as a
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`promising therapeutic approach for the treatment of Duchenne muscular dystrophy
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`(DMD). Numerous preclinical in vitro and in vivo studies had confirmed the safety
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`and efficacy of exon skipping for DMD. By August 31, 2011, early results from
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`human clinical trials of two AOs targeting exon 51 of the dystrophin gene were
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`reported as encouraging. Both treatments were reported as of August 31, 2011 to
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`cause exon skipping and to increase dystrophin levels in DMD patients. By
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`August 31, 2011, researchers were also investigating and developing AOs targeting
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`exon 53 that were reported to have the potential to treat a large number of DMD
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`patients.
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`13. By August 31, 2011, prior art references including Popplewell had
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`identified a superior, hotspot target region within exon 53. It had been shown that
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`many AOs targeting this hotspot target region effectively induced exon 53 skipping
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`in in vitro and in vivo assays. Popplewell highlighted a number of these exon 53
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`targeting AOs as “worthy of consideration for any upcoming clinical trial.”
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`Among the most effective prior art AOs targeting this hotspot region was a 25-mer
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`AO targeting the (+35+59) sequence in exon 53—just one nucleotide shifted from
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`the target region of the AOs claimed in the ’361 patent—which was singled out by
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`Popplewell as a “viable” candidate for clinical trials.
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`14. Oligomers are synthetic molecules. During drug development of
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`synthetic molecules, it is common to synthesize many variant compounds that are
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`closely related to compounds that show promising activity. The purpose is to
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`search for compounds that might have better properties and make successful pre-
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`clinical and clinical development more likely. By August 31, 2011, it was
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`common knowledge that testing dozens or even hundreds of different compounds
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`was the norm for companies pursuing nucleic-acid-based drugs.
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`15. By August 31, 2011, based on known effective exon 53 targeting
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`compounds, a POSA would have been motivated to use routine screening
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`techniques to identify and test additional AOs that were close chemical relatives to
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`such known active AOs. Using the prior art 25-mer AO targeting the (+35+59)
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`sequence in exon 53 as a starting point, a POSA would have designed a one-
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`nucleotide stepped array and tested the resulting AOs for their ability to induce
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`exon 53 skipping. Using such an approach, a POSA would have immediately
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`identified and chosen for testing an AO consisting of SEQ ID NO: 57 (targeting
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`the (+36+60) sequence of exon 53) as claimed in the ’361 patent.
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`16. A POSA would have reasonably expected that an AO targeting the
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`(+36+60) sequence of exon 53 would induce skipping of exon 53. The (+36+60)
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`target sequence falls within the hotspot target region of exon 53 identified in
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`Popplewell. The numerous effective prior art AOs (including those identified in
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`Popplewell) with target regions throughout the hotspot target region of exon 53
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`(+30+65) would have provided a reasonable expectation that an AO targeting the
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`(+36+60) region of exon 53 would be similarly effective in causing skipping of
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`exon 53. The (+36+60) AO lacks any distinguishing feature relative to the
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`previously tested AOs that would suggest that it would have a significantly
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`different activity from those previously tested AOs.
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`17. A POSA would have expected that the (+36+60) AO would have
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`skipping efficacy similar to the many closely related AOs that had been tested
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`previously and would have tested the (+36+60) AO because thorough testing of
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`related AOs reassures that no potentially better AO has been overlooked.
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`18. The data disclosed in the ’361 patent demonstrates how similar in
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`efficacy the claimed AOs are to the prior art AOs. For instance, the ’361 patent
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`specification shows that an AO consisting of SEQ ID NO: 57 was no better than an
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`AO targeting the same (+35+59) sequence of exon 53 that was disclosed in the
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`prior art. The similar skipping efficiencies for these two AOs would have been
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`unsurprising to a POSA, given that the target region of an AO consisting of SEQ
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`ID NO: 57 (+36+60) is shifted by just one nucleotide as compared to the target
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`region of the prior art AO targeting the (+35+59) sequence.
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`19. Two prominent classes of AOs as of August 31, 2011 were PMOs and
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`2’-OMePSs. Both of these classes of AOs bind their targets by Watson-Crick base
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`pairing and share similar mechanisms of action. Indeed, one of the two exon 51
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`targeting AOs undergoing clinical trials for DMD as of August 31, 2011 was a
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`PMO (being developed by Sarepta) and the other was a 2’-OMePS (being
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`developed by Prosensa, now BioMarin). By August 31, 2011, a POSA would have
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`been aware that Sazani provides the structure of Sarepta’s exon 51 PMO (including
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`its 5’ triethylene glycol moiety) and reports on its favorable safety profile.
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`Because both the PMO and 2’-OMePS share common mechanisms and were both
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`showing promise in the clinic for the treatment of DMD, a POSA making an AO
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`for the treatment of DMD would have been motivated to generate PMOs and 2’-
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`OMePSs and would have had reasonable expectation that these AOs could be used
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`to safely and effectively induce skipping of exon 53.
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`20. Thus, as discussed below, it is my opinion that the challenged claims
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`of the ’361 patent would have been obvious to a POSA as of August 31, 2011.
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`V. BACKGROUND AND THE STATE OF THE ART
`From DNA to Protein
`A.
`21. Deoxyribonucleic acid (“DNA”) is the hereditary material in humans
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`and other organisms. (Ex. 1068 [Alberts], 191–192.) DNA comprises smaller
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`portions (“genes”), each of which encodes a protein that carries out various
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`functions in the body. (Id., 301.) The process of encoding a protein from a gene
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`involves three main steps: transcription, splicing, and translation. (Id.) Each step
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`is explained below.
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`22. DNA is a long polymer made up of four different building blocks
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`(referred to as “nucleotides”). (Id., 193.) Each nucleotide comprises a five-carbon
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`sugar (deoxyribose), a phosphate group, and one of four nitrogen-containing bases.
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`(Id.) The base is selected from adenine (A), guanine (G), cytosine (C), and
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`thymine (T). (Id.) The four base symbols, A, G, C, and T commonly denote the
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`four nucleotides as well. (Id.) Figure 1 depicts the four nucleotides found in
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`DNA:
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`U.S. Patent No. 9,708,361
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`O
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`N
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`NH
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`NH2
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`Guanine (G)
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`NH
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`O
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`Thymine (T)
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`O
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`N
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`H
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`
`
`N
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`N
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`H
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`HH
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`O
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`-
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`O
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`O
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`P
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`-
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`O
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`O
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`O
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`H
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`OH
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`H
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`-
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`O
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`O
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`P
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`-
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`O
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`O
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`H
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`OH
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`H
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`H
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`H
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`NH2
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`N
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`N
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`Adenine (A)
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`N
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`N
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`H
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`NH2
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`N
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`O
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`O
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`H
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`OH
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`H
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`H
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`H
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`O
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`Cytosine (C)
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`N
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`H
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`H
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`H
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`O
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`O
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`H
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`OH
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`H
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`
`
`
`
`O
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`P
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`-
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`O
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`O
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`P
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`-
`
`O
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`-
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`O
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`-
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`O
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`Figure 1. Nucleotides in DNA
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`23.
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`In DNA, nucleotides are covalently linked via phosphodiester
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`linkages between the 5’ carbon of one nucleotide and the 3’ hydroxyl group of the
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`next nucleotide. (Id.) The sugar-phosphate “backbone” forms the structural
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`framework of DNA. As shown in Figure 2, one end of DNA has a free phosphate
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`group and is referred to as the 5’-end of DNA. (Id.) The other end has a free -OH
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`group and is referred to as the 3’-end of DNA. (Id.)
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`U.S. Patent No. 9,708,361
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`O
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`N
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`NH
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`NH2
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`NH2
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`N
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`O
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`N
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`H
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`H
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`H
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`O
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`H
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`H
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`H
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`H
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`OH
`3'-end
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`O
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`O
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`O
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`N
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`H
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`NH
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`O
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`
`
`N
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`N
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`N
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`H
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`NH2
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`N
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`HH
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`O
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`O
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`H
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`O
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`H
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`O
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`P
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`-
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`O
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`5'-end
`
`O
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`P
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`-
`
`O
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`O
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`-
`
`O
`
`H
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`O
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`H
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`O
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`P
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`-
`
`O
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`N
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`N
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`H
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`H
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`H
`
`O
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`O
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`H
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`O
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`H
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`O
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`P
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`-
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`O
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`
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`Figure 2. Linked Nucleotides in DNA
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`24. One chain of polynucleotides can form a double helix with another
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`chain of polynucleotides because a base from one chain can pair with its
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`complementary base from the other chain (also referred to as “Watson-Crick” base
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`pairing). (Id., 194.) As shown in Figure 3 below, in DNA, A pairs with T while G
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`pairs with C.
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`Figure 3. Complementary Base Pairing in DNA (Ex. 1068, 194, Fig. 4-4)
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`25. To encode a protein, cells copy (or “transcribe”) a portion of DNA
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`into a precursor messenger RNA (“pre-mRNA”). (Id., 302, 317.) The pre-mRNA
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`transcript contains the same genetic information as its DNA counterpart, except
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`that it is in a different chemical form. (Id., 302.) Like DNA, RNA is a polymer
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`made up of nucleotides containing a five-carbon sugar (ribose) and one of four
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`nucleobases. RNA has the same bases as DNA except that it contains uracil (U)
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`instead of thymine (T). (Id.) Like T in DNA, U in RNA makes a complementary
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`base pairing with A. (Id.)
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`26. Each pre-mRNA transcript contains segments that ultimately encode a
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`protein (“exons”), interspersed with additional segments that are deleted (or
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`“spliced”) from the transcript (“introns”). (Id., 317.) Concurrently with
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`transcription of pre-mRNA, a complex referred to as the spliceosome recognizes
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`and binds to certain parts of the transcript, cuts out introns, and stitches exons
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`together. (Id., 319–320.) The resulting product is referred to as messenger RNA
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`(“mRNA”). (Id., 304, 317.)
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`27. The spliceosome binds to two ends of each intron (referred to as
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`“donor” and “acceptor” regions). Other spicing components such as serine and
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`arginine rich proteins (“SR proteins”) bind to select regions within exons known as
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`exonic splicing enhancers (“ESEs”). (Ex. 1057, 1611–1612.) Binding of SR
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`proteins to ESEs was understood to be essential for exon inclusion and interfering
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`with this binding with AOs would have been expected to result in exon skipping.
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`(Id., 1612.)
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`28.
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`In translating an mRNA molecule to a protein, proteins within cells
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`read the sequence of nucleotides in the mRNA molecule in groups of three
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`nucleotides (“codons”). (Ex. 1068, 336.) Each group of three nucleotides
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`corresponds to (1) one of twenty amino acids or (2) a stop codon (i.e., a signal that
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`terminates the translation of mRNA to protein). (Id.) In principle, an mRNA
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`molecule can be read in three possible reading frames. (Id.) In reality, only one of
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`the three reading frames has the ability to encode a correct protein (“open reading
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`frame”). (Id.) Generally, each mRNA molecule contains a special signal that
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`12
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`allows for cells to read the mRNA molecule in a correct reading frame. (Id., 348–
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`U.S. Patent No. 9,708,361
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`349.) If, however, the mRNA molecule contains a mutation such as a deletion, the
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`reading frame may be disrupted (“out-of-frame” or “frameshift” mutations). In
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`that case, the translation of mRNA to protein may terminate prematurely due to
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`inadvertent introduction of a stop codon and lead to a truncated protein or no
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`protein at all. In contrast, an “in-frame” mutation can result from, for example, the
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`addition or deletion of three nucleotides (or a multiple of three nucleotides). In this
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`case, the open reading frame is preserved and subsequent codons after the mutation
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`will not have a disrupted reading frame.
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`B. Duchenne Muscular Dystrophy (DMD)
`29. DMD is an X-linked recessive muscular dystrophy that affects 1 in
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`3,500–6,000 newborn males and causes progressive muscle weakness,
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`cardiomyopathy, and respiratory failure. (Ex. 1027 [Bushby 2010], 77; Ex. 1028
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`[Kinali 2009], 918; Ex. 1067 [Mann 2002], 644–645.) DMD patients lack
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`functional dystrophin, a protein which is required to maintain the membrane
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`integrity of muscle fibers. (Id.)
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`30.
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`In DMD, dystrophin protein is not produced, typically because a
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`frame-shift mutation in the dystrophin (or DMD) gene disrupts the triplet reading
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`frame, leading to premature termination of translation. (Ex. 1028 [Kinali 2009],
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`918–919; Ex. 1029 [Hoffman 2007], 2721.) As shown in Figure 4 below,
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`dystrophin plays an important role in the muscle fiber’s structure and function,
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`U.S. Patent No. 9,708,361
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`forming a complex with other proteins that stabilizes the membrane of muscle
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`cells. (Ex. 1040 [Nakamura 2009], 495.)
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`Figure 4. Dystrophin (Ex. 1069 [Wood 2013], 170, Fig. 1)
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`31. The dystrophin (or DMD) gene contains 79 exons. (Ex. 1030 [Wilton
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`2008], 255.) Although mutations can occur anywhere in the dystrophin gene, it
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`was known by August 31, 2011 that mutations in DMD patients are concentrated
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`in regions between exon 43 and exon 55. (Ex. 1036 [Muntoni 2010], 358.)
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`14
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`Researchers by August 31, 2011 understood that AOs targeting exon 51 or exon 53
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`had the potential to treat relatively large numbers of DMD patients. (Ex. 1021
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`[Popplewell], 102–103; Ex. 1036 [Muntoni 2010], 358, Table 1.)
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`C. Exon Skipping Antisense Oligomers (AOs) as DMD Therapies
`Exon Skipping AOs
`1.
`In the 1990s, Kole and colleagues demonstrated that AOs can induce
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`32.
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`alternative splicing. (Ex. 1070 [Dominski and Kole 1993].) For instance, certain
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`mutations to β-globin genes generate alternative splice sites. These result in an
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`incorrect, alternatively spliced transcript. (Id. [Dominski and Kole 1993], 8673.)
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`AOs complementary to these aberrant splice sites masked the alternative sites,
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`forcing the splicing machinery to recognize normal splice sites, thereby restoring
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`correct splicing. (Id. [Dominski and Kole 1993], 8675; see also Ex. 1071
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`[Dominski 1994], Abstract; Ex. 1072 [Sierakowska 1996], Abstract.)
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`33.
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`Inducing exon skipping is similar in principle (Figure 5). A sequence
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`in the pre-mRNA is targeted by an AO such that the binding of the AO to the pre-
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`mRNA interferes with splicing. This interference results in one or more exons in
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`the pre-mRNA being excluded from the mRNA transcript. (Ex. 1030 [Wilton
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`2008], 256.)
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`15
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`Figure 5. AO Inducing Skipping of Exon 3
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`Natural Precedents for Exon Skipping AOs in DMD
`2.
`34. Truncated but semi-functional dystrophins are expressed in so-called
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`“revertant fibers” in many DMD patients. (Ex. 1031 [Arechavala-Gomeza 2007],
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`798–799.) These individual dystrophin positive fibers were believed to arise via
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`alternative splicing of dystrophin pre-mRNAs. (Id.)
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`35. Mutations in the dystrophin gene were also known to cause a milder
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`form of muscular dystrophy called Becker muscular dystrophy (BMD). (Ex. 1028
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`[Kinali 2009], 919; Ex. 1030 [Wilton 2008], 254; Ex. 1031 [Arechavala-Gomeza
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`2007], 798; Ex. 1067 [Mann 2002], 645.) In BMD patients (unlike in DMD
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`patients), in-frame mutations allow the expression of internally deleted, but largely
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`functional, dystrophin. (Id.) BMD patients experience milder symptoms than
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`DMD patients, ranging from borderline DMD to no symptoms at all. (Id.)
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`36. Exon skipping therapy in DMD has a natural precedent in the
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`revertant fibers found in many DMD patients and in the truncated but functional
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`dystrophin of BMD patients. (Ex. 1031 [Arechavala-Gomeza 2007], 798–799.)
`16
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`Exon Skipping AOs in DMD
`3.
`37. As of August 31, 2011, AOs investigated for exon skipping therapy
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`were typically single stranded and were 20–30 bases in length. (Ex. 1035 [Wilton
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`2007], 1290, Table 1; Ex. 1040 [Nakamura 2009], 495; Ex. 1041 [Yokota 2009],
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`32; Ex. 1021 [Popplewell], 109; Ex. 1042 [Aartsma-Rus 2009], 548.) Exon
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`skipping AOs can bind (or “hybridize”) to a target sequence in the pre-mRNA
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`under physiological conditions via Watson-Crick base paring. Binding of the AO
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`to the pre-mRNA alters splicing of the pre-mRNA such that one or more exon(s) in
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`the pre-mRNA are “skipped” in the mRNA transcript. (Ex. 1029 [Hoffman 2007],
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`2719–2720.) The resulting restoration of the reading frame produces truncated,
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`semi-functional dystrophin protein. (Ex. 1031 [Arechavala-Gomeza 2007], 799;
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`Ex. 1032 [Ginjaar 2000], 796; see generally Ex. 1033 [Wilton 2005].) Figure 6
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`below depicts the mechanism of skipping exon 53 in the dystrophin gene:
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`17
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`U.S. Patent No. 9,708,361
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`Figure 6. Mechanism of Exon Skipping AO (adapted from Ex. 1043
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`[Aartsma-Rus 2009], 873, Fig. 1)
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`Preclinical and Clinical Studies
`a)
`38. By August 31, 2011, numerous preclinical studies, including DMD
`
`animal models, had confirmed exon skipping AOs to be a viable treatment strategy
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`for DMD. (See Ex. 1031 [Arechavala-Gomeza 2007], 800–801, Table 1
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`(summarizing various in vitro and in vivo studies evaluating the exon skipping
`
`efficacy of PMOs and 2’-OMePSs).)
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`39. Following the 2007 publication of the pre-clinical testing and
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`selection of Sarepta’s exon 51 targeting AO (Ex. 1031 [Arechavala-Gomeza 2007],
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`and by August 31, 2011, two companies had initiated clinical trials evaluating two
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`AOs targeting exon 51. Prosensa was developing drisapersen (f/k/a PRO051), a
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`2’-OMePS, and Sarepta was developing eteplirsen (f/k/a AVI-4658), a PMO. In
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`2007, it was reported that in a proof-of-concept study PRO051 induced skipping of
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`18
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`exon 51 and resulted in production of truncated dystrophin protein in four DMD
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`patients. (Ex. 1037 [van Deutekom 2007], Abstract, Figure 4.) Similarly
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`encouraging results were reported in 2009 for AVI-4658. (Ex. 1028 [Kinali 2009],
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`Abstract, Figure 3.) A study published in April 2011 reported that PRO051
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`resulted in functional improvement in DMD patients. (Ex. 1038 [Goemans 2011],
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`Abstract.) Shortly thereafter, on August 13, 2011, positive results from a Phase II
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`trial evaluating AVI-4658 were published, further establishing exon skipping
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`therapy as a viable approach for treating DMD. (Ex. 1039 [Cirak 2011], Abstract.)
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`b) Design of Exon Skipping AOs Targeting the
`Dystrophin Transcript
`40. By August 31, 2011, researchers had identified length as important in
`
`the design of AOs. For some AOs, increasing their length improved skipping
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`efficiency, whereas for other AOs, increasing AO length decreased skipping
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`efficiency. (Ex. 1046 [Adams 2007], 3 (“While ‘longer is better’ in many
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`instances of single AO-induced exon skipping, this does not hold true in all
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`cases.”), 5; Ex. 1057 [Aartsma-Rus 2007], 1613 (“Our own recent studies indicate
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`that the efficiency of some AONs that induce very low levels of exon skipping can
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`be enhanced by increasing AON length, whereas increasing the length of an
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`already efficient AON did not enhance AON efficiency and occasionally even
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`19
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`
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`reduced exon skipping levels.”); Ex. 1042 [Aartsma-Rus 2009], 552; Ex. 1030
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`U.S. Patent No. 9,708,361
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`[Wilton 2008], 258.)
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`41.
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`It was understood as of August 31, 2011 that as AO length increased,
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`so too did the possibility of specific and non-specific off-target effects.
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`Researchers were also mindful of considerations such as ease of synthesis and
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`manufacturing costs. (See, e.g., Ex. 1040 [Nakamura 2009], 495 (“Development
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`of appropriate AOs requires consideration of several characteristics of AOs, such
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`as the chemical specificity, affinity, nuclease resistance, stability, safety, and ease
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`of synthesis[.]”).) As a general matter, as AO length increases, so do
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`manufacturing costs and synthesis challenges. For these reasons, when two AOs
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`showed similar skipping efficiency, the shorter one was often favored.
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`42. By August 31, 2011, researchers routinely used modified nucleobases,
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`chemical backbones, and intersubunit linkages to enhance the stability and potency
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`of AOs, while preserving their ability to bind to their target pre-mRNA sequences
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`via Watson-Crick base pairing. (Ex. 1044 [Chan 2006], 535–536; Ex. 1040
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`[Nakamura], 495–496; Ex. 1045 [Summerton 1997].) Two of the most common
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`classes of AOs used as of August 31, 2011 were “2’-OMePSs” and “PMOs,”
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`shown in Figure 7 below.
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`20
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`Phosphorothioate-linked 2’-O-methyl
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`Phosphorodiamidate morpholino
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`ribose oligomer (“2’-OMePS”)
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`oligomer (“PMO”)
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`Figure 7. 2’-OMePS and PMO Modifications (adapted from Ex. 1040
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`[Nakamura 2009], 496, Fig. 1)
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`43. For example, Nakamura 2009 noted that “[2’-OMePS] and PMO are
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`the most frequently utilized because of their suitable properties.” (Ex. 1040
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`[Nakamura 2009], 495). Similarly, Aartsma-Rus 2009 stated: “The most
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`commonly used chemistries for exon-skipping antisense oligonucleotides are 2’-O-
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`methyl RNA with a negatively charged phosphorothioate backbone (2’-OMePS)
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`and uncharged phosphorodiamidate morpholino oligomers (PMO) . . . .” (Ex. 1043
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`[Aartsma-Rus 2009], 873.) Sazani further recognized: “The favorable safety
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`profile of the uncharged PMOs has been remarkably consistent and predictable, as
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`21
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`more than 460 patients have been safely dosed with PMO in clinical trials for a
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`variety of indications.” (Ex. 1022 [Sazani 2010], 155.)
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`44.
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`2’-OMePSs have a structure similar to RNA but the 2’-OH position of
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`the ribose ring has been methylated, and instead of using a phosphodiester link
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`between nucleotides, one of the non-bridging oxygen atoms of the phosphate group
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`is substituted with a sulfur atom to create a phosphorothioate linkage. PRO051,
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`one of two exon 51 clinical candidates being evaluated for the treatment of DMD
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`as of August 31, 2011, was a 2’-OMePS that was 20 bases in length. (Ex. 1037
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`[van Deutekom 2007], 2679; Ex. 1043 [Aartsma-Rus 2009], 874, Table 1.)
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`45. As of August 31, 2011, it was understood that 2’-OMePSs could
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`contain either uracil or thymine bases, and that either base would be
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`complementary to an adenine base found in pre-mRNA and would pair with
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`adenine in the same way via Watson-Crick base pairing. (Ex. 1048 [Aartsma-Rus
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`2005], 286, Table 1 (describing 2’-OMePSs with U’s); Ex. 1055 [Summerton
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`1999], 144, Fig. 1 (showing 2’-OMePS modification and indicating bases are
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`selected from A, C, G, T, and U).)
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`46. PMOs, a type of “morpholino,” have a six-membered morpholinyl
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`moiety instead of a ribose. PMO subunits are linked through uncharged
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`phosphorodiamidate linkages. (Ex. 1040 [Nakamura 2009], 496.) The second
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`exon 51 targeting AO undergoing clinical trials as of August 31, 2011, AVI-4658,
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