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`UNITED STATES PATENT AND TRADEMARK OFFICE
`_____________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_____________________
`
`
`BUTAMAX™ ADVANCED BIOFUELS LLC
`Petitioner
`v.
`GEVO, INC.
`Patent Owner
`
`_____________________
`
`CASE IPR: Unassigned
`_____________________
`
`DECLARATION OF DENNIS J. THIELE, Ph.D.
`
`1
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`
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`

`

`
`
`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`TABLE OF CONTENTS
`
`B. 
`
`C. 
`
`I. 
`Overview .......................................................................................................... 2 
`II.  My background and qualifications .................................................................. 5 
`III.  List of documents considered in formulating my opinion .............................. 8 
`IV.  Person of ordinary skill in the art .................................................................. 10 
`V. 
`The '565 patent ............................................................................................... 11 
`VI.  Basis of my analysis with respect to written description .............................. 12 
`VII.  The full scope of claims 1-19 of the '565 patent is not disclosed in the '952
`and '209 provisional applications .................................................................. 12 
`A. 
`The provisional applications do not describe the full scope of
`inactivated GRX3 and/or GRX4 as encompassed by claims 1-19 of
`the '565 patent ...................................................................................... 13 
`The provisional applications do not describe all of the claimed
`nucleotide deletions, insertions, or combinations of deletions and
`insertions into endogenous GRX3 and/or GRX4 genes as
`encompassed by claims 1-19 of the '565 patent .................................. 19 
`The provisional applications do not fully describe the full genus of
`yeast genera as encompassed by claims 1-19 of the '565 patent ......... 25 
`VIII.  Basis of my analysis with respect to anticipation .......................................... 26 
`IX.  Flint anticipates claims 1-8 and 11-19 of the '565 patent .............................. 26 
`A. 
`Claim 1 ................................................................................................ 27 
`B. 
`Claims 2-8 and 11-19 .......................................................................... 30 
`1. 
`Claim 2 ...................................................................................... 31 
`2. 
`Claim 3 ...................................................................................... 32 
`3. 
`Claim 4 ...................................................................................... 33 
`4. 
`Claim 5 ...................................................................................... 33 
`5. 
`Claim 6 ...................................................................................... 34 
`6. 
`Claim 7 ...................................................................................... 34 
`7. 
`Claim 8 ...................................................................................... 34 
`8. 
`Claim 11 .................................................................................... 35 
`9. 
`Claim 12 .................................................................................... 35 
`i
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`
`X. 
`
`10.  Claim 13 .................................................................................... 36 
`11.  Claim 14 .................................................................................... 36 
`12.  Claim 15 .................................................................................... 36 
`13.  Claim 16 .................................................................................... 37 
`14.  Claim 17 .................................................................................... 38 
`15.  Claim 18 .................................................................................... 38 
`16.  Claim 19 .................................................................................... 39 
`Basis of my analysis with respect to obviousness ......................................... 40 
`A.  Overview of Fe Regulation and the Roles of Fe-S Cluster Proteins in
`Yeast .................................................................................................... 41 
`Comparison of claims 1-4, 6-8 and 11-19 to Anthony in view of Puig
`and Ojeda ............................................................................................. 46 
`1. 
`Claim 1 ...................................................................................... 46 
`2. 
`Claims 2-4 and 6-8 .................................................................... 52 
`3. 
`Claims 11-14 ............................................................................. 53 
`4. 
`Claims 15 and 16....................................................................... 54 
`5. 
`Claims 17 and 18....................................................................... 56 
`6. 
`Claim 19 .................................................................................... 56 
`Comparison of claim 5 to Anthony in view of Puig, Ojeda and the
`'376 publication ................................................................................... 57 
`Comparison of claim 9 to Anthony in view of Puig, Ojeda and van
`Maris .................................................................................................... 58 
`Comparison of claim 10 to Anthony in view of Puig, Ojeda and
`Overkamp ............................................................................................ 60 
`The prior art does not teach away from modifying a yeast containing
`recombinant overexpressed DHAD .................................................... 61 
`Gevo's alleged unexpected results would have been entirely expected
`for recombinantly expressed DHAD ................................................... 65 
`XI.  Conclusion ..................................................................................................... 67 
`
`
`B. 
`
`B. 
`
`C. 
`
`D. 
`
`E. 
`
`F. 
`
`
`
`ii
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`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`I, Dennis J. Thiele, hereby declare as follows:
`
`I.
`
`Overview
`1.
`
`I am over the age of eighteen (18) and otherwise competent to make
`
`this declaration.
`
`2.
`
`I have been retained as an expert witness on behalf of Butamax™
`
`Advanced Biofuels, LLC ("Butamax") for the above-captioned inter partes review
`
`("IPR"). I am being compensated for my time in connection with this IPR at my
`
`standard consulting rate. I understand that the Petition for IPR involves U.S. Patent
`
`No. 8,273,565 ("the
`
`'565 patent"), BMX1001, which resulted from U.S.
`
`Application No. 13/246,693 ("the '693 application"), filed on September 27, 2011.
`
`The '565 patent issued on September 25, 2012, from the '693 application, which is
`
`a division of U.S. Patent Appl. No. 13/228,342, filed September 8, 2011, now U.S.
`
`Patent No. 8,071,358; and of U.S. Patent Appl. No. 12/953,884, filed November
`
`24, 2010, now U.S. Patent No. 8,017,376, which claimed the benefit of U.S.
`
`Provisional Application No. 61/263,952 ("the '952 application"), BMX1010, filed
`
`on November 24, 2009, and U.S. Provisional Application No. 61/350,209,
`
`BMX1011, filed on June 1, 2010. It is my understanding that the earliest claimed
`
`priority date of the '565 patent is November 24, 2009, the filing date of the '952
`
`application. I further understand that, according to the United States Patent and
`
`
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`2
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`Trademark Office ("USPTO") records, the '565 patent is currently assigned to
`
`Gevo, INC. ("Gevo").
`
`3.
`
`In preparing this Declaration, I have reviewed the '565 patent and
`
`considered each of the documents cited herein, in light of general knowledge in the
`
`art as of the earliest claimed priority date of the '565 patent. In formulating my
`
`opinions, I have relied upon my experience in the relevant art. In formulating my
`
`opinions, I have also considered the viewpoint of a person of ordinary skill in the
`
`art ("POSA"), as described below at ¶17.
`
`4.
`
`Claim 1 of the '565 patent, from which claims 2-19 ultimately depend,
`
`generally encompasses a
`
`recombinant microorganism
`
`that
`
`recombinantly
`
`overexpresses a polynucleotide encoding a dihydroxy acid dehydratase. Claim 1
`
`further specifies that the recombinant microorganism is engineered to comprise at
`
`least one inactivated monothiol glutaredoxin selected from the group consisting of
`
`monothiol glutaredoxin-3 (GRX3), monothiol glutaredoxin-4 (GRX4), and GRX3
`
`and GRX4 wherein the inactivated GRX3 and/or GRX4 results from the deletion
`
`of one or more nucleotides in the coding region of an endogenous GRX3 and/or
`
`GRX4 gene, the insertion of one or more nucleotides into the coding region of an
`
`endogenous GRX3 and/or GRX4 gene, or a combination of such a deletion and
`
`
`
`3
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`

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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`insertion. My conclusions regarding these claims are summarized in the following
`
`paragraphs 5-7.
`
`5.
`
`Claims 1-19 of the '565 patent are not entitled to the benefit of the
`
`filing dates of the '952 and '209 provisional applications.
`
`6.
`
`Flint (BMX1003) discloses each and every limitation of claims 1-8
`
`and 11-19 of the '565 patent.
`
`7.
`
`A POSA would have had a reason to arrive at the claimed invention
`
`with a reasonable expectation of success.
`
`a. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claims 1-4, 6-8 and 11-19 of the '565 patent by
`
`combining the disclosures of Anthony (BMX1005), Puig (BMX1006),
`
`and Ojeda (BMX1007).
`
`b. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 5 of the '565 patent by combining the disclosures of
`
`Anthony (BMX1005), Puig (BMX1006), Ojeda (BMX1007), and the
`
`'376 publication (BMX1015).
`
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`4
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`c. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 9 of the '565 patent by combining the disclosures of
`
`Anthony (BMX1005), Puig (BMX1006), Ojeda (BMX1007), and van
`
`Maris (BMX1008).
`
`d. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 10 of the '565 patent by combining the disclosures
`
`of Anthony (BMX1005), Puig (BMX1006), Ojeda (BMX1007), and
`
`Overkamp (BMX1009).
`
`e. Gevo's arguments that (i) the art taught away from the claimed yeast,
`
`and (ii) the claimed yeast had some alleged unexpected property were
`
`directed to a native yeast. These arguments are therefore inapplicable
`
`to the claims in the '565 patent, which are directed to a recombinant
`
`yeast.
`
`II. My background and qualifications
`8. My qualifications and credentials are fully set forth in my curriculum
`
`vitae, attached as BMX1023. I am an expert in the fields of molecular biology and
`
`yeast genetics, and one emphasis of my work focuses on understanding the
`
`
`
`5
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`regulation of iron and copper homeostasis in yeast. I am knowledgeable about the
`
`experimental techniques used in the fields of molecular biology and yeast genetics.
`
`I have been an expert in these fields since 1984. For the past 34 years, I have
`
`accumulated significant training and experience in the fields of molecular biology
`
`and yeast genetics, as well as related fields.
`
`9.
`
`I received a Bachelor's Degree in Biology from the State University of
`
`New York, College at Fredonia, Fredonia, New York in 1978. I received a Master's
`
`Degree in Microbiology in 1981 and a Ph.D. in Microbiology in 1983 from
`
`Rutgers University, New Brunswick, New Jersey.
`
`10. From 1984 to 1987, I was a Staff Fellow at the National Cancer
`
`Institute in Bethesda, Maryland. From 1987 to 1992, I was an Assistant Professor
`
`of Biological Chemistry at the University of Michigan Medical School, Ann Arbor,
`
`Michigan. From 1992 to 1996, I was an Associate Professor at the University of
`
`Michigan Medical School. From 1996 to 2003, I was a Professor at the University
`
`of Michigan Medical School.
`
`11. Since 2003, I have been a Professor in the Department of
`
`Pharmacology and Cancer Biology at the Duke University School of Medicine in
`
`Durham, North Carolina. In addition, since 2006, I have been the George Barth
`
`Geller Distinguished Professor of Pharmacology and Cancer Biology at the Duke
`
`
`
`6
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`University School of Medicine, and from 2006 to 2012 I served as the Vice Chair
`
`of the Department of Pharmacology and Cancer Biology at the Duke University
`
`School of Medicine.
`
`12.
`
`I have published more than 100 papers in the areas of yeast genetics
`
`and molecular biology including metal homeostasis in yeast. I serve as a reviewer
`
`for professional journals in my field and have been a member of the Editorial
`
`Boards of The Journal of Biological Chemistry, Genes and Nutrition, Eukaryotic
`
`Cell and Virulence. I am a member of multiple professional societies including the
`
`Genetics Society of America, the American Society for Microbiology, the
`
`American Association for the Advancement of Science and the American Society
`
`for Biochemistry and Molecular Biology. In addition, I have been elected a Fellow
`
`of the American Academy of Microbiology and of the American Association for
`
`the Advancement of Science. I collaborate with several prominent researchers in
`
`the fields of molecular biology and yeast genetics.
`
`13.
`
`I am the corresponding author of Puig, S., et al. "Coordinated
`
`Remodeling of Cellular Metabolism during Iron Deficiency through Targeted
`
`mRNA Degradation," Cell 120:99-110 (2005) ("Puig") which is cited in the
`
`Petition for Inter Partes Review (attached to the Petition as BMX1006). The work
`
`
`
`7
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`reported in this article was conducted under my supervision in my laboratory at the
`
`Duke University School of Medicine.
`
`14.
`
`In addition to my educational training, professional experiences, and
`
`research experiences described above, I attend multiple scientific conferences each
`
`year, and I am invited to speak at scientific seminars several times a year. I have
`
`been the Keynote Lecturer at the Gordon Research Conference on the Cell Biology
`
`of Metals held in 2009, the Gordon Research Conference on Stress Proteins also
`
`held in 2009, and the Federation of American Societies for Experimental Biology
`
`(FASEB) Trace Elements Summer Conference held in 2010.
`
`15. Accordingly, I am an expert in the fields of molecular biology and
`
`yeast genetics.
`
`III. List of documents considered in formulating my opinion
`16.
`In formulating my opinion, I have considered
`
`the following
`
`documents:
`
`Exhibit No.
`
`BMX1001
`
`BMX1003
`
`BMX1004
`
`BMX1005
`
`
`
`Document
`
`U.S. Patent No. 8,273,565
`
`PCT Application Publication No. WO 2011/1033000 A2
`("Flint")
`
`U.S. Prov. Appl. No. 61/305,333 ("the '333 application")
`
`U.S. Appl. Publ. No. 2010/0081179 ("Anthony")
`
`8
`
`

`

`
`
`BMX1006
`
`BMX1007
`
`BMX1008
`
`BMX1009
`
`BMX1010
`
`BMX1011
`
`BMX1012
`
`BMX1013
`
`BMX1014
`
`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`Puig, S., et al., "Coordinated Remodeling of Cellular
`Metabolism during Iron Deficiency through Targeted
`mRNA Degradation," Cell 120:99-110 (2005) ("Puig")
`
`Ojeda, L., et al., "Role of Glutaredoxin-3 and
`Glutaredoxin-4 in the Iron Regulation of the Aft1
`Transcriptional
`Activators
`in
`Saccharomyces
`cerevisiae," J. Biol. Chem. 281(26): 17661-17669
`(2006) ("Ojeda")
`
`van Maris et al., "Directed Evolution of Pyruvate
`Decarboxylase-Negative Saccharomyces
`cerevisiae,
`Yielding a C2-Independent, Glucose-Tolerant, and
`Pyruvate-Hyperproducing Yeast," Appl. Environ.
`Microbiol. 70(1):159-66 (2004) ("van Maris")
`
`Overkamp et al., "Metabolic Engineering of Glycerol
`Production
`in Saccharomyces
`cerevisiae" AEM
`68(6):2814-2821 (2002) ("Overkamp")
`
`U.S. Prov. Appl. No. 61/263,952 ("the '952 provisional
`application")
`
`U.S. Prov. Appl. No. 61/350,209 (" the '209 provisional
`application")
`
`File Wrapper for U.S. Appl. No. 13/246,693
`
`"Cytosolic Monothiol
`et al.,
`Mühlenhoff, U.,
`Glutaredoxins Function in Intracellular Iron Sensing
`and Trafficking via Their Bound Iron-Sulfur Cluster,"
`Cell Metabolism, 12:373-385 (2010) ("Mühlenhoff")
`
`Pujol-Carrion, N., et al., "Glutaredoxins Grx3 and Grx4
`regulate nuclear localisation of Aft1 and the oxidative
`stress response in Saccharomyces cerevisiae," J. Cell
`Sci. 119(2):4554-4564 (2006) ("Pujol-Carrion")
`
`BMX1015
`
`U.S. Appl. Pub. No. 2009/0163376
`
`
`
`9
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`Order in Reexam Control. No. 95/001,870
`
`Mercier, A. and Labbé, S., "Both Php4 Function and
`Subcellular Localization are Regulated by Iron via a
`Multistep Mechanism Involving the Glutaredoxin Grx4
`and
`the Exportin Crm1," Journal of Biological
`Chemistry 284: 20249-202261 (2009) ("Mercier")
`
`U.S. Appl. Pub. No. 2007/0092957
`
`Rutherford , J.C., et al., "Aft1p and Aft2p Mediate Iron-
`responsive Gene Expression in Yeast through Related
`Promoter Elements," Journal of Biological Chemistry
`278(30): 27636-27643 (2003) ("Rutherford 2003")
`
`Li, H., et al., "The Yeast Iron Regulatory Proteins
`Grx3/4 and Fra2 Form Heterodimeric Complexes
`Containing a [2FE-2S] Cluster with Cysteinyl and
`Histidyl Ligation," Biochemistry 48: 9569-9581 (2009)
`("Li")
`
`Rutherford, J.C., et al., "A second iron-regulatory
`system in yeast independent of Aft1p," PNAS 98(25):
`14322-14327 (2001) ("Rutherford 2001")
`
`BMX1016
`
`BMX1017
`
`BMX1018
`
`BMX1019
`
`BMX1020
`
`BMX1021
`
`BMX1022
`
`U.S. Appl. Pub. No. 2010/0197519
`
`
`
`
`
`IV. Person of ordinary skill in the art
`17.
`In formulating my opinions, I have considered the viewpoint of a
`
`person of ordinary skill in the art ("POSA"). I understand that a POSA is one who
`
`is presumed to be aware of all pertinent art, thinks along conventional wisdom in
`
`the art, and is a person of ordinary creativity. With regard to the '565 patent, a
`
`POSA typically would have a Ph.D. in the life sciences or a similar related
`
`
`
`10
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`discipline, and have familiarity, training, and experience in molecular biology,
`
`microbial genetics and/or microbial metabolism. Alternatively, a POSA typically
`
`would have at least a scientific background such as a Bachelor's degree in the life
`
`sciences (e.g., biology, microbiology, molecular biology or biochemistry) or a
`
`similar related discipline, and have substantial familiarity, training, and experience
`
`in molecular biology, microbial genetics and/or microbial metabolism. A POSA
`
`also would have had knowledge of the scientific literature concerning molecular
`
`biology, microbial (e.g. yeast) genetics and microbial metabolism, that was
`
`available at a time point of interest, including knowledge about experimental
`
`techniques available in the art.
`
`V. The '565 patent
`18.
`I understand that this declaration is being submitted together with a
`
`Petition for IPR of claims 1-19 of the '565 patent.
`
`19.
`
`I have considered the disclosure of the '565 patent in light of the
`
`earliest claimed priority date of the '565 patent, which I understand to be
`
`November 24, 2009.
`
`20. According to the Abstract, the '565 patent specification is directed to
`
`"recombinant microorganisms comprising one or more dihydroxyacid dehydratase
`
`(DHAD)-requiring biosynthetic pathways and methods of using said recombinant
`
`
`
`11
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`microorganisms to produce beneficial metabolites derived from said DHAD-
`
`requiring biosynthetic pathways." (BMX1001, Abstract.)
`
`21. The '565 patent claims recombinant yeast microorganisms with a
`
`recombinantly overexpressed DHAD and an inactivated GRX3 and/or GRX4, as
`
`well as a method of producing isobutanol by cultivating the claimed yeast.
`
`(BMX1001, 91:15 through 91:26 and 92:58 through 92:63.)
`
`VI. Basis of my analysis with respect to written description
`22.
`It is my understanding that in order to meet the written description
`
`requirement, a patent specification must describe the claimed invention in
`
`sufficient detail that a POSA can clearly conclude that the inventor invented
`
`(possessed) the full scope of the claimed invention. It is my understanding that a
`
`priority application, such as a provisional application, must also meet this standard.
`
`It is my understanding that if a priority application does not meet this standard,
`
`then the claims of a patent undergoing analysis are not entitled to the benefit of the
`
`priority application's filing date.
`
`VII. The full scope of claims 1-19 of the '565 patent is not disclosed in the
`'952 and '209 provisional applications
`23. Claims 1-19 of the '565 patent broadly encompass any recombinant
`
`yeast comprising a recombinantly overexpressed polynucleotide encoding any
`
`DHAD and engineered to comprise any inactivated GRX3 and/or GRX4 protein,
`
`
`
`12
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`and which further broadly encompass: (1) any deletion of one or more nucleotides
`
`in the coding region of an endogenous GRX3 and/or GRX4 gene, or (2) any
`
`insertion of one or more nucleotides into the coding region of an endogenous
`
`GRX3 and/or GRX4 gene, or (3) any combination of such deletions and insertions,
`
`that result in the inactivated protein, such that the protein is not expressed or is
`
`expressed in a completely non-functional form. As discussed below, in view of the
`
`disclosures of the '952 and '209 provisional applications, a POSA would not have
`
`understood the applicants to have invented the recombinant yeast microorganisms
`
`encompassed by the claims of the '565 patent as of the '952 and '209 provisional
`
`application filing dates.
`
`A. The provisional applications do not describe the full scope of
`inactivated GRX3 and/or GRX4 as encompassed by claims 1-19 of
`the '565 patent
`24. Claims 1-19 of the '565 patent are directed to recombinant yeast
`
`microorganisms and a method of using a recombinant yeast microorganism
`
`comprising an "inactivated" GRX3 and/or GRX4.
`
`25. A POSA reading the '952 and '209 provisional applications would
`
`understand the applications as being primarily directed to expression and
`
`
`
`13
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`overexpression of GRX3 and/or GRX4.1 For example, ¶¶[00155] and [00156] of
`
`the '952 application and ¶¶[00179] and [00180] of the '209 provisional application,
`
`which are under the sections describing ways to enhance DHAD activity, discuss
`
`alleged knowledge regarding GRX3 and GRX4 in yeast and list nine specific
`
`"embodiments" associated with GRX3 and/or GRX4 expression. Eight of these
`
`
`1 See, e.g, BMX1010 at ¶¶[0023] and [00116] and BMX1011 at ¶¶ [0023]
`
`and [00122], disclosing GRX3 and GRX4 as a "chaperone protein" that when
`
`expressed can assist the folding of a DHAD exhibiting cytosolic activity;
`
`BMX1010 and BMX1011 at ¶[0028], disclosing overexpression of one or more
`
`genes including GRX3 and GRX4 as leading to increased iron levels in the cytosol
`
`and mitochondria for availability in producing Fe-S cluster-containing proteins in
`
`the cytosol, which BMX1011 further discloses includes cytosolic DHAD;
`
`BMX1010 at ¶¶[0030] and [00189] and BMX1011 at ¶¶[0033] and [00212],
`
`disclosing overexpression of GRX4 for increasing glutathione levels in the cytosol.
`
`See also, BMX1010 at ¶¶[00154], [00155], [00156], [00158], claim 33, claim 55,
`
`and claim 66, and BMX1011 at ¶¶[00178], [00179], [00180], [00182], claim 33,
`
`claim 55, and claim 66.
`
`
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`14
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`embodiments discuss overexpression of GRX3 and/or GRX4.2 Indeed, the header
`
`of the section including these GRX3/GRX4 embodiments in the '209 provisional
`
`application, the later-filed of the two provisional applications, is entitled
`
`"Enhancing DHAD Activity by Increased GRX3/GRX4 Activity and/or
`
`Expression." As such, even at the later priority date of the '209 provisional
`
`application, it would have been apparent to a POSA reading the '209 application
`
`that the applicants considered increasing GRX3 and GRX4 expression to be the
`
`focus of any change in GRX3 and GRX4 activity, and not deletion or
`
`"inactivation."
`
`26. Furthermore, ¶[00156] of the
`
`'952 provisional application and
`
`¶[00180] of the '209 provisional application state that the embodiments disclosed
`
`in those paragraphs "can also be combined with increases in the extracellular iron
`
`2 (1) "GRX3 is overexpressed," (2) "GRX4 is overexpressed," (3) "GRX3
`
`and GRX4 are overexpressed," (4) "GRX3, GRX4, or GRX3 and GRX4 are
`
`deleted or attenuated," (5) "GRX3 and Aft1 are overexpressed," (6) "GRX4 and
`
`Aft1 are overexpressed," (7) "GRX3 and Aft2 are overexpressed," (8) "GRX4 and
`
`Aft2 are overexpressed," and (9) "One or both of: Aft1, Aft2 is overexpressed
`
`either alone or in combination with: GRX3 or GRX4." BMX1010 at ¶[00156] and
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`BMX1011 at ¶[00180].
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`15
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`concentration to provide increased iron in the cytosol or mitochondria of the cell."
`
`As such, ¶[00156] of the '952 application and ¶[00180] of the '209 application
`
`confusingly describe achieving the same effect of increased iron in the cytosol or
`
`mitochondria as a result of either "overexpression" or "deletion or attenuation" of
`
`GRX3 and/or GRX4. As such, it would have been apparent to a POSA reading the
`
`provisional applications that the applicants had not yet settled on what they
`
`believed to be the invention when they filed the provisional applications.
`
`27. Based on the requirement that the yeast of the claims comprise
`
`inactivated GRX3 and/or GRX4 that do not have any activities, the claims broadly
`
`encompass: (1) a yeast in which the GRX3 and/or GRX4 proteins are not
`
`expressed, (2) a yeast in which GRX3 and/or GRX4 proteins are expressed in
`
`completely non-functional forms, and (3) a yeast in which one of GRX3 or GRX4
`
`protein is not expressed, while the other is expressed in a completely non-
`
`functional form, provided such deletions, insertions, or combinations of deletions
`
`and insertions that result in complete inactivation of GRX3 and/or GRX4 protein
`
`do not occur in the regulatory regions associated with the endogenous genes.
`
`28. The '565 patent and the provisional applications do not define the term
`
`"inactivated" as recited in claim 1 of the '565 patent.
`
`
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`29. The term "inactivated" is not defined in the provisional applications,
`
`
`
`and expression of GRX3 and/or GRX4 proteins in completely non-functional
`
`forms is not described. There are no working examples of recombinant yeast in
`
`which GRX3 and/or GRX4 are deleted. At best, the '952 provisional application
`
`sparsely discloses that yeast may be engineered to "delete" or "attenuate" GRX3
`
`and/or GRX4 genes, while the '209 provisional application sparsely discloses that
`
`yeast may be engineered to "delete," "reduce," or "attenuate" GRX3 and/or GRX4
`
`genes.3 Reduction and attenuation of GRX3 and/or GRX4 genes would be
`
`understood to accomplish less than complete inactivation of all GRX3 and/or
`
`GRX4 activities. Therefore, since "inactivated" means the absence of all GRX3
`
`and/or GRX4 activity, it is my understanding that "inactivated" GRX3 and/or
`
`GRX4 would not result from yeast that have been engineered to "reduce" or
`
`"attenuate" GRX3 and/or GRX4 genes.
`
`
`3 BMX1010 at ¶¶[0028], [00154], [00156], [00157], [00158], and claim 56,
`
`and BMX1011 at ¶¶[00178], [00180], [00181], [00182], and claim 56 generically
`
`disclose deletion and/or attenuation of GRX3 and/or GRX4 genes. BMX1011 at
`
`¶[0031] also generically discloses deletion, reduction, and/or attenuation of GRX3
`
`and/or GRX4.
`
`
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`17
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`30.
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`It is my understanding that the term "inactivated" with respect to
`
`
`
`GRX3 and/or GRX4 was discussed by the applicants and the Examiner during
`
`prosecution of U.S. Application No. 13/246,693 ("the '693 application"). In that
`
`discussion the applicants indicated the meaning of "inactivate" is "to render
`
`inactive so that GRX3 and GRX4 protein have no activity thereof" in contrast to
`
`partially functional GRX3 and GRX4 proteins. File Wrapper for the '693
`
`application, Examiner-Initiated Interview Summary dated May 17, 2012
`
`(BMX1012). As such, it is my understanding that "inactivated" means that the
`
`GRX3 and/or GRX4 protein lacks all activity and excludes GRX3 and/or GRX4
`
`proteins having reduced, attenuated or partial activities.
`
`31. A POSA would understand the generic disclosure of yeast engineered
`
`to "delete" GRX3 and/or GRX4 genes to only encompass, at best, a complete
`
`deletion of the GRX3 and/or GRX4 genes, such that the corresponding proteins are
`
`not expressed.
`
`32. For example, the '952 provisional application at ¶[00268] and the '209
`
`provisional application at ¶[00299] disclose that "when expression is to be
`
`repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be
`
`eliminated by known deletion techniques." Therefore, the '952 and '209 provisional
`
`
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`18
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
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`applications do not disclose expression of GRX3 and/or GRX4 proteins that are
`
`non-functional as a result of partial deletions of the endogenous genes.
`
`33.
`
`In view of the disclosures of the '952 and '209 provisional applications
`
`as discussed above, a POSA would not have understood the applicants to have
`
`invented recombinant yeast microorganisms comprising, among other things, (1) a
`
`yeast in which the GRX3 and/or GRX4 proteins are not expressed, (2) a yeast in
`
`which GRX3 and/or GRX4 proteins are expressed in completely non-functional
`
`forms, and (3) a yeast in which one of GRX3 or GRX4 proteins is not expressed,
`
`while the other is expressed in a completely non-functional form at the filing dates
`
`of the '952 and '209 provisional applications.
`
`B.
`
`The provisional applications do not describe all of the claimed
`nucleotide deletions, insertions, or combinations of deletions and
`insertions
`into endogenous GRX3 and/or GRX4 genes as
`encompassed by claims 1-19 of the '565 patent
`34. Claims 1-19 of the '565 patent encompass any deletion of one or more
`
`nucleotides, any insertion of one or more nucleotides, or any combination of any
`
`deletion and any insertion of one or more nucleotides in an endogenous yeast
`
`GRX3 and/or GRX4 gene that results in any "inactivated" GRX3 and/or GRX4
`
`protein, in which, as discussed above, the protein is not expressed or is expressed
`
`in a completely non-functional form.
`
`
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`19
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
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`35. The deletions, insertions, or combinations of deletions and insertions
`
`
`
`that result in inactivation of GRX3 and/or GRX4 protein are not limited by the
`
`claims to any portion of the coding regions of the endogenous GRX3 and/or GRX4
`
`genes. As such, it i