UNITED STATES PATENT AND TRADEMARK OFFICE
`
`_________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_________________
`
`
`BLUEBIRD BIO, INC.,
`Petitioner
`
`v.
`
`SLOAN KETTERING INSTITUTE FOR CANCER RESEARCH,
`Patent Owner
`
`_________________
`
`Patent No. 7,541,179
`_________________
`
`
`DECLARATION OF JÖRG BUNGERT, Ph.D.
`
`
`
`Page 1 of 100
`
`BLUEBIRD EXHIBIT 1002
`
`

`

`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`Table of Contents
`
`Page
`Introduction—U.S. Patent No. 7,541,179 ....................................................... 1
`I.
`Qualifications ................................................................................................... 2
`II.
`Summary of Opinions ...................................................................................... 4
`III.
`IV. Person of Ordinary Skill in the Art .................................................................. 5
`V.
`Technological Overview .................................................................................. 6
`A.
`Inherited Hemoglobin Disorders and Gene Therapies .......................... 8
`B.
`A Predicate to Gene Therapy:
`Understanding the Regulation of Hemoglobin Genes ........................ 11
`Development of Vectors for Gene Therapy ........................................ 16
`C.
`VI. Overview of the ’179 Patent .......................................................................... 20
`VII. Claim Construction ........................................................................................ 22
`VIII. The Priority Date for the ’179 Patent ............................................................ 23
`IX. Overview of the Prior Art .............................................................................. 28
`A.
`Chad M. May, “Therapeutic Hemoglobin
`Synthesis in Beta-Thalassemic Mice Expressing
`Lentivirus-Encoded Human Beta-Globin,” Cornell
`University (2001) (“May Thesis”) (Ex. 1004)..................................... 28
`Chad May, et al., “Therapeutic
`Haemoglobin Synthesis in β-Thalassaemic
`Mice Expressing Lentivirus-Encoded Human β-Globin,”
`Nature, 406(6791) (2000) (“May Article”) (Ex. 1005) ....................... 29
`C. May et al., “Lentiviral-Mediated Transfer of the
`Human β-Globin Gene and Large Locus Control Region
`Elements Permit Sustained Production of Therapeutic Levels
`of β-Globin in Long-Term Bone Marrow Chimeras,” Mol.
`Therapy, 1(5) (2000), (“May Abstract”) (Ex. 1006) ........................... 32
`X. Ground 1: The May Thesis Teaches
`All of the Limitations of Claims 1, 19, and 22 of the ’179 Patent ................ 34
`A.
`Claim 1 ................................................................................................ 34
`
`B.
`
`i
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`Page 2 of 100
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`1.
`2.
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`[1.pre] “A recombinant vector comprising” ............................. 34
`[1.a] “a nucleic acid encoding a functional
`globin operably linked to a 3.2-kb nucleotide
`fragment which consists essentially of three
`contiguous nucleotide fragments obtainable from
`a human β-globin locus control region (LCR),” ....................... 35
`[1.b] “the three fragments being a BstXI
`and SnaBI HS2-spanning nucleotide fragment
`of said LCR, a BamHI and HindIII HS3-spanning
`nucleotide fragment of said LCR and a BamHI and
`BanII HS4-spanning nucleotide fragment of said LCR,” ......... 37
`[1.c] “said vector providing
`expression of the globin in a mammal in vivo.” ....................... 37
`Claim 19: “The vector of claim 1,
`wherein the functional globin is a β-globin.” ...................................... 38
`Claim 22: “The vector of claim 1,
`wherein the vector is a lentiviral vector” ............................................ 39
`XI. Ground 2: The May Article Teaches All
`of the Limitations of Claims 1, 19, and 22 of the ’179 Patent ...................... 39
`A.
`Claim 1 ................................................................................................ 39
`1.
`[1.pre] “A recombinant vector comprising” ............................. 39
`2.
`[1.a] “a nucleic acid encoding a functional
`globin operably linked to a 3.2-kb nucleotide
`fragment which consists essentially of three
`contiguous nucleotide fragments obtainable from
`a human β-globin locus control region (LCR),” ....................... 40
`[1.b] “the three fragments being a BstXI
`and SnaBI HS2-spanning nucleotide fragment
`of said LCR, a BamHI and HindIII HS3-spanning
`nucleotide fragment of said LCR and a BamHI and
`BanII HS4-spanning nucleotide fragment of said LCR,” ......... 42
`[1.c] “said vector providing
`expression of the globin in a mammal in vivo.” ....................... 50
`Claim 19: “The vector of claim 1,
`wherein the functional globin is a β-globin.” ...................................... 51
`
`B.
`
`C.
`
`B.
`
`3.
`
`4.
`
`3.
`
`4.
`
`ii
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`Page 3 of 100
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`

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`C.
`
`B.
`
`C.
`
`Claim 22: “The vector of claim 1,
`wherein the vector is a lentiviral vector” ............................................ 52
`XII. Ground 3: The May Article Teaches or Suggests All of
`the Limitations of Claims 1, 19, and 22 of the ’179 Patent ........................... 52
`XIII. Ground 4: The May Abstract Teaches or Suggests All of the
`Limitations of Claims 1, 10, 19, and 22 of the ’179 Patent ........................... 55
`A.
`Claim 1 ................................................................................................ 56
`1.
`[1.pre] “A recombinant vector comprising” ............................. 56
`2.
`[1.a] “a nucleic acid encoding a functional
`globin operably linked to a 3.2-kb nucleotide
`fragment which consists essentially of three
`contiguous nucleotide fragments obtainable from
`a human β-globin locus control region (LCR),” ....................... 56
`[1.b] “the three fragments being a BstXI
`and SnaBI HS2-spanning nucleotide fragment
`of said LCR, a BamHI and HindIII HS3-spanning
`nucleotide fragment of said LCR and a BamHI and
`BanII HS4-spanning nucleotide fragment of said LCR,” ......... 58
`[1.c] “said vector providing
`expression of the globin in a mammal in vivo.” ....................... 68
`Claim 10: “The vector of claim 1,
`wherein the functional globin is human β-globin.” ............................. 68
`Claim 19: “The vector of claim 1,
`wherein the functional globin is a β-globin.” ...................................... 69
`Claim 22: “The vector of claim 1,
`wherein the vector is a lentiviral vector” ............................................ 69
`XIV. Conclusion ..................................................................................................... 70
`
`
`
`D.
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`3.
`
`4.
`
`iii
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`Page 4 of 100
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`List of Materials Considered1
`
`Ex. 1001 U.S. Patent No. 7,541,179 to Sadelain et al. (“the ’179 patent”)
`
`Ex. 1004
`
`Ex. 1005
`
`Ex. 1006
`
`May, “Therapeutic Hemoglobin Synthesis in Beta-Thalassemic
`Mice Expressing Lentivirus-Encoded Human Beta-Globin,” Cornell
`University (2001) (“the May Thesis”)
`
`May, et al., “Therapeutic Haemoglobin Synthesis in β-Thalassaemic
`Mice Expressing Lentivirus-Encoded Human β-globin,” Nature,
`406:82-86 (2000) (“the May Article”)
`
`May, et al., “Lentiviral-Mediated Transfer of the Human β-Globin
`Gene and Large Locus Control Region Elements Permit Sustained
`Production of Therapeutic Levels of β-Globin in Long-Term Bone
`Marrow Chimeras,” Mol. Therapy, 1(5):S248-49 (2000) (“the May
`Abstract”)
`
`Ex. 1007
`
`Perutz, et al., “Hemoglobin Structure and Respiratory Transport,”
`Sci. Am., 239(6): 92-125 (1978)
`
`Ex. 1008
`
`Thein & Rochette, “Disorders of Hemoglobin Structure and
`Synthesis,” in Principles of Mol. Med. 179 (Jameson, ed., 1998)
`Ex. 1009 Bank, et. al, “Disorders of Human Hemoglobin,” Science,
`207:486-93 (1980)
`
`
`1 Non-patent publication citations are to the original page numbers of the
`
`publication, and citations to U.S. patents are to the column:line number of the
`
`patents. The only exception concerns to Exs. 1032 and 1037-39, as Petitioner
`
`utilizes an asterisk (*) to denote citation to the exhibit page.
`
`iv
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`Page 5 of 100
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`

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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`Ex. 1010
`
`He & Russell, “Expression, Purification, and Characterization of
`Human Hemoglobins Gower-I (ζ2ε2), Gower-2 (α2ε2), and
`Portland-2 (ζ2β2) Assembled in Complex Transgenic-Knockout
`Mice, Blood, 97(4):1099-1105 (2001)
`Ex. 1011 Bunn, “Pathogenesis and Treatment of Sickle Cell Disease,” N.
`Engl. J. Med., 337(11):762-69 (1997)
`
`Ex. 1012
`
`Hardison, et al., “Locus Control Regions of Mammalian β-globin
`Gene Clusters: Combining Phylogenetic Analyses and
`Experimental Results to Gain Function Insights, Gene, 205:73-94
`(1997)
`
`Ex. 1013
`
`Civin, et al., “Sustained, Retransplantable, Multilineage
`Engraftment of Highly Purified Adult Human Bone Marrow Stem
`Cells In Vivo,” Blood, 88(11):4102-09 (1996)
`Ex. 1014 High, “Gene Therapy in Haematology and Oncology,” Lancet,
`356:S8 (2000)
`
`Ex. 1015
`
`Ex. 1016
`
`Ellis, et al., “Evaluation of β-globin Gene Therapy Constructs in
`Single Copy Transgenic Mice,” Nucleic Acids Res.,
`25(6):1296-1302 (1997)
`
`Li, et al., “Nucleotide Sequence of 16-Kilobase Pairs of DNA 5’ to
`the Human ε-Globin Gene,” J. Biol. Chem., 260(28):14901-10
`(1985)
`
`Ex. 1017
`
`Mishima, et al., “The DNA Deletion in an Indian δβ-thalassaemia
`Begins One Kilobase From the Aγ Globin Gene and Ends in an L1
`Repetitive Sequence,” Br. J. Haemotol., 73:375-79 (1989)
`Ex. 1018 Vosberg, “Molecular Cloning of DNA: An Introduction Into
`Techniques and Problems,” Hum. Genet. 40(1):1-72 (1977)
`Ex. 1019 Roberts, “Restriction Enzymes and Their Isoschizomers,” Nucleic
`Acids Res., 15(Suppl.):r189-r217 (1987)
`
`Ex. 1020
`
`Zufferey, et al., “Multiply Attenuated Lentiviral Vector Achieves
`Efficient Gene Delivery in Vivo,” Nature Biotech., 15:871-75
`(1997)
`
`v
`
`Page 6 of 100
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`

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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`Ex. 1021
`
`Ex. 1022
`
`Ex. 1023
`
`Miyoshi, et al., “Transduction of Human CD34+ Cells that Mediate
`Long-Term Engraftment of NOD/SCID Mice by HIV Vectors,”
`Science, 283:682-86 (1999)
`
`Sadelain, et. al., “Generation of a High-titer Retroviral Vector
`Capable of Expressing High Levels of the Human β-Globin Gene,”
`Proc. Natl. Acad. Sci. USA, 92:6728-32 (1995)
`
`Bouhassira, et al., “Transcriptional Behavior of LCR Enhancer
`Elements Integrated at the Same Chromosomal Locus by
`Recombinase-Mediated Cassette Exchange,” Blood 90(9):3332-44
`(1997)
`
`Ex. 1024
`
`Fraser, et al., “Each Hypersensitive Site of the Human β-Globin
`Locus Control Regions Confers a Different Developmental Pattern
`of Expression on the Globin Genes,” Genes Dev., 7:106-113 (1993)
`
`Ex. 1025
`
`Engel, “Developmental Regulation of Human β-Globin Gene
`Transcription: A Switch of Loyalties?,” Trend. Genet., 9(9):304-09
`(1993)
`Ex. 1026 Roberts & Macelis, “REBASE – Restriction Enzymes and
`Methylases,” Nucleic Acids Res., 26(1):338-350 (1998)
`Ex. 1027 Roberts & Macelis, “REBASE – Restriction Enzymes and
`Methylases,” Nucleic Acids Res., 27(1):312-13 (1999)
`Ex. 1028 Roberts & Macelis, “REBASE – Restriction Enzymes and
`Methylases,” Nucleic Acids Res., 28(1):306-07 (2000)
`Ex. 1029 Roberts & Macelis, “REBASE – Restriction Enzymes and
`Methylases,” Nucleic Acids Res., 29(1):268-69 (2001)
`
`Ex. 1030
`
`Sequence Manipulation Suite (last visited October 11, 2022)
`(Website)
`Ex. 1031 Restriction Mapper, April 20, 2001 Wayback Machine Capture (last
`visited October 11, 2022) (Website)
`
`Ex. 1032
`
`Prosecution History of the ’179 Patent
`(U.S. Patent Application No. 10/188,221)
`
`vi
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`Ex. 1034 U.S. Provisional Application 60/301,861 to Sadelain
`
`Ex. 1035 U.S. Provisional Application 60/302,852 to Sadelain
`
`Ex. 1037
`
`SciMago, Nature (last visited October 11, 2022) (Website)
`
`Ex. 1038
`
`SciMago, Molecular Therapy (last visited October 11, 2022
`(Website)
`
`Ex. 1040
`Steele, “Editorial,” Mol. Therapy, 1(5):S1 (2000)
`Ex. 1041 Glorioso, “Highlights from the Third Annual ASGT Meeting,” Mol.
`Therapy, 2(2):96-100 (2000)
`
`Ex. 1042
`
`“Author Index,” Mol. Therapy, 1(5):S345-61 (2000)
`
`Himanen, et al., “A Recombinant Sickle Hemoglobin Triple Mutant
`With Independent Inhibitory Effects on Polymerization,” J. Biol.
`Chem., 271(41):25152-56 (1996) (“Himanen”)
`
`
`Ex. 1047
`
`
`
`
`vii
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`I, Jörg Bungert, Ph.D., declare as follows:
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`I.
`
`Introduction—U.S. Patent No. 7,541,179
`1.
`I have been retained by bluebird bio, Inc. (“Petitioner”) to be an
`
`independent expert consultant in this proceeding before the United States Patent
`
`and Trademark Office regarding U.S. Patent No. 7,541,179 (“the ’179 patent”)
`
`(Ex. 1001), which I understand is assigned to Sloan Kettering Institute for Cancer
`
`Research (“Patent Owner”). I have been asked to consider whether: (1) whether
`
`applications to which the ’179 patent claims priority, provisional application No.
`
`60/301,861 (filed on June 29, 2001) and 60/302,852 (filed on July 2, 2001),
`
`provide adequate support for the subject matter of claims 1, 19, and 22; (2) the
`
`May Thesis teaches all limitations of claims 1, 19, and 22 of the ’179 patent;
`
`(3) the May Article teaches all limitations of claims 1, 19, and 22 of the ’179
`
`patent; (4) the May Article teaches or suggests all limitations of claims 1, 19, and
`
`22 of the ’179 patent; and (5) the May Abstract teaches or suggests all limitations
`
`of claims 1, 10, 19, and 22 of the ’179 patent.
`
`2.
`
`I am being compensated at my normal consulting rate of $300 per
`
`hour. My compensation does not depend in any way on the nature of my findings,
`
`the presentation of my findings in testimony, or the outcome of this or any other
`
`proceeding. I have no other interest in this proceeding.
`
`1
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`II. Qualifications
`3. My qualifications are summarized below, and they are also set forth in
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`more detail in my curriculum vitae, attached as Ex. 1003.
`
`4.
`
`I am a Professor in the Department of Biochemistry and Molecular
`
`Biology at the University of Florida in Gainesville, Florida. I have approximately
`
`30 years of research experience concerning globin gene regulation.
`
`5.
`
`I received a Bachelor of Science in Biology from the Christian
`
`Albrecht University of Kiel (located in Kiel, Germany) in 1986. I also obtained a
`
`Master of Science in Biology and Molecular Genetics from the Philipps University
`
`of Marburg (located in Marburg, Germany) in 1991. I then stayed on at the same
`
`institution to pursue a Ph.D. in Molecular Genetics, which I received in 1993.
`
`6.
`
`After completing my education, I worked as a Post-Doctoral Fellow in
`
`the Laboratory of Dr. James Douglas Engel at Northwestern University until 1996.
`
`I then served as a research assistant professor in the Department of Biochemistry,
`
`Molecular, and Cell Biology at Northwestern University from 1996-1998. In
`
`1998, I moved to the Department of Biochemistry and Molecular Biology at the
`
`University of Florida, where I still serve as a professor.
`
`7.
`
`I have published more than 70 articles in peer-reviewed journals in
`
`my career, including several papers on analyzing how the β-globin locus control
`
`region (“LCR”) affects the transcriptional regulation and expression of the globin
`
`2
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`

`genes. Those articles appeared in many preeminent journals for my field,
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`including Molecular Therapy and Nature.
`
`8.
`
`Throughout my career, I have engaged with both academic and
`
`industrial researchers concerning, among other things, how the β-globin LCR
`
`operates and regulates gene expression. I have served as either chair or co-chair
`
`for the American Heart Association Study Section, have been a member of the
`
`American Society for Gene and Cell Therapy, and am presently a member of the
`
`American Society for Hematology. I have also been a reviewer for numerous
`
`journals and institutions, and an editorial board member for several refereed
`
`academic journals. Currently, I serve as the Director of the University of Florida
`
`Master of Science Program in Biochemistry and Molecular Biology.
`
`9.
`
`Over the course of my career, I have been privileged to receive
`
`several honors and awards for my research, including being selected as a member
`
`for multiple NIH study sections. I have also been recognized by the University of
`
`Florida, receiving the Exemplary Teacher Award for 17 consecutive years.
`
`Moreover, on March 1, 2020, I was given a grant for my research regarding the
`
`structure and function of the human β-globin LCR by the American Society of
`
`Hematology.
`
`3
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`III. Summary of Opinions
`10. The opinions expressed in this declaration are based on the documents
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`I reviewed, my professional judgment, as well as my education, experience, and
`
`knowledge, including how the β-globin LCR operates and regulates gene
`
`expression.
`
`11.
`
`In forming my opinions expressed in this declaration, I reviewed the
`
`following documents and materials: (1) the ’179 patent (Ex. 1001); (2) the May
`
`Thesis (Ex. 1004); (3) the May Article (Ex. 1005); (4) the May Abstract (Ex. 1006);
`
`(5) the materials in the List of Materials Considered; and (6) any other materials I
`
`refer to in this Declaration in support of my opinions.
`
`12. My opinions are guided by how a person of ordinary skill in the art
`
`(which I define in the next section) would have understood the claims and the
`
`specification of the ’179 patent at the time of the alleged invention. I have been
`
`asked by counsel to consider the art as of July 1, 2002, for Grounds 1 through 3,
`
`and June 29, 2001, for Ground 4.2 My opinions expressed below would not change
`
`
`2 Throughout this declaration, I also rely on other documents, including for
`
`background information and to show what was generally known in the field as of
`
`the relevant dates. I am not citing any such document to show it teaches one or
`
`more features recited in the claims of the ’179 patent (even though it may teach
`
`4
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`if the relevant time was determined to be somewhat different (e.g., either
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`somewhat earlier or later).
`
`13.
`
`In my opinion: (1) the May Thesis teaches all limitations of claims 1,
`
`19, and 22 of the ’179 patent; (2) the May Article teaches all limitations of claims
`
`1, 19, and 22 of the ’179 patent; (3) the May Article teaches or suggests all
`
`limitations of claims 1, 19, and 22 of the ’179 patent; and (4) the May Abstract
`
`teaches or suggests all limitations of claims 1, 10, 19, and 22 of the ’179 patent.
`
`IV. Person of Ordinary Skill in the Art
`14. Based on my review of the ’179 patent, the types of problems
`
`encountered in the art, prior-art solutions to those problems, the rapidity with
`
`which innovations were made, the sophistication of the technology, and the
`
`educational level of active workers in the field, I believe a person of ordinary skill
`
`in the art at the time of the alleged invention would have had at least an advanced
`
`degree (e.g., a Master’s or Ph.D.) in biochemistry, biotechnology, protein
`
`chemistry, genetics, molecular and structural biology, bioengineering, or similar
`
`disciplines. A person of ordinary skill in the art would have also had several years
`
`of post-graduate training or related experience in one or more of these areas.
`
`
`one or more such features). Instead, I rely on the May Thesis, the May Article, and
`
`the May Abstract for the claimed features.
`
`5
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`Based on these years of post-graduate training or related experience, a person of
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`ordinary skill in the art would have had an understanding of vector design and the
`
`effect of LCR fragments on gene expression, including how the LCR regulates
`
`gene expression.
`
`15. All of my opinions in this declaration are from the perspective of a
`
`person of ordinary skill in the art, as I have defined it here, during the relevant
`
`timeframe (i.e., late 1990s-early 2000s). During this timeframe, I possessed at
`
`least the qualifications of a person of ordinary skill in the art, as defined above, and
`
`trained individuals who would qualify as persons of ordinary skill in the art. My
`
`opinions expressed below would not change if the level of skill were determined to
`
`be somewhat different (e.g., either somewhat higher or lower).
`
`V. Technological Overview
`16.
`In this section, I present a brief overview of several key technologies
`
`that were widely known before June 29, 2001, and relate to the issues discussed in
`
`the next section. This section is not intended to be technically comprehensive, but
`
`rather provides a foundation for better understanding the ’179 patent, the prior art,
`
`and certain terminology.
`
`17. As a general matter, red blood cells are responsible for the critical
`
`(and life-preserving) task of carrying oxygen to the body’s organs and tissues.
`
`(Ex. 1007 at 92.) And they will also transport carbon dioxide from the body’s
`
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`organs and tissues back to the lungs. (Id.) In particular, there is a protein in red
`
`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`
`blood cells—hemoglobin—that is critical to this function. (Id.) Hemoglobin has a
`
`tetrameric structure—meaning, it contains four subunits (also known as “globin
`
`chains”). (Ex. 1008 at 179.) Each of these globin chains have a heme group with
`
`an iron ion capable of binding oxygen. (Ex. 1007 at 92.) The various globin
`
`chains are identified by Greek letters (e.g., α, β, γ, δ, ε, ζ) in order to differentiate
`
`them. (Ex. 1008 at 179; Ex. 1009 at 486; Ex. 1012 at 73.)
`
`18. There are a variety of hemoglobin types that exist. In fact, humans
`
`express different hemoglobin types, such as embryonic, fetal, and adult
`
`hemoglobin, at various stages of their development, with two notable switches
`
`occurring: (1) from embryonic to fetal, and (2) from fetal to adult. (Ex. 1008 at
`
`179; Ex. 1009 at 486; Ex. 1012 at 73.) The most common type of hemoglobin
`
`found in adults is Hemoglobin A (“HbA”), which consists of two α- and two
`
`β-globin chains. (Ex. 1008 at 179-80, Fig 20-1; Ex. 1009 at 486.) An illustration
`
`of a red blood cell (on the right) made up of HbA hemoglobin is shown below.
`
`(Ex. 1011 at 762-63, Fig. 1 (adapted).)
`
`7
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
`19. Embryonic hemoglobin is normally found in the first several weeks of
`
`gestation, and it contains two ζ-globin chains and two ε-globin chains (Hb
`
`Gower-1), two α-globin chains and two ε-globin chains (Hb Gower-2), two
`
`ζ-globin chains and two γ-globin chains (Hb Portland-1), or two ζ-globin chains
`
`and two β-globin chains (Hb Portland-2). (Ex. 1008 at 180, Fig. 20-1; Ex. 1009 at
`
`486; Ex. 1010 at 1099.) Of these, Hb Gower-1 is the primary embryonic
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`hemoglobin, while the other three are found at relatively low levels. (Ex. 1010 at
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`1099.)
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`20. Fetal hemoglobin, or Hemoglobin F (“HbF”), is normally found in
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`fetuses and newborn babies. (Ex. 1008 at 180, Fig. 20-1; Ex. 1009 at 486.) HbF
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`has the same α-globin chains as adult hemoglobin, but two γ chains instead of the
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`typical β chains. (Ex. 1008 at 180, Fig. 20-1; Ex. 1009 at 486; Ex. 1012 at 74.)
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`A.
`Inherited Hemoglobin Disorders and Gene Therapies
`21. Hemoglobin disorders, or hemoglobinopathies, are genetically
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`inherited disorders in which there is either (1) abnormal production of hemoglobin,
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`or (2) structural variation in hemoglobin. (Ex. 1008 at 179; Ex. 1009 at 487.)
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`These types of hemoglobinopathies are known as thalassemia and sickle cell
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`anemia, respectively. (Ex. 1008 at 179; Ex. 1009 at 487.) A specific example of
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`the first type is β-thalassemia. Βeta-thalassemias are hemoglobinopathies that
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`negatively impact the amount of β-globin chain produced, ultimately resulting in
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`chronic low hemoglobin production (resulting in excess α-globin, which
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
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`precipitates in the differentiating red blood cells and prevents maturation and
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`release of these cells into the blood stream). (Ex. 1008 at 179, 182; Ex. 1009 at
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`487.) Because patients with β-thalassemia have less hemoglobin, they also have
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`less healthy red blood cells—which in turn means less oxygen is reaching the rest
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`of the tissues in their bodies. (Ex. 1008 at 179, 182; Ex. 1009 at 487.) Low (or
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`mutant) hemoglobin or low number of red blood cells results in a condition known
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`as anemia, but β-thalassemias can also cause stunted growth, and, if untreated,
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`even death. (Ex. 1008 at 179-81; Ex. 1009 at 487.)
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`22. Hemoglobinopathies that result in a structural change of a globin
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`chain, such as sickle cell anemia, can result in chronic anemia leading to recurrent
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`episodes of severe chronic pain and tissue damage. (Ex. 1008 at 186.) Over long
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`periods of time, this can result in stroke, kidney failure, and other issues. (Id. at
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`186-87.)
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`23.
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`In the case of sickle cell anemia, normal HbA is instead replaced by a
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`mutant form called sickle hemoglobin (“HbS”), which is comprised of mutated
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`β-globin chains containing a single amino acid substitution at position 6.
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`(Ex. 1008 at 185-87; Ex. 1011 at 762-63, Fig. 1.) At low oxygen concentrations,
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`this mutation results in polymerization of HbS in red blood cells, distorting the
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`cell. (Ex. 1008 at 187; Ex. 1011 at 762-63, Fig. 1.) The image reproduced below
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
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`demonstrates the sickling phenomenon; that is, the shift from the normal “circular”
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`shape of red blood cells to the classic “sickle” shape (which has sometimes been
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`described as an “elongated banana”). A classic “sickle” cell is depicted below.
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`(Ex. 1011 at 762-63, Fig. 1 (adapted).)
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`
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`24. By the late 1990s, β-thalassemia patients were being primarily treated
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`with regular blood transfusions in order to keep their hemoglobin levels
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`sufficiently high. (Ex. 1008 at 180-81; Ex. 1009 at 487.) Because these regular
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`blood transfusions can cause iron to accumulate to toxic levels in the bloodstream
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`and specific organs, patients also had to undergo chelation therapy in order to
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`regulate their iron levels. (Ex. 1008 at 180-81; Ex. 1009 at 487.) Management for
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`treating sickle cell disease is usually aimed at avoiding painful episodes (i.e., pain
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`relief), relieving symptoms, and preventing complications. (Ex. 1008 at 188.)
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`25. While most patients will reach adulthood if well treated, both quality
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
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`of life and life expectancy are severely impaired by complications. (Ex. 1008 at
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`180-81, 185, 186-88.) Therapies for treating β-thalassemia and sickle cell disease
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`can have serious complications. (Id.) Repeated blood transfusions, even when
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`paired with chelation therapy, can lead to excess levels of iron in the heart,
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`ultimately resulting in death. (Id. at 180-81; Ex. 1009 at 487.)
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`B. A Predicate to Gene Therapy:
`Understanding the Regulation of Hemoglobin Genes
`26. As early as the 1980s, it was thought that the ideal treatment for
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`hemoglobinopathies would be a gene therapy. (Ex. 1009 at 492; Ex. 1012 at 74.)
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`Gene therapy for hemoglobinopathies would involve placing a globin gene (along
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`with any necessary regulatory sequences) onto a DNA molecule known as a
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`“retroviral vector.” (Ex. 1009 at 492; Ex. 1014 at S8.) Retroviral vectors are the
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`vehicle in gene therapy that will carry a gene of choice (and accompanying
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`sequences necessary for the expression of that gene) into the recipient cells so that
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`those elements may be integrated. (Ex. 1014 at S8.)
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`27. For gene therapy treatment of hemoglobinopathies, the retroviral
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`vector would be transferred (or “transduced”) into recipient bone marrow cells that
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`have been removed from the patient. (Ex. 1009 at 492; Ex. 1014 at S8.) The
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`patient’s bone marrow is made up of hematopoietic progenitor and stem cells—
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`cells designed to turn into blood cells. (Ex. 1013 at 4102, 4108.) Once the
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
`
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`retroviral vector is in the bone marrow cells, it mediates integration of the globin
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`gene (and associated regulatory sequences) into the cellular DNA of these cells and
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`allows for the expression of normal hemoglobin. (Ex. 1009 at 492; Ex. 1014 at
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`S8.)
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`28. This overall process is illustrated below:
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`
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`(Ex. 1014, S8.) The benefit of this therapy is that it circumvents the need for
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`constant blood transfusions, thereby avoiding a regimen with potentially fatal
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`consequences. (Ex. 1008 at 180-81; Ex. 1009 at 487, 492.)
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`29.
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`In order to develop a successful gene therapy for hemoglobinopathies,
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`a thorough understanding of how globin genes are regulated is critical. (Ex. 1012
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`at 75.) And during the 1980 to 1990 time period, that understanding grew
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`exponentially. (Ex. 1012 at 75; Ex. 1015 at 1296.) Research in this area focused
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
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`on the β-globin LCR, which may also be thought of as the master regulator of all
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`β-type globin genes during development. (Ex. 1012 at 75.)
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`30. The LCR was initially discovered in the late 1980s and has five
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`hallmark hypersensitive sites (“HS” sites)—numbered HS1 through HS5֫—to
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`deoxyribonuclease I or DNase I (an enzyme capable of cleaving DNA). (Id.) At
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`the time, hypersensitivity to DNAse I was also known to be associated with a more
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`open chromatin structure, which allows transcription factors to bind and drive gene
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`expression. (Id.) Without the LCR, human β-globin is poorly expressed. (Id. at
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`75-76.)
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`31. By 1985, a genetic map of the LCR region had been published and
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`deposited in the GenBank3 genetic sequence database by at least 1989 under the
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`entry accession number “HUMHBB”—followed later by accession numbers
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`“U01317” in 1994 and “NG_000007.l” in 2001. (Ex. 1016 at 14903-05, Fig. 2;
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`Ex. 1017 at 376; Ex. 1032 at *254, 312-14.)
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`32. Having a map of the entire sequence of the LCR region is critical to
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`engineering retroviral vectors for gene therapy. The map allows researchers to
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`3 GenBank is a public repository where researchers are expected to deposit all
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`generated sequences.
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`identify specific sites where the LCR may be cut so that fragments of the LCR can
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`Declaration of Jörg Bungert, Ph.D.
`U.S. Patent No. 7,541,179
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`be inserted into the retroviral vector, as I discuss in more detail below. I
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`understand that the Applicants for the ’179 patent acknowledged the importance of
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`the map, noting that at the relevant time period “sequence information providing
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`the location of the restriction sites with the LCR was readily accessible to one
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`skilled in the art using conventional sequence analysis techniques.” (Ex. 1032 at
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`*245-46.)
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`33. Of the five HS sites (HS1-HS5) in the LCR, HS2, HS3, and HS4 were
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`(and still are) considered to be the most important for enhancement of globin gene
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`expression. (Ex. 1012 at 88-89.) By the 1990s, LCR fragments composed of only
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`HS2, HS3, and HS4 had been shown to be capable of producing a high level of
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`globin gene expression. (Id.) Because of this, the HS2, HS3, and HS4 regions
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`were determined to be critical elements for hemoglobin gene regulation. (Id.)
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`Moreover, within each of HS2, HS3, and HS4, there was a smaller region, also
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`known as a “core” element, that was determined to be the minimum DNA
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`sequence necessary for enhancing globin gene expression. (Id. at 76; Ex. 1015 at
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`1296.)
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`34. The core HS2, HS3, and HS4 regions are each approximately 200 to
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`400 bp in size and ha