Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 1 of 45
`
`DAVID G. MANGUM (4085)
`C. KEVIN SPEIRS (5350)
`KRISTINE EDDE JOHNSON (7190)
`MICHAEL R. MCCARTHY (8850)
`PARSONS BEHLE & LATIMER
`One Utah Center
`201 South Main Street, Suite 1800
`Salt Lake City, UT 841111
`Telephone: (801) 532-1234
`Facsimile: (801) 536-6111
`ecf@parsonsbehle. com
`
`BENJAMIN G. JACKSON (admitted pro hac vice)
`MATHEW GORDON (12526)
`MYRIAD GENETICS, INC.
`320 Wakara Way
`Salt Lake City, UT 84108
`bjackson@myriad.com
`mgordon@myriad.com
`Attorneys for Plaintiffs
`
`IN THE UNITED STATES DISTRICT COURT FOR THE
`DISTRICT OF UTAH, CENTRAL DIVISION
`
`UNIVERSITY OF UTAH RESEARCH
`FOUNDATION, et. al,
`
`DECLARATION OF MARK
`ALLAN KAY, M.D., PH.D.
`
`Plaintiffs,
`
`vs.
`
`AMBRY GENETICS CORPORATION,
`
`Case No. 2:13-cv-00640-RJS
`
`Defendant.
`
`Judge Robett J. Shelby
`
`UNIVERSITY OF UTAH RESEARCH
`FOUNDATION, et. al.,
`
`Plaintiffs,
`
`vs.
`
`GENE BY GENE, LTD.,
`
`Defendant.
`
`GeneDX 1015, pg. 1
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 2 of 45
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`I, Mark Allan Kay, hereby declare that:
`
`I.
`
`1.
`
`QUALIFICATIONS AND BACKGROUND
`
`I am cunently a tenmed professor and Head of the Division of Human Gene
`
`Therapy at Stanford University School of Medicine. I an1 the Dennis Farrey Family Professor in
`
`the Depmtments of Pediatrics and Genetics at Stanford University. I am also the Associate Chair
`
`for Basic Resemd1 in the Depmtment of Pediatrics at Stanford University School of Medicine.
`
`My qualifications, expettise, and list of publications are set forth in my cunicuhun vitae, which
`
`is attached as Exhibit A.
`
`2.
`
`I received my Ph.D. in Developmental Genetics and my M.D. fi.·om Case Westem
`
`Reserve University in 1986 and 1987, respectively. I completed my intemship and residency in
`
`the Depmtment of Pediatrics at the Baylor College ofMedicine, Houston, Texas in 1990.
`
`Between1990 and 1993, I joined the Department of Molecular m1d Human Genetics at Baylor
`
`College of Medicine as a medical genetics fellow where I completed clinical training to be Bom·d
`
`eligible in both Clinical Medical Genetics and Biochemical Genetics. Dming those three years, I
`
`also completed my post-doctoral research on gene therapy for hepatic deficiencies at Baylor
`
`College ofMedicine, Houston, Texas.
`
`3.
`
`I was triple-boarded: Pediatrics from 1990 until1997; Clinical Genetics, and
`
`Clinical Biochemical Genetics fi.·om 1993 tmtil 2003. In my medical practice, I have seen many
`
`patients for diagnosis, recurrence risk, and/or treatment of genetic disorders between 1990 and
`
`1998. I have been and continue to be involved in Phase I I II clinical trials in gene therapy.
`
`4.
`
`My resem·ch focuses primm·ily on developing gene transfer technologies for gene
`
`therapy of genetic and acquired diseases of the liver. The second major focus of my resem·ch
`
`includes the role of small RNAs in mammalian gene regulation.
`
`2
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`GeneDX 1015, pg. 2
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 3 of 45
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`5.
`
`I keep abreast of ongoing research developments in the area ofmoleculru· biology
`
`and gene therapy by regular pemsal of the relevant literature and my service on the editorial
`
`boru·ds of several different scientific joumals. In pruticulru·, I run or have been on the editorial
`
`boards of numerous scientific jomnals, including Gene Therapy, Human Gene Therapy, and
`
`Molecular Therapy. I run currently the Associate Editor of Human Gene Therapy and continue
`
`to be on the editorial boards of other scientific joumals. Based on this work, I am familiar with
`
`the review process associated with publishing scientific articles. That process can involve
`
`multiple rounds of edits, including requests for authors to nm additional experiments to generate
`
`more data to include in the manuscript before publication. In my experience, it is common for
`
`the content of manuscripts to change considerably between when an article is first submitted and
`
`when it is published.
`
`6.
`
`I have been retained by attomeys for Plaintiffs to provide consultation and expelt
`
`opinions regarding United States Patent Nos. 5,747,282 (the '" 282 patent"); 5,837,492 (the '"492
`
`patent"); 5,753,441 (the '" 441 patent"); 6,033,857 (the '" 857 patent"); 6,951,721 (the '" 721
`
`patent"); and 5,654, 155 (the " ' 155 patent") (collectively, the "patents-in-suit") in support of
`
`Plaintiffs' Motion for a Preliminruy Injunction. I have been asked to provide opinions in
`
`response to the declru·ations of Dr. Anne Bowcock ("Bowcock Declru·ation") and Dr. Simon
`
`Gregory ("Gregory Declaration"), both dated August 14, 2013.
`
`7.
`
`This declaration and the opinions set forth herein are based on Defendants'
`
`Opposition to Plaintiffs' Motion for Preliminary Injunction, the Bowcock Declru·ation, the
`
`Gregory Declru·ation, along with the references and other documents cited therein, documents
`
`cited herein, and my personal education, professional experience and general knowledge of the
`
`3
`
`GeneDX 1015, pg. 3
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 4 of 45
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`field as of the early-to-mid-1990s and thereafter. A full list of the materials I considered in
`
`preparing tllis declaration is attached as Exhibit B.
`
`8.
`
`In the past four years, I have testified at trial or by deposition in the following
`
`matter: Tekmira Phrumaceuticals Corp. and Protiva Biotherapeutics, Inc. v. Alnylrun
`
`Phrumaceuticals, Inc. and Alcana Technologies, Inc., Civil Action No. 11-10-10-BLS2 (Mass.
`
`Super. Ct.).
`
`9.
`
`I am being compensated at my notmal consulting rate of$650 per hour for non-
`
`testimonial work, and $850 per hour for testimony at depositions, heru·ing and trial. My
`
`compensation is no way dependent on the outcome of this litigation.
`
`10.
`
`I continue my investigation and study. I may review additional doctm1ents and
`
`information the pa1ties provide or rely on after the submission of my declru·ation. IfDefendants'
`
`experts provide additional opinions not expressed in their repotts, I may supplement my repott to
`
`respond to them. Therefore, I may expand or modify my opinions as my investigation and study
`
`continues, and supplement my opinions in light of any additional infotmation I review, any
`
`matters Defendants raise, or any opinions Defendru1ts' experts may provide.
`
`11 .
`
`I reserve the right to make demonstratives for use at trial or hearings that include
`
`figures or text fi·om any of the materials I have considered, as well as any other infonnation that I
`
`believe may assist me in explaining issues relevant to this case.
`
`II.
`
`TECHNOLOGY BACKGROUND
`
`A.
`
`DNA
`
`12.
`
`DNA, which stands for deoxyribonucleic acid, is a type of chemical compound
`
`called a nucleic acid. At its most basic level, a DNA molecule is composed of several chemical
`
`elements, namely Carbon, Hydrogen, Oxygen, Nitrogen, and Phosphorus. These chenlical
`
`elements make up repeating units that ru·e connected to form a strand or polymer of the DNA
`
`4
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`GeneDX 1015, pg. 4
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 5 of 45
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`molecule. These repeating units of DNA are known a.s nucleotides. The standard nucleotides in
`
`vertebrate DNA contain four different bases: Adenine, Thymine, Cytosine, and Guanine. These
`
`bases are linked together by chemical bonds via a sugar-phosphate backbone. As sh01thand for
`
`convenience, scientists often denote nucleotides by the first letter of the names of their bases:
`
`"A" for Adenine; "G" for Guanine; "T" for Thymine; and "C" for Cytosine. Presented below are
`
`depictions of the chemical stmcnu·es of the four nucleotides:
`
`H2N
`
`0
`
`NH
`({ J {
`~ J-NH2
`
`N
`
`N
`H
`
`N
`
`N
`H
`Adenine
`
`f:
`"CNH
`N~O N~O
`
`0
`
`H
`
`H
`Cytosi ne
`
`Guanine
`
`Thymjne
`
`13.
`
`A molecule ofDNA is typically represented by the linear order of its nucleotides,
`
`i.e., its "nucleotide sequence" or simply - its "sequence." The nucleotide sequence defmes
`
`important stmcture and chemical propet1ies of a pat1icular DNA molecule based on the linear
`
`order of nucleotides in that patti culm· DNA molecule. The structure atld chemical propet1ies of a
`
`pat1iculm· DNA molecule can help establish its function.
`
`14.
`
`Generally, DNA exists as a double helix, which consists of two intertwined
`
`strands of DNA. This structure is made possible because each base in one strand is paired via
`
`hydrogen bonds with another base in the other, complementary strand (Adenine pairs with
`
`Thymine and Cytosine pairs wjth Guanine).
`
`15.
`
`DNA as it is fotmd in the hmnan body, i.e., native DNA, is one integral
`
`component of cln-omosomes. Chromosomes are complex stmcnu·es that catTY genes and which
`
`are located in most cells of the human body. Historically, the term "gene" has been used to
`
`5
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`GeneDX 1015, pg. 5
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 6 of 45
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`describe the tmit that is responsible for the inheritance of a discrete trait, such as the color of peas
`
`in a peapod. In molecular tenns, a gene includes the aggregate of several segments of a
`
`chromosome.
`
`16.
`
`Generally speaking, a gene is a segment of DNA that codes for a protein, as
`
`described more fully below.
`
`17.
`
`A single-stranded DNA molecule has "directionality," i.e., the two ends of the
`
`molecule are chemically different. The "beginning" of a DNA molecule is called the 5' ("5
`
`prime")-end and the "end" of the molecule is called the 3' ("3 prime")-end.
`
`18.
`
`Typically, the sequences at the 5'- and sometimes the 3'- ends of the strand of
`
`native DNA coding for a protein conespond to regulat01y elements that control when a cell
`
`activates a gene. For example, a regulatory element can detennine in which tissue types or tmder
`
`what conditions a gene may be tmned on ("expressed"). The native DNA of a gene also contains
`
`regions that can, as described in more detail below, code for protein molecules. Protein-coding
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`segments of native DNA are contained in "exons." In humans and other higher organisms,
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`protein-coding exonic DNA sequences are typically intenupted by intervening DNA sequences
`
`known as "introns" that do not code for proteins, but may contain regulatory elements.
`
`19.
`
`Native DNA sequence alterations that cause disease conditions (often tem1ed
`
`"mutations") can be found in exons, introns, or regulat01y elements, although it is often easier for
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`geneticists to identify disease-causing mutations in exons and, hence, mutation screens often
`
`concentrate on examining DNA sequences containing exons.
`
`B.
`
`RNA
`
`20.
`
`Like DNA, RNA- which stands for ribonucleic acid- is a chemical compotmd.
`
`Unlike DNA, however, the fom bases that make up RNA are Guanine, Cytosine, Uracil, and
`
`Adenine. Thus, instead of the base Thymine, RNA contains Uracil. Common abbreviations of
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`6
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`GeneDX 1015, pg. 6
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 7 of 45
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`the bases ofRNA are: "G" for guanine; "C" for Cytosine, "U" for Uracil, and "A" for Adenine.
`
`Each base together with one sugar and one phosphate molecule makes up one repeating unit
`
`known as a nucleotide of the RNA. Also like DNA, RNA is fmmed by a stnmd of bases that are
`
`linked together via a sugar-phosphate backbone. The structures of the sugar-phosphate backbone
`
`of RNA and DNA, however, are different from each other- while RNA contains a ribose sugar,
`
`the sugar component of DNA is a deoxyribose. Because of these differences in structure,
`
`whereas native DNA forms a double helix, RNA usually exists as a single strand. Moreover,
`
`DNA is much more stable than RNA.
`
`21.
`
`RNA is generated in the body fi:om native DNA in a process called
`
`"transcription." During transcription ofRNA from DNA, a discrete segment of the DNA
`
`tmwinds, and the bases of the DNA molecule act as "clamps" that hold the bases of the newly
`
`fom1ing RNA in place while the chemical bonds of the sugar-phosphate backbone are fanned.
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`Tins process is mediated by a structure in the cell known as the RNA polymerase.
`
`22.
`
`A newly transcribed RNA molecule (a "transcript"), or precursor messenger RNA
`
`("pre-mRNA"), is processed to result in a matme messenger RNA ("mRNA"). Pre-mRNA
`
`contains nucleotides that are eliminated during a process called "splicing." The segments of the
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`pre-mRNA spliced out are the introns while the remaining segments, exons, are ligated together,
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`or joined to fonn the intact mRNA molecule. In addition, a chemical "cap" is added to the 5 '(cid:173)
`
`end of the RNA. A "tail" of nucleotides that contain the base Adenine is added to the 3 '-end.
`
`The finalmRNA molecule contains only exons, a cap, and a poly-adenosine tail. The following
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`figure is a simplified illustration of the process of pre-nlRNA splicing to form mature mRNA. In
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`many cases, the first (5') and last (3') exons (or the end sequences of the first and last exons) do
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`not contribute to encoding the protein (green shading). These non-coding exons can influence the
`
`7
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`GeneDX 1015, pg. 7
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 8 of 45
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`ftmctional properties of the mRNA such as its activity and/or how long it can last in a cell before
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`it is degraded. The intemal exons (red) have the sequences that are ultimately translated (see
`
`below) into a protein. The number of exons and introns varies for each gene. Even the DNA
`
`fi:agment that encodes a gene can produce different mRNAs with different combinations of
`
`exons through a process of altemative splicing.
`
`pre-mRNA
`
`Exon
`
`Exon
`
`C.
`
`Proteins
`
`23.
`
`Proteins are generally large, complex molecules that play many critical roles in
`
`the body. They do most of the "work" in the body and are required for the structure, ftmction,
`
`and regulation of the body's tissues and organs. Proteins are made up ofhundreds or thousands
`
`of smaller units called amino acids, which are attached to one another in long chains. There are
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`20 different amino acids that can be combined to make a protein. The sequence of amino acids
`
`dete1mines each protein's unique 3-dimensional stlucture and its specific ftmction.
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`24.
`
`Proteins are translated fi·om mRNA through a process called "u·anslation."
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`During u·anslation, mRNA serves as a template to assemble a protein. Three consecutive bases
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`in an mRNA molecule constitute a " codon," which codes for one ofthe 20 amino acids. Pairing
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`interactions take place between an mRNA molecule and another RNA molecule known as tRNA,
`
`which serves as an adaptor during protein translation. Specifically, sets of three nucleotides in
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`the coding region of an mRNA, react with three nucleotides in a tRNA in such a way as to cause
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`the amino acid linked to the tRNA molecule to be chemically transfened to the growing
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`8
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`GeneDX 1015, pg. 8
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31/13 Page 9 of 45
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`polypeptide chain destined to become a protein. The bases of the mRNA serve as "clamps" to
`
`hold the amino acids in place while the chemical bonds between the individual amino acids are
`
`f01med. Dming translation, the mRNA template, the tRNA, the newly f01ming polypeptide
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`chain, and the next amino acid reside in a multi-protein complex called a ribosome. Once a
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`protein is translated it typically undergoes post-translational or chemical modifications that are
`
`important for the protein' s fi.mction.
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`25.
`
`The genetic code describes which codons code for which amino acids. For
`
`example, the codon Adenine-Thymine-Guanine encodes the amino acid Methionine. Thus, the
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`chemical composition of an mRNA molecule detetmines the amino acid composition of a
`
`protein.
`
`D.
`
`eDNA
`
`26.
`
`Complementary DNA, or " eDNA," is commonly synthesized from a mature
`
`mRNA in a reaction catalyzed by a protein known as reverse transcriptase. eDNA received its
`
`name because each base in the eDNA can bind to a base in the mRNA from which the eDNA is
`
`synthesized. In other words, it is " complementary" to the mRNA from which it is synthesized.
`
`27.
`
`eDNA is stmctmally different from native DNA. First, eDNA made from an
`
`mRNA does not contain introns in contrast to native DNA, which contains intronic sequences.
`
`Second, eDNA can contain sequences that conespond to the poly-adenosine tail of mRNA,
`
`which does not exist in native DNA. Third, because it is not associated with proteins as with
`
`native DNA and because it lacks a 5' cap, no protein can be produced from an isolated eDNA
`
`molecule without introduction ofregulat01y sequences. Fomih, the sugar-phosphate backbone of
`
`native DNA is usually chemically modified, e.g., by methylation. In contrast, the sugar(cid:173)
`
`phosphate backbone of eDNA is not modified. Finally, as discussed above, isolated eDNA can
`
`9
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`GeneDX 1015, pg. 9
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 10 of 45
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`serve as a probe, as a tm·get for a probe, and as a template for a polymerase chain reaction
`
`("PCR"), aU of which native mRNA cannot do.
`
`28.
`
`eDNA is also functionally different from native DNA First, native DNA contains
`
`regulatmy sequences. These regulatory sequences are not present in eDNA because they are not
`
`present in the mRNA from which the eDNA was synthesized. Second, because eDNA does not
`
`contain inu·onic sequences, mRNA can be transcribed from eDNA without the need for splicing.
`
`Third, inu·oducing a eDNA alone into a cell does not give rise to protein production from that
`
`eDNA Fomth, native DNA and chromosomal proteins fmm a functional unit; isolated or
`
`synthetic eDNA, however, is not associated with chromosomal protein and can thus be used as a
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`moleculm· tool in various biotechnological applications.
`
`E.
`
`Primers
`
`29.
`
`A primer is an artificial single-stranded DNA molecule, usually between 15 and
`
`30 nucleotides long, that binds specifically to the target nucleotide sequence. The sequence of the
`
`primer is complementmy to the target sequence such that the bases of the primer and the bases of
`
`the target sequence bind to each other_
`
`30. When its sequence is chosen carefully, a prin1er will bind to a unique location of
`
`the native DNA tm·get, allowing primers to be used for methods that relate to a pmticulm· DNA
`
`sequence. These methods include producing multiple copies of a specific DNA segJnent for
`
`DNA sequencing reactions or other moleculm· characterization.
`
`F.
`
`Polymerase Chain Reaction
`
`31.
`
`Because native DNA gene sequences are generally only present in two copies in
`
`each cell (one copy inherited fi·om each parent), and m olecular testing methods such as DNA
`
`sequencing reactions often require m any copies of an input DNA molecule to generate reliable
`
`results, geneticists need a method of amplifying DNA products.
`
`10
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`GeneDX 1015, pg. 10
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 11 of 45
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`32.
`
`The most widely used DNA amplification method is the polymerase chain
`
`reaction (PCR).1 PCR involves mixing an input DNA sequence known as the template with a
`
`thetmostable DNA polymerase enzyme, a pool of all fom DNA nucleotides (A, C, T and G), and
`
`a great excess of two distinct single-stranded primers. One primer is complementruy to one end
`
`of the region to be amplified on one strru1d of the template DNA molecule and the other primer is
`
`complementary to the other end of the region to be amplified on the other strand of the template
`
`DNA.
`
`33.
`
`PCR involves several steps in a cyclical reaction. First, the mixtme is heated so
`
`that the bonds linking the two strands of the template DNA molecule are overcome and the
`
`strands sepru·ate ("denatmation"). Second, the mixture is cooled enough to allow one copy of
`
`each primer to bind to its complementary template DNA sequence ("annealing"). Third, the
`
`DNA polymerase adds nucleotides to the 3 ' -end of each of the primers in an order
`
`complementruy to the template DNA. This extension reaction results in the generation of a copy
`
`of each strand of the template DNA ("amplifying"). The number of copied DNA molecules
`
`doubles with each PCR cycle. A typical PCR nms for 20-30 cycles and results in the
`
`accumulation of millions of copies of the template DNA for the interval spanned by the two
`
`pnmers.
`
`34.
`
`The '282 patent discloses the use ofPCR to amplify sequences of the BRCA1
`
`gene for screening by allele-specific probes ('282 patent at col. 15, 11. 21-25) and for sequencing
`
`BRCA1 segments ('282 patent at col. 16, ll. 23-25). The '492 patent discloses similar uses of
`
`PCR for study of the BRCA2 gene. (See '492 patent at col. 14, ll. 7-11 ; id. at col. 15, 11. 10-12.)
`
`1 Basic infonnation about PCR is discussed in Sambrook et al., Moleculru· Cloning (Cold Spting
`Harbor Laboratmy Press 1989), at Chapter 14.
`
`11
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`GeneDX 1015, pg. 11
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`

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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 12 of 45
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`3 5.
`
`I note that Dr. Gregory characterizes the genetic marker D 17S85 5 as
`
`"compris[ing] two strands of single-stranded DNA." (Gregmy Decl. ~ 79.) He also states that
`
`" [t]he two strands can be used as a pair of primers in a polymerase chain reaction." (Id.) I
`
`disagree with both parts of this characterization. First, rather than describing D17S855 as "two
`
`strands of single-stranded DNA," because the strands are complementruy to each other, it is more
`
`appropriate to describeD 17S855 as a double-strru1ded DNA molecule. Second, it was well
`
`understood by a person of ordinary skill in the early 1990s that PCR involves the use of single(cid:173)
`
`stranded primers that are not complementruy to each other to mnplify tru·get DNA located
`
`between the prin1ers. The result is an mnplified PCR product that is double-stranded and
`
`incorporates the sequence of the first primer (forwru·d primer) followed by the mnplified target
`
`sequence, m1d further followed by the sequence of the second primer (reverse p1imer).
`
`Described differently, the p1imers are complementruy to the ends of the target DNA to be
`
`mnplified. The end result is mnplification of sequences of the target DNA intemal to the
`
`primers~ the PCR product contains the primer sequences and the target DNA sequence.
`
`36.
`
`The concept described by Dr. Gregmy- sepru·ating a double-stranded DNA
`
`marker into two complementary single strands of DNA- would not result in an mnpli.fied PCR
`
`product. The single DNA strru1ds would either exactly align and re-anneal with one another or
`
`anneal to the exact smne position along the tru·get DNA. In either case there would be no tru·get
`
`DNA located between the "primers" to mnplify.
`
`37.
`
`Dr. Bowcock made similru· statements about D17S855 and another marker,
`
`D 17S932. (Bowcock Decl. at~ 83 .) I disagree with her position that these double-stranded
`
`DNA molecules could have been used as a pair of single-stranded PCR primers for the same
`
`reasons.
`
`12
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`GeneDX 1015, pg. 12
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`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 13 of 45
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`G.
`
`Discovery of the BRCA1 Gene
`
`38.
`
`Discovery of the BRCA1 gene was a highly competitive effmt that involved
`
`multiple groups of scientists with diverse expettise in addition to the inventors of the '282 patent.
`
`(See Ex. C, Shattuck Decl. ~ 9). This discovery effol1 took place in the early 1990s, when it had
`
`been dete1mined that a gene linked to chromosome 17q21, coined "BRCA1," was likely
`
`responsible for a large number of familial breast and ovarian cancer. 2 (See '282 patent at coL 3,
`
`11. 13-29.)
`
`39. Myriad's identification of the BRCA1 gene, as described in the '282 patent,
`
`required the application of a positional cloning approach. (Shattuck Decl., ~~ 4, 5.; See generally
`
`'282 patent ; id at Example 1-8.)
`
`40. Myriad had to take a positional cloning approach because the BRCA1 protein had
`
`not been identified. Had the protein been known, they may have been able to use different
`
`methods to identify its mRNA and ultimately the gene. Those were methods like
`
`imrounoprecipitation of the newly made protein while still attached to the ribosome, prokaryotic
`
`cloning and expression cloning, and overlapping deletion fragments, which I understand were
`
`discussed as conventional prior art techniques for isolating and sequencing a known protein in a
`
`case called In re Kubin that Defendants mention in their opposition brief. BRCA1 thus had to be
`
`identified using positional cloning techniques, which rely upon linkage analyses of fan1ilial data
`
`to search for the location of the disease-causing gene.
`
`41.
`
`After the BRCA1 gene was mapped to chromosome 17 in 1990, intensive study of
`
`sets of large families with multiple individuals afflicted with breast and ovarian cancer was
`
`needed to nanow the candidate region of chromosome 17 thought to contain BRCA1. (Shattuck
`
`2 J. M. Hall, et al., Linkage of Early-Onset Familial Breast Cancer to Chromosome 17q21, 250
`Sci. 1684-89 (1990) (Gregmy Ex. H .).
`
`13
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`GeneDX 1015, pg. 13
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`

`
`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 14 of 45
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`Decl. ~~ 9-14.) Myriad had large and inf01mative family data available due to work by Dr.
`
`Skolnick. (See Ex. D (Skolnick Dec.)~~ 2-18; '282 patent at col. 8, 11. 15-37.) Having accmate
`
`multi-generational pedigrees and availability of DNA samples from these families is
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`instnunental to the positional cloning approach. Families that are not as large or that contain an
`
`incorrect diagnosis (including sporadic or non inherited fmms of breast cancer- which can
`
`sometimes be difficult to differentiate) or a false paternity may not be as useful. One wrinkle in
`
`the linkage analysis approach for BRCA1 is that certain families with familial breast cancer
`
`showed no linkage to chromosome 17. This suggested a second BRCA gene localized to a
`
`different chromosome (ultimately identified as BRCA2).
`
`42.
`
`The inventors started with a region that encompassed millions of base pairs. By
`
`using a specifically large and informative kindred and other families, the inventors were able to
`
`localize BRCA1 to a section of chromosome 17 spanning about 600,000 base pairs. (Jd ~ 12.)
`
`Tins result came in the face of conflicting results fi-om other scientists that had localized BRCA1
`
`to other parts of clu-omosome 17, as described below. (See also id ~ 7.)
`
`43.
`
`Before candidate breast cancer genes could be isolated for additional study, the
`
`inventors needed to create a "physical map" of the putative BRCAl gene interval on
`
`chromosome 17 by obtaining a set of smaller, overlapping cloned DNA molecules that covered
`
`the BRCA1 region. (Shattuck Decl. ~~ 17-21.) The inventors used a vari.ety of DNA cloning
`
`vectors-PI and BAC clones-in addition to the yeast artificial chromosome systems (YACs).
`
`(Jd. ~ 17; '282 patent at col. 9, 11. 44-49.) As described further below, the use ofPl and BAC
`
`clones was not as well tested at that time.
`
`44. With a set of clones containing pieces of the BRCA1 region, the inventors were
`
`then able to put the pieces together and test them as carrdidate BRCAl genes. (Shattuck Dec!.~~
`
`14
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`GeneDX 1015, pg. 14
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`

`
`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 15 of 45
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`18-20.) The identification of gene fragments from cloned copies of chromosomal DNA was a
`
`challenging process and a likely explanation for the inventor's success in these efforts was the
`
`use of a novel gene selection teclmique. (See id ~~ 22-26.)
`
`45.
`
`To do this, the inventors isolated mRNA and conve1ted it to eDNA using different
`
`primer pairs for PCR. Then they hybridized the synthesized eDNA to genomic segments that
`
`were believed to contain the BRCA1locus. Thilty-nine independent candidate gene fragments
`
`by tllis hybridization selection technique were isolated and ultimately used to identify the full
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`length eDNA ofBRCAl. (See Shattuck Dec., ~~ 22-26.)
`
`46.
`
`The fact that BRCA1 ultimately contained 24 exons (22 coding exons) witl1 a
`
`long open-reading frame distributed over 100,000 base pairs made the ultimate identification of
`
`the complete eDNA challenging. (See Shattuck Dec., ~~ 27-29.) A single DNA sequence error
`
`could have resulted in total misunderstanding oftl1e coding reading fi·ame of the eDNA. In
`
`addition, the fact that there were multiple mRNA transcripts derived from multiple splicing
`
`variants (alternative splicing) transcribed fi·om the BRCAl gene made the eventual identification
`
`of the full eDNA sequence, e.g., mRNA, even more difficult. In fact, without knowing all the
`
`alternative splice forms, it would be likely that the full eDNA sequence would not have been
`
`conectly assembled and identified, leading to the inability to identify impmtant disease-causing
`
`mutations.
`
`4 7.
`
`Proving that a candidate gene fragment identified by the inventors was indeed
`
`responsible for an inherited fonn of breast and ovarian cancer required the identification ofDNA
`
`sequence mutations that occmred only in family members affected by breast or ovarian cancer.
`
`(See Shattuck Decl. ~ 27.) The inventors were able to find such mutations in five fan1ilies with
`
`strong lllstories of inherited breast and ovarian cancer. (See Shattuck Decl. ~ 15.)
`
`15
`
`GeneDX 1015, pg. 15
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`

`
`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 16 of 45
`
`48.
`
`Subsequent studies revealed that the BRCA1 gene did not behave in the same
`
`manner as other cancer-causing genes. (See Shattuck Decl. ~~ 30-34.) It was expected that
`
`somatic mutations in BRCA1 would be discovered in sporadic breast cancer. Unlike other tumor
`
`suppressor genes, they were not. These findings highlighted the unexpected natme of the
`
`BRCA1 gene and provided additional evidence that the process of identifying the gene was a
`
`substantial scientific accomplishment. (See id ~ 34.)
`
`H.
`
`Discovery of the BRCA2 Gene
`
`49.
`
`Identification of the BRCA2 gene, as described in the ' 492 patent, also involved
`
`an approach that was similar to that described above for the identification of the BRCA1 gene
`
`(See Ex. E, Tavtigian Decl. ~ 4; see generally '492 patent.)
`
`50.
`
`The inventors of the '492 patent analyzed 19 families, with multiple members
`
`affected by breast cancer, to refme the localization of the BRCA2 gene to a region on
`
`clu·omosome 13. (' 492 patent at col. 34, 1. 19-col. 36, 1. 55 & tbl.l.) This analysis allowed the
`
`inventors to reduce the candidate interval on clu·omosome 13 carrying the BRCA2 gene to 1.5
`
`million base pairs in length. (Id at col. 36, 11. 40-49.)
`
`51. With the nanowed BRCA2 candidate interval in hand, the inventors were then
`
`able to construct a physical map of cloned DNA covering the BRCA2 candidate inte1val. (' 492
`
`patent at col. 36, 1. 61-col. 37, 1. 27 & fig.l.) As was the case for the BRCA 1 discovery effort,
`
`this physical map used a combination of yeast rutificial chromosomes and other DNA cloning
`
`vectors to ens me that an accmate physical map was obtained. (See id ; see also Shattuck Decl. ~
`
`17.)
`
`52.
`
`The inventors used a range of challenging gene discove1y techniques to identify
`
`genes that were located in the BRCA2 candidate interval. ('492 patent at col. 37, 1. 34-col. 44, 1.
`
`16
`
`GeneDX 1015, pg. 16
`
`

`
`Case 2:13-cv-00640-RJS Document 103 Filed 08/31113 Page 17 of 45
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`5.) These experiments involved the analysis of thousands of clones containing fi.·agments of
`
`BRCA2 candidate genes. (See, e.g., id at col. 41, 11. 55-66.)
`
`53.
`
`In order to test whether candidate BRCA2 genes might indeed be responsible for
`
`inherited breast cancer, the inventors screened candidate DNA sequences for sequence changes
`
`that could adversely affect the fi.mction of a protein coded for by that candidate. (' 492 patent at
`
`col. 39, 1. 63 to col. 40, 1. 28.) More comprehensive study performed after the inventors had
`
`sequenced the entire protein-coding pmtion of the BRCA2 gene identified mutations that
`
`segregated with familial breast cancer in nine of the eighteen families screened. (Id at col. 46, 11.
`
`7-62 & tbl.3.) These studies provided convincing evidence that the inventors conectly identified
`
`BRCA2, the gene responsible for familial breast cancer on chromosome 13. The protein coded
`
`for by BRCA2 was unusually large- being composed of 3418 amino acids- and bore no
`
`significant sequence similarities to other known proteins. (Id. at col. 45, 11. 11-20.)
`
`Ill. LEGAL FRAMEWORK
`
`54.
`
`In expressing opinions on legal issues, I have applied the following legal
`
`standards as described to me by cmmsel for Plaintiffs.
`
`55.
`
`I understand that issued patents are presumed valid and that the party challenging
`
`invalidity bears the burden to prove by clear and convincing evidence that a patent is invalid.
`
`A.
`
`Anticipation
`
`56.
`
`I understand that a patent claim is anticipated by a prior art reference (i.e., it is n