·1· ·UNITED STATES PATENT AND TRADEMARK OFFICE
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`·2· · · · · · ·_________________
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`·3· ·BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`·4· · · · · · ·_________________
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`·5· ·BLUEBIRD BIO, INC.
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`·6· · · · · Petitioner
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`·7· ·vs.
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`·8· ·SLOAN KETTERING INSTITUTE FOR CANCER RESEARCH,
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`·9· · · · · Patent Owner
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`10· · · · · · ·_________________
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`11· · · · · · ·Patent No. 8,058,061
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`12· · · · · · ·_________________
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`13
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`14· · · · · · · · · · · July 10, 2023
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`15
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`16
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`17· · · · · Video-Recorded Deposition of
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`18· · · · · · · ·Jörg Bungert, Ph.D.
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`19
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`20
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`21
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`22· ·Stenographically Reported By:
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`23· ·Mark Richman, RPR, CSR, CCR, CM
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`24· ·Job No. J9814137
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`25
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`SKI Exhibit 2055
`Page 1 of 254
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`
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`·2· · · · · · ·_________________
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`·3· ·BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`·4· · · · · · ·_________________
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`·5· ·BLUEBIRD BIO, INC.
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`·6· · · · · Petitioner
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`·7· ·vs.
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`·8· ·SLOAN KETTERING INSTITUTE FOR CANCER RESEARCH,
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`·9· · · · · Patent Owner
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`10· · · · · · ·_________________
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`11· · · · · · ·Patent No. 8,058,061
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`12· · · · · · ·_________________
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`13
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`14· · · · · · · · · · · July 10, 2023
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`15
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`16
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`17· · · · · Video-Recorded Deposition of
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`18· · · · · · · ·Jörg Bungert, Ph.D.
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`19
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`20
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`21
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`22· ·Stenographically Reported By:
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`23· ·Mark Richman, RPR, CSR, CCR, CM
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`24· ·Job No. J9814137
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`25
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`SKI Exhibit 2055
`Page 1 of 254
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`·1
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`·3· · · · · · · · · · · ·JULY 10, 2023
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`·4· · · · · · · · · · · ·9:41 A.M. EST
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`·5
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`·7· · · · · · Video-Recorded Deposition of
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`·8· ·JÖRG BUNGERT, PH.D., at the offices of Paul
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`·9· ·Hastings LLP, 200 Park Avenue, New York, New
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`10· ·York, before Mark Richman, Certified
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`11· ·Shorthand Reporter, Certified Court
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`12· ·Reporter, Registered Professional Reporter,
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`13· ·and a Notary Public within and for the State
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`14· ·of New York.
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`SKI Exhibit 2055
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`·1· ·A P P E A R A N C E S:
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`·2· ·PAUL HASTINGS LLP
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`·3· ·Attorneys for Bluebird Bio
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`·4· · · · · 200 Park Avenue
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`·5· · · · · New York, New York 10166
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`·6
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`·7· ·BY:· MAX YUSEM, ESQ.
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`·8· · · · KRYSTINA HO, ESQ.
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`·9
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`10· ·FOX ROTHSCHILD
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`11· ·Attorneys for San Rocco Therapeutics as
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`12· ·Representative for MSK
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`13· · · · · 101 Park Avenue, 17th floor
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`14· · · · · New York, NY 10178
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`15
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`16· ·BY:· · MICHAEL GLYNN, ESQ.
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`17· · · · · JOE CHEN, ESQ.
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`18· · · · · HOWARD SUH, ESQ.
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`19
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`20· ·ALSO PRESENT:
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`21· ·SILVIO FACCHIN, Videographer
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`·1· · · · · ·J. BUNGERT - 7.10.23
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`·2· · · · THE VIDEOGRAPHER:· This is the
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`·3· ·media labeled number 1 of the video
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`·4· ·recorded deposition of Dr. Jörg
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`·5· ·Bungert in the matter of Bluebird Bio
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`·6· ·v. Sloan Kettering Institute for
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`·7· ·Cancer Research.
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`·8· · · · We are now going on the record.
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`·9· ·The time is 9:41 a.m.
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`10· · · · Counsel please state their
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`11· ·appearances for the record.
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`12· · · · MR. YUSEM:· Yes.· Max Yusem for
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`13· ·Paul Hastings and Bluebird.
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`14· · · · MS. HO:· Krystina Ho for Paul
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`15· ·Hastings and Bluebird.
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`16· · · · DR. GLYNN:· Michael Glynn from
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`17· ·Fox Rothschild, joined by my
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`18· ·colleagues, Joe Chen and Howard Suh,
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`19· ·also from Fox Rothschild representing
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`20· ·San Rocco Therapeutics.
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`21· · · · THE VIDEOGRAPHER:· Will the court
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`22· ·reporter please swear in the witness.
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`23· · · · //
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`24· · · · //
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`25· · · ·//
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`SKI Exhibit 2055
`Page 4 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · · · · ·JÖRG BUNGERT, PH.D., called as a
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`·3· · ·witness, having been first duly sworn
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`·4· · ·by the Notary Public (Mark Richman),
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`·5· · ·was examined and testified as
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`·6· · ·follows:
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`·7· · · · · EXAMINATION BY DR. GLYNN:
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`·8· · Q.· · Good morning.
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`·9· · A.· · Good morning.
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`10· · · · · Perhaps before we begin, in
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`11· ·preparation of this meeting, I noticed
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`12· ·that there were some minor errors in the
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`13· ·declaration and they don't affect my
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`14· ·conclusions but I would like to hand
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`15· ·them to you.
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`16· · · · · MR. YUSEM:· We've got copies too.
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`17· · · · · MS. HO:· Yes, pass them out?
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`18· · · · · DR. GLYNN:· Yes, let's distribute
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`19· · ·copies as well.
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`20· · Q.· · These are corrected versions of
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`21· ·figures?
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`22· · A.· · Yes, of the visualizations of the
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`23· ·analysis of the restriction fragments.
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`24· · Q.· · And what is different about these
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`25· ·figures with respect to the figures in
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`SKI Exhibit 2055
`Page 5 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·your declaration?
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`·3· · A.· · Yes.· Let me go to the... so one
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`·4· ·change is with regard to HS2.· So if you
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`·5· ·go to page 47 of the declaration or 55
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`·6· ·of a 100, you notice that --
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`·7· · Q.· · I'm sorry, what page?· I'm sorry,
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`·8· ·what page?
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`·9· · A.· · Page 47.
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`10· · Q.· · 47.
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`11· · A.· · Or 55 out of 100, declaration
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`12· ·179.
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`13· · Q.· · I went too far, sorry.
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`14· · A.· · It's the one before.
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`15· · Q.· · Got it.
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`16· · A.· · So that's the first one.· And you
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`17· ·noticed in here that we change the
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`18· ·position of the HS2 site so we
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`19· ·erroneously used the ones at 493 and we
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`20· ·corrected that to use the ones at 765
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`21· ·and we didn't consider any of these
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`22· ·fragments or I didn't consider any of
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`23· ·these fragments for the analysis. I
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`24· ·mean for the final selection of
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`25· ·fragments that we used.
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`SKI Exhibit 2055
`Page 6 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · Q.· · I'm not following you.
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`·3· · A.· · So in this, in this figure you
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`·4· ·see below in the original version we
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`·5· ·list six fragments and they were ordered
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`·6· ·according to the length.· And the first
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`·7· ·two fragments of HS2, AvaI, HS2i, you
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`·8· ·see these two fragments?
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`·9· · Q.· · Mm-hmm.
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`10· · A.· · And so in the revised version we
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`11· ·changed using the HS2 that is a closer
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`12· ·to the core.· We meant, I meant to use
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`13· ·the restriction enzyme that is closer to
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`14· ·the core.
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`15· · Q.· · Okay.· Okay, I understand.· So in
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`16· ·the original figure it's using the HS2
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`17· ·site at 493?
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`18· · A.· · That's correct.
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`19· · Q.· · And you changed this to use the
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`20· ·XhoII site at 653?
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`21· · A.· · That's correct.
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`22· · Q.· · All right.· Is that the only
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`23· ·difference?
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`24· · A.· · This is the only difference in
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`25· ·this figure here.
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`SKI Exhibit 2055
`Page 7 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · Q.· · Okay.
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`·3· · A.· · And then in the next figure, I
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`·4· ·made the mistake to include one
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`·5· ·additional fragment which in this
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`·6· ·original figure is the NdeI sub 1
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`·7· ·fragment and we -- I should not have
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`·8· ·included that one because it is too
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`·9· ·large for the fragments that we
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`10· ·considered for this analysis based on
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`11· ·the May article.
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`12· · Q.· · So you're excluding from this
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`13· ·figure the NdeI?
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`14· · A.· · NdeI.
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`15· · Q.· · Okay.· Any other changes?
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`16· · A.· · No.
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`17· · Q.· · Okay.· When did you -- when did
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`18· ·you notice these errors in the
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`19· ·declaration?
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`20· · A.· · Just in preparation for this
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`21· ·deposition before I met with the
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`22· ·counsel.
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`23· · Q.· · Have you been deposed before?
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`24· · A.· · Excuse me?
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`25· · Q.· · Have you been deposed before?
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`SKI Exhibit 2055
`Page 8 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · No.· This is the first time.
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`·3· · Q.· · This is the first time?· Okay.
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`·4· ·So you understand that you are under
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`·5· ·oath.
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`·6· · A.· · Mm-hmm.
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`·7· · Q.· · To give truthful and complete
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`·8· ·testimony?
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`·9· · A.· · Mm-hmm.
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`10· · Q.· · All right.· I'll be asking you
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`11· ·questions today.· You are required to
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`12· ·give answers.· And if any of the
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`13· ·questions that I ask you are unclear to
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`14· ·you, please let me know and I'll do my
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`15· ·best to rephrase in a way that's clear
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`16· ·to you.
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`17· · · · · We will be taking breaks from
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`18· ·time to time.· If you find that you need
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`19· ·a break, please let me know and we'll
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`20· ·take a break either when that question
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`21· ·and answer is complete or depending when
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`22· ·that line of questioning is complete and
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`23· ·we'll take a break as soon as
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`24· ·practicable.
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`25· · · · · Is that all right?
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`SKI Exhibit 2055
`Page 9 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · That's all right.
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`·3· · Q.· · Okay.· Other things, just keep in
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`·4· ·mind the talking over which we covered.
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`·5· ·That can, that complicates things for
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`·6· ·the record.
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`·7· · · · · Answers should be verbal, right.
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`·8· ·Nods and things like that also aren't
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`·9· ·conducive for the deposition atmosphere,
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`10· ·so.
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`11· · · · · All right.· I think with that in
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`12· ·mind we will start.
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`13· · · · · How did you identify those, the
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`14· ·mistakes that we just covered?· You just
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`15· ·happened to notice them as you were
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`16· ·going through?
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`17· · A.· · Well, I just went over and it
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`18· ·just didn't make sense to me that we
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`19· ·would use fragments where we used the
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`20· ·further upstream HS2 sites so I went
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`21· ·back in the restriction map that I did
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`22· ·and then I noticed that we should have
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`23· ·used these in the corrected version.
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`24· · Q.· · Oh, is that because if you used
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`25· ·the upstream one then you had another
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`SKI Exhibit 2055
`Page 10 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·XhoII site cutting in the middle and
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`·3· ·that wouldn't make sense?
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`·4· · A.· · No, not because of that.· Because
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`·5· ·we were looking for fragments that had
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`·6· ·the size that were outlined in the May
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`·7· ·article in Figure 1 according to the
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`·8· ·size.· And so if we would have used the
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`·9· ·other one, it wouldn't be in that range.
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`10· · Q.· · Okay.· All right.· As we go
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`11· ·through your discussion today, you
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`12· ·understand that the time frame that
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`13· ·we're talking about with respect to the
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`14· ·knowledge of a person of ordinary skill
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`15· ·in the art or what's in the art is the
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`16· ·2001 time frame?
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`17· · A.· · And earlier.
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`18· · Q.· · And earlier, yes.
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`19· · A.· · Mm-hmm.
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`20· · Q.· · Up to and including, you know,
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`21· ·mid 2001, right?
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`22· · A.· · Mm-hmm.
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`23· · Q.· · Okay.· In, in that time frame, up
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`24· ·and including 2001 or mid 2001, what
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`25· ·methods would a person of ordinary skill
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`SKI Exhibit 2055
`Page 11 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·in the art use to clone or subclone
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`·3· ·segments of DNA?
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`·4· · A.· · I would say the preferential
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`·5· ·method would be to use restriction
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`·6· ·enzymes.
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`·7· · Q.· · Okay.· Are there other methods?
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`·8· · A.· · There are other methods.
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`·9· · Q.· · Okay.· And what are they?
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`10· · A.· · You could use PCR, other methods.
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`11· · Q.· · Okay.· Have you used PCR to clone
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`12· ·fragments?
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`13· · A.· · I've used PCR.
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`14· · Q.· · Okay.· And a person of ordinary
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`15· ·skill in the art would have known that
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`16· ·PCR is a way of cloning these fragments,
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`17· ·right?
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`18· · A.· · With respect to this particular
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`19· ·situation, a POSA, or a person of
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`20· ·ordinary skill in the arts, would have
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`21· ·looked at this publication, would have
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`22· ·looked at previous publications and
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`23· ·would have assumed that in this
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`24· ·particular situation restriction enzymes
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`25· ·were used.
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · Q.· · Based on what?
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`·3· · A.· · Based on previous publications
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`·4· ·that generated LCR fragments, and I cite
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`·5· ·the Ellis paper, I cite the Bouhasirra
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`·6· ·paper, based on another publication from
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`·7· ·the Sadelain group in 1990 where they
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`·8· ·use restriction enzymes to clone
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`·9· ·together these fragments for the
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`10· ·Lentiviral factor.
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`11· · · · · So based on that, I think a POSA
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`12· ·would assume that this was done with
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`13· ·restriction enzymes.
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`14· · Q.· · When you've cloned fragments from
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`15· ·the LCR you used PCR, right?
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`16· · A.· · We used it in a very different
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`17· ·context.· We used, we make constructs --
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`18· ·we used it in a different context.· We
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`19· ·generated PCR fragments and cloned
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`20· ·together fragments that we used for a
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`21· ·recombination in yeast so the context is
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`22· ·very different.
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`23· · · · · We didn't care about the ends of
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`24· ·these fragments because during the
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`25· ·recombination process they wouldn't be
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`SKI Exhibit 2055
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·incorporated into the, into the final
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`·3· ·product.
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`·4· · Q.· · If you're cloning with
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`·5· ·restriction sites, are there particular
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`·6· ·qualities that a person of ordinary
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`·7· ·skill in the art would look like -- look
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`·8· ·for when that person is evaluating which
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`·9· ·restriction enzymes to, to use or which
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`10· ·sites to use?
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`11· · A.· · I'm not sure about the question.
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`12· ·Can you repeat or rephrase?
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`13· · Q.· · Sure.· In -- when you're cloning
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`14· ·or subcloning a segment of DNA, there is
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`15· ·often a variety of restriction enzyme
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`16· ·sites or restriction sites to choose
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`17· ·from, and a person of ordinary skill in
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`18· ·the art is going to make a decision
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`19· ·about which enzymes or sites they want
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`20· ·to use and maybe which ones they don't
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`21· ·use in the first instance, right?
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`22· · A.· · Mm-hmm.
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`23· · Q.· · All right.· So are there
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`24· ·qualities that a person of ordinary
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`25· ·skill in the art would look for in the
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`SKI Exhibit 2055
`Page 14 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·restriction enzymes or restriction
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`·3· ·enzyme sites that would be preferable?
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`·4· · A.· · Yes.· And I outlined this in a
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`·5· ·declaration.· That preferably you would
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`·6· ·not use enzymes that cut too often.
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`·7· ·Preferably you would not use enzymes
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`·8· ·that cut outside the recognition site
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`·9· ·because that makes the fragment harder
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`10· ·to define.· So these are criteria I
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`11· ·think that you would, that you would use
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`12· ·in general.
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`13· · Q.· · Are there other criteria?
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`14· · A.· · Well, with respect to the claim
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`15· ·here in the May article, you would look
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`16· ·for restriction enzymes that match the
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`17· ·fragments that are outlined in the May
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`18· ·article.
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`19· · Q.· · Okay.· But right now we're
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`20· ·talking generally.
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`21· · A.· · Okay.
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`22· · Q.· · For restriction enzymes.
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`23· · · · · Are there other, are there other
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`24· ·characteristics that you would look for,
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`25· ·perhaps sticky ends versus blunt ends?
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`SKI Exhibit 2055
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · I mean that is just the
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`·3· ·technology, a technological question
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`·4· ·about how to put these fragments
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`·5· ·together.
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`·6· · Q.· · Sure.
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`·7· · A.· · Sometimes you use sticky and
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`·8· ·sometimes you use blunt ends.
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`·9· · Q.· · Right, blunt-end cloning being a
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`10· ·bit more inefficient?
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`11· · A.· · In terms of what?
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`12· · Q.· · In terms of ligating, ligating
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`13· ·efficiency.
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`14· · A.· · I think by that time conditions
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`15· ·for sticky end ligation and blunt end
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`16· ·ligation were both out, so doable.
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`17· · Q.· · Certainly both were doable?
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`18· · A.· · Yes.
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`19· · Q.· · Would you have a preference for
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`20· ·an enzyme that cut once versus having to
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`21· ·use a different enzyme on both sides of
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`22· ·the fragment?
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`23· · A.· · Do you mean that would I prefer
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`24· ·using an enzyme that cuts once and then
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`25· ·use another enzyme to release the
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`SKI Exhibit 2055
`Page 16 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·fragment or use an enzyme that cuts
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`·3· ·twice and release as fragment?
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`·4· · Q.· · Yes, the second one.· Would you
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`·5· ·prefer if the enzyme cut twice, once on
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`·6· ·each side of the fragment so that you
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`·7· ·only have to use one restriction site --
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`·8· ·or one restriction enzyme, excuse me?
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`·9· · A.· · That depends on the situation and
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`10· ·the context.
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`11· · Q.· · So there's no preference one way
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`12· ·or the other?
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`13· · A.· · It depends on the context.
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`14· · Q.· · And we were talking about cloning
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`15· ·here.· What do you understand me to mean
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`16· ·by that?· I want to make sure we're
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`17· ·using the same language.
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`18· · A.· · Sure.· By cloning I mean that you
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`19· ·create a DNA construct that contains
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`20· ·multiple DNA elements and that you use
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`21· ·for further experiments.
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`22· · Q.· · Okay.· So you're isolating DNA
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`23· ·from one source and putting it into
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`24· ·another source, and assembling it into
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`25· ·another source?
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`Page 17 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · That's correct.
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`·3· · Q.· · Right.· And that's true of course
`
`·4· ·whether it's, that the source that
`
`·5· ·you're assembling it into is for
`
`·6· ·homologous recombination, right, to
`
`·7· ·incorporate something into, you know,
`
`·8· ·into yet another source, right, using --
`
`·9· ·or for expression studies and the like,
`
`10· ·kind of like what we're talking about
`
`11· ·here?
`
`12· · A.· · What --
`
`13· · · · · MR. YUSEM:· I'll object to the
`
`14· · ·form of the question.
`
`15· · A.· · Yes.· So it always depends on the
`
`16· ·context.· So the way you put things
`
`17· ·together for recombination may be
`
`18· ·totally different than the way you put
`
`19· ·things together for generating an
`
`20· ·expression construct.
`
`21· · Q.· · If you're assembling an LCR that
`
`22· ·you're then going to use in expression
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`23· ·construct and that expression construct
`
`24· ·is going to be expressed from a vector,
`
`25· ·maybe a lentiviral vector versus that
`
`SKI Exhibit 2055
`Page 18 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·construct being used for homologous
`
`·3· ·recombination and integrated into a
`
`·4· ·target DNA, why would a person of
`
`·5· ·ordinary skill in the art when
`
`·6· ·assembling the LCR consider different
`
`·7· ·methods for the assembly?
`
`·8· · A.· · Because for that assembly the
`
`·9· ·ends of the restriction fragments would
`
`10· ·matter versus for the homologous
`
`11· ·recombination, because the homologous
`
`12· ·recombination does not incorporate the
`
`13· ·restriction enzymes that you add during
`
`14· ·the PCR.
`
`15· · Q.· · Maybe my question wasn't clear.
`
`16· ·If you're using homologous recombination
`
`17· ·to incorporate the LCR and whatever gene
`
`18· ·is being expressed from it into a
`
`19· ·target, that LCR is going to contain,
`
`20· ·you know, all the -- you know, the
`
`21· ·fragments that you put into it, whatever
`
`22· ·method that you use, right?
`
`23· · · · · And the same goes for an
`
`24· ·expression construct that's not used for
`
`25· ·homologous recombination but maybe a
`
`SKI Exhibit 2055
`Page 19 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·lentiviral expression reconstruct,
`
`·3· ·right?
`
`·4· · · · · That LCR when you assemble it is
`
`·5· ·still the LCR, is going to contain the
`
`·6· ·elements that you put together along
`
`·7· ·with the remnant of the assembly, right?
`
`·8· · A.· · I don't agree with that and the
`
`·9· ·reason I don't agree with that is we
`
`10· ·didn't use PCR to assemble fragments of
`
`11· ·the LCR, to generate an LCR that we use
`
`12· ·for expression construct.· We use PCR to
`
`13· ·generate constructs that we use to
`
`14· ·remove elements from the LCR or to
`
`15· ·replace elements from the LCR.
`
`16· · · · · It was an analytical method.· We
`
`17· ·did not, for us it was not important
`
`18· ·where the ends of the restriction sites
`
`19· ·were.
`
`20· · · · · And I want to also clarify that
`
`21· ·if we talk about PCR versus restriction
`
`22· ·fragments that in the May article there
`
`23· ·is no mention of PCR.· There's reference
`
`24· ·to other articles before and all of
`
`25· ·these articles use restriction enzymes
`
`SKI Exhibit 2055
`Page 20 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·to generate fragments for the LCR.
`
`·3· · Q.· · Nonetheless, you would agree
`
`·4· ·that, generally speaking, when a person
`
`·5· ·of ordinary skill in the art is looking
`
`·6· ·to assemble or clone segments of DNA,
`
`·7· ·they would consider both the use of
`
`·8· ·restriction endonucleases and PCR?
`
`·9· · A.· · I would say a POSA by looking at
`
`10· ·the May article would have concluded
`
`11· ·that restriction enzymes were used based
`
`12· ·on the article, based on previous
`
`13· ·publications and would not have
`
`14· ·considered PCR.
`
`15· · Q.· · That wasn't my question.· My
`
`16· ·question was, a person of ordinary skill
`
`17· ·in the art at the relevant time frame,
`
`18· ·2001 and before, would understand that
`
`19· ·when cloning or subcloning segments of
`
`20· ·DNA, that, that one could use
`
`21· ·restriction enzymes and one could also
`
`22· ·use PCR, correct?
`
`23· · A.· · That depends on the context.· So
`
`24· ·in this particular context here a POSA
`
`25· ·would have assumed that restriction
`
`SKI Exhibit 2055
`Page 21 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·fragments were used.
`
`·3· · Q.· · It's not your testimony, is it,
`
`·4· ·that in this context a person of
`
`·5· ·ordinary skill in the art would be
`
`·6· ·unable to use PCR to assemble the LCR,
`
`·7· ·correct?
`
`·8· · A.· · That is not correct.
`
`·9· · Q.· · It's your testimony that a person
`
`10· ·of ordinary skill in the art would not
`
`11· ·be able to use PCR to assemble an LCR?
`
`12· · A.· · I don't think I mentioned PCR at
`
`13· ·all in my declaration.
`
`14· · Q.· · That's true, you did not.· And
`
`15· ·you didn't mention PCR because the
`
`16· ·claims themselves cite restriction
`
`17· ·fragments, right?
`
`18· · · · · MR. YUSEM:· Object to form.
`
`19· · A.· · I didn't mention the PCR because
`
`20· ·there was no reason for me to assume
`
`21· ·that a POSA would have thought that
`
`22· ·these fragments were generated by PCR.
`
`23· ·I was a POSA at that time.· I worked in
`
`24· ·this area.· I would not have assumed
`
`25· ·that they would use PCR to generate an
`
`SKI Exhibit 2055
`Page 22 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·LCR fragment.
`
`·3· · Q.· · A person of ordinary skill in the
`
`·4· ·art could use PCR when doing that,
`
`·5· ·right?· It's within their capability?
`
`·6· · A.· · A POSA would be able to use PCR
`
`·7· ·but a POSA that would have looked at the
`
`·8· ·May article would have assumed, based on
`
`·9· ·the information given in this paper and
`
`10· ·the information that was cited by this
`
`11· ·paper, other papers that used cloning to
`
`12· ·generate an LCR fragment that expresses
`
`13· ·ß-globin in all these previous
`
`14· ·publications restriction enzymes were
`
`15· ·used, as far as I know.
`
`16· · Q.· · All right.· But you answered a
`
`17· ·different question so I just want to, I
`
`18· ·just want to ask you the question again
`
`19· ·and get the answer to the question.
`
`20· · · · · A person of ordinary skill in the
`
`21· ·art in the time frame of mid 2001 and
`
`22· ·before could use PCR to assemble the
`
`23· ·LCR, correct?
`
`24· · A.· · But a POSA looking --
`
`25· · Q.· · Please, please answer -- please
`
`SKI Exhibit 2055
`Page 23 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·answer that question.· We'll get to
`
`·3· ·talking about the article later, I
`
`·4· ·promise.· But please answer the question
`
`·5· ·that I'm asking you.
`
`·6· · A.· · Yes, and I would say a POSA --
`
`·7· ·what a POSA in attempting to generate an
`
`·8· ·LCR fragment would have preferred using
`
`·9· ·restriction enzymes given all the
`
`10· ·information at that time.
`
`11· · Q.· · Okay.
`
`12· · A.· · A POSA would not have considered
`
`13· ·using PCR, putting these elements
`
`14· ·together to drive ß-globin expression.
`
`15· · Q.· · But that is not the question I
`
`16· ·asked.· A person of ordinary skill in
`
`17· ·the art had the knowledge, the ability
`
`18· ·to use PCR in this context, right?
`
`19· · A.· · A person of ordinary skill in the
`
`20· ·art would have looked in this paper
`
`21· ·knowing that PCR exists.· A POSA would
`
`22· ·know that PCR exists.· But in this
`
`23· ·particular situation, given the
`
`24· ·information provided in this paper and
`
`25· ·given previous publications, would have
`
`SKI Exhibit 2055
`Page 24 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·assumed that these fragments were
`
`·3· ·generated by restriction enzyme
`
`·4· ·digestion.
`
`·5· · Q.· · Dr. --
`
`·6· · A.· · And another reason for that,
`
`·7· ·another reason for that --
`
`·8· · Q.· · -- that is answering a different
`
`·9· ·question.· Please --
`
`10· · · · · MR. YUSEM:· Sorry, Dr. Bungert,
`
`11· · ·were you done with your answer?
`
`12· · · · · THE WITNESS:· Excuse me?
`
`13· · · · · MR. YUSEM:· Were you done with
`
`14· · ·your answer?
`
`15· · · · · THE WITNESS:· I would just add
`
`16· · ·one, one more thing.
`
`17· · Q.· · Please do.
`
`18· · A.· · And that is if you look at the
`
`19· ·information given in the May article,
`
`20· ·you notice that they not only provide
`
`21· ·the position of the core hypersensitive
`
`22· ·sites, they also provide the position of
`
`23· ·the fragments that were used, correct?
`
`24· · Q.· · I'm not here to answer questions,
`
`25· ·I'm sorry.
`
`SKI Exhibit 2055
`Page 25 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · Okay, yes.· So you can see that
`
`·3· ·they not only provide the position of
`
`·4· ·the hypersensitive sites, but also the
`
`·5· ·position of these fragments.· And the
`
`·6· ·way these fragments are positioned, for
`
`·7· ·example, the idea was using larger
`
`·8· ·fragments, the idea was having the core
`
`·9· ·and the franking sequences, you notice
`
`10· ·that some of these were very close to
`
`11· ·the course.· That would be another
`
`12· ·reason for a POSA to assume that they
`
`13· ·were not generated by PCR, in my
`
`14· ·opinion.
`
`15· · Q.· · Couldn't you just make PCR
`
`16· ·primers in those positions?
`
`17· · A.· · But to me it wouldn't make sense.
`
`18· ·To me it would not have made sense.
`
`19· · Q.· · Okay.· To you --
`
`20· · A.· · If I would have generated an LCR
`
`21· ·with the core and flanking sequences, I
`
`22· ·would not have chosen a PCR fragment
`
`23· ·that originates at the hypersensitive
`
`24· ·site.
`
`25· · Q.· · Okay.· A person of ordinary skill
`
`SKI Exhibit 2055
`Page 26 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·in the art was aware that of the
`
`·3· ·techniques that could be used to clone
`
`·4· ·or subclone fragments, PCR was one of
`
`·5· ·them, right?
`
`·6· · A.· · I think a POSA would have
`
`·7· ·excluded using PCR.· He would be aware
`
`·8· ·of it, but he would have excluded using
`
`·9· ·PCR to generate an LCR fragment that
`
`10· ·drives high ß-globin expression.
`
`11· · Q.· · Let's forget about LCRs for a
`
`12· ·second and just talk about cloning
`
`13· ·generally.· Can we do that?
`
`14· · A.· · Yes.
`
`15· · Q.· · Okay.· In the time frame of mid
`
`16· ·2001 and before, a person of ordinary
`
`17· ·skill in the art was aware that in
`
`18· ·addition to the use of restriction
`
`19· ·endonucleases PCR was an option for
`
`20· ·cloning and subcloning DNA fragments,
`
`21· ·right?
`
`22· · A.· · I would still maintain that even
`
`23· ·if someone would not work on the LCR but
`
`24· ·in another topic, the preferentially
`
`25· ·technique to assemble regulatory
`
`SKI Exhibit 2055
`Page 27 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·elements into an LCR like configuration
`
`·3· ·would be restriction enzyme digestion.
`
`·4· · Q.· · Not the question I asked.· But
`
`·5· ·I'll try one more time.
`
`·6· · · · · It's true, isn't it, that a
`
`·7· ·person of ordinary skill in the art in
`
`·8· ·this time frame, mid 2001, would be
`
`·9· ·aware that one could use restriction
`
`10· ·endonucleases to subclone or clone
`
`11· ·fragments of DNA and one could use PCR
`
`12· ·to subclone or clone fragments of DNA,
`
`13· ·right?
`
`14· · A.· · A POSA would know about the
`
`15· ·different technologies available and
`
`16· ·when assembling the regulatory element
`
`17· ·like the LCR would have used restriction
`
`18· ·enzymes.
`
`19· · Q.· · Let's talk about --
`
`20· · A.· · That's my opinion.
`
`21· · Q.· · Okay.· Let's talk about
`
`22· ·restriction enzymes and how techniques
`
`23· ·for using those to assemble multiple
`
`24· ·pieces like in the LCR.· Maybe you
`
`25· ·could, maybe you can explain that.· So
`
`SKI Exhibit 2055
`Page 28 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·if you're looking to clone multiple
`
`·3· ·fragments of DNA using restriction
`
`·4· ·endonucleases, how would you do it, just
`
`·5· ·generally?· Please, I'm not talking
`
`·6· ·about looking at the May article right
`
`·7· ·now.· If you want to clone multiple
`
`·8· ·fragments together, what are the
`
`·9· ·techniques you would use?
`
`10· · A.· · First, I would look at the
`
`11· ·database.· I would look for restriction
`
`12· ·map.· I would look for where these
`
`13· ·restriction enzymes are relative to the
`
`14· ·elements that I would like to subclone.
`
`15· ·And then I would design a strategy to
`
`16· ·use particular restriction fragments to
`
`17· ·clone these together.
`
`18· · Q.· · Okay.· And how would you develop
`
`19· ·that strategy?· What would that entail?
`
`20· · A.· · Well, I would look at restriction
`
`21· ·enzymes that encompass the regulatory
`
`22· ·element and any other additional
`
`23· ·elements I would like to include.· Then
`
`24· ·I would isolate these fragments and
`
`25· ·would ligate them together in expression
`
`SKI Exhibit 2055
`Page 29 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·vector or whatever context.
`
`·3· · Q.· · Would you have any particular
`
`·4· ·strategy when it comes to selecting the
`
`·5· ·restriction enzymes?
`
`·6· · A.· · I would use restriction enzymes
`
`·7· ·that do not cut too often preferably.
`
`·8· ·That is not always possible.· I would
`
`·9· ·prefer enzymes that would not ---that
`
`10· ·cut outside the recognition sequence. I
`
`11· ·would prefer using enzymes that have
`
`12· ·been used before in cloning techniques.
`
`13· ·I would use enzymes that are available
`
`14· ·at that time, whatever.
`
`15· · · · · So all these things I would take
`
`16· ·into consideration.
`
`17· · Q.· · And you'd consider, for example,
`
`18· ·whether the enzymes that you're using
`
`19· ·had compatible sticky ends?
`
`20· · A.· · Yes, sometimes I would consider
`
`21· ·this.· I would maybe prefer enzymes that
`
`22· ·don't have sticky ends but sometimes
`
`23· ·that is not possible.
`
`24· · Q.· · So you would just look for blunt
`
`25· ·ended?
`
`SKI Exhibit 2055
`Page 30 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · A.· · I would look for both.
`
`·3· · Q.· · Okay, both?
`
`·4· · A.· · Mm-hmm.
`
`·5· · Q.· · Okay.· And if some are blunt and
`
`·6· ·some are sticky, then what do you do?
`
`·7· · A.· · If it's at the same position, I
`
`·8· ·would use the sticky end.· If it's, if
`
`·9· ·it's at a different position and I need
`
`10· ·to include other elements that the
`
`11· ·blunt-end restriction enzyme includes, I
`
`12· ·would use the blunt end.
`
`13· · Q.· · Okay.· And when you're putting
`
`14· ·the fragments together, if you need to
`
`15· ·assemble these fragments and some have
`
`16· ·sticky ends that aren't compatible or
`
`17· ·some have, one has a blunt end and one
`
`18· ·has a sticky end, what's the strategy
`
`19· ·then?
`
`20· · A.· · Then you make the other one
`
`21· ·blunt.
`
`22· · Q.· · All right.· So you just blunt
`
`23· ·both?
`
`24· · A.· · Mm-hmm.
`
`25· · Q.· · Okay.· And you're okay with
`
`SKI Exhibit 2055
`Page 31 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·losing whatever sequence you're
`
`·3· ·blunting, right?
`
`·4· · A.· · It depends.· It depends on the
`
`·5· ·context.
`
`·6· · Q.· · Okay.· But one option is, one
`
`·7· ·option is to generate blunt ends and do
`
`·8· ·blunt-end cloning?
`
`·9· · A.· · If there is no other possibility,
`
`10· ·then that's what you do in a particular
`
`11· ·context.
`
`12· · Q.· · Okay.· In the situations where
`
`13· ·you would use PCR to clone, because you
`
`14· ·said you have, right, you used PCR to
`
`15· ·clone and subclone fragments, what are
`
`16· ·the advantages of using PCR in those
`
`17· ·situations over restriction
`
`18· ·endonucleases?
`
`19· · A.· · So if you can either restriction
`
`20· ·enzymes or PCR, I would use restriction
`
`21· ·enzymes.
`
`22· · Q.· · Okay.· But that's not what I
`
`23· ·asked.
`
`24· · A.· · The advantage of restriction
`
`25· ·enzymes is that you don't introduce
`
`SKI Exhibit 2055
`Page 32 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· ·mutations.· If you use PCR there's a
`
`·3· ·possibility that you introduce
`
`·4· ·mutations.· Nucleotide changes.
`
`·5· · Q.· · I understand.· I understand.
`
`·6· ·Because, because the -- during, during
`
`·7· ·the PCR itself?
`
`·8· · A.· · Yes.
`
`·9· · Q.· · Okay.· In these situations where
`
`10· ·you did use PCR, are you aware of any
`
`11· ·advantages to using PCR or is there just
`
`12· ·disadvantages like that mutations?
`
`13· · A.· · So ideally you use restriction
`
`14· ·enzymes to preserve the nucleotide
`
`15· ·sequences.· I understand we talked about
`
`16· ·blunt end where you have maybe small
`
`17· ·changes but with PCR you will maybe
`
`18· ·disturb that more.· So having that's
`
`19· ·another advantage of restriction
`
`20· ·enzymes.
`
`21· · · · · Another advantage, as I said, is
`
`22· ·you avoid generating mutations by PCR
`
`23· ·and, yeah, so in this particular
`
`24· ·situation the POSA would prefer using
`
`25· ·restriction enzymes.
`
`SKI Exhibit 2055
`Page 33 of 254
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`·1· · · · · · ·J. BUNGERT - 7.10.23
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`·2· · Q.· · When using PCR, are you aware of
`
`·3· ·any strategies that a person of ordinary
`
`·4· ·skill in the art in the relevant time
`
`·5· ·frame would use in order to address the
`
`·6· ·possibility of mutations arising during
`
`·7· ·the PCR?
`
`·8· · A.· · Technology --
`
`·9· · Q.· · Sure.
`
`10· · A.· · -- it is --· yes, I mean you
`
`11· ·could sequence.
`
`12· · Q.· · Sure, you could sequence.
`
`13· · A.· · But if you use restriction
`
`14· ·enzymes you don't have to really
`
`15· ·sequence because if you clone it out you
`
`16· ·don't introduce any mutations or
`
`17· ·nucleotide changes.
`
`18· · Q.· · Are you using --
`
`19· · A.· · So that is an additional step you
`
`20· ·w
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