S
`EXHIBIT NO-.
`i-r2"M C
`
`RECOMBINANT DNA ADVISORY COMMITTEE
`
`Minutes of Meeting
`
`February 10, 2003
`
`U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
`Public Health Service
`National Institutes of Health
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`Minutes of the RecornL ant DNA Advisory Committee - 2/10/03
`
`CONTENTS
`
`I.
`
`Welcome and Opening Remarks: Review of Purpose and Objectives.................................................
`
`if.
`
`Case Preseitabon
`
`and Molecular Analyses
`
`.........................................................................................
`
`A. RAC Discussion
`
`.............................................................................................................................
`
`B.
`
`Public Cummert
`
`.......................
`
`........... ...........................................................................................
`
`III.
`
`Pediatric T-AL.L ...... ......................
`
`.... ..........................................
`
`.........................................................
`
`.
`
`A. RAC DincLission ..............................................................................................................................
`
`B.
`
`Public Comment..............................................................................................................................
`
`U.
`
`Retroviruses
`
`and Insertional Mutagenesis
`
`............................................................................................
`
`A. RAC Discussion
`
`..............................................................................................................................
`
`B.
`
`Public Commert..
`
`... ..................
`
`.......................................................................................................
`
`V.
`
`LMO Genes
`
`in Leukemogenosis
`
`.......................................................
`
`.......................
`
`..................
`
`...........
`
`VI,
`
`Defective Oynokine Signaling
`
`in X-Linked SCID ....................................................................................
`
`A. RAC Di,;cussion
`
`..............................................................................................................................
`
`VII.
`
`Points To Consider................................................................................................................................
`
`VIII. FDA Actions
`
`in Response
`
`to the Second SAE......................................................................................
`
`IX.
`
`Public Questions ard Comments..........................................................................................................
`
`X.
`
`Into Hematopoietic Cells: Points To Consider.............................
`
`Retroviral-Mediated Gene Transfer
`Letter F,-cm the RAC to Investigators
`
`A.
`
`in January 2003 .................................................................
`
`B. RAC [)n:ussiol:
`
`.............................................................................................................................
`
`C. Cornmin.€a Motion 1 .......................................................................................................................
`
`XI.
`
`RAC Recornrnenda'
`
`ions ........................................................................................................................
`
`A. Comm tton Motion 2.........................................................................................................................
`
`XII. Closing Remarks and Adjournment
`
`......................................................................................................
`
`Attachment
`
`I.
`
`Committee Rostar ...... ... .......... ..................................................................................
`
`A-I-i
`
`AttachmentII.
`
`/,ttondees
`
`..... .............
`
`.............................................................................................
`
`A-Il-i
`
`Attachment
`
`III.
`
`Abbreviations and Acronyms
`
`...................................................................................A-Ill-i
`
`Note: The latest
`
`I Inman Gene Transfer Protocol List can be found at the Office of Biotechnology
`
`Activities' Web site at <wvjw4.od.nih.gov/oba/rac/protocol.pdf
`
`>.
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`Minutes of the Recombinant DNA Advisory Committee - 2/10/03
`
`U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
`NATIONAL INSTITUTES OF HEALTH
`RECOMBINANT DNA ADVISORY COMMITTEE
`MINUTES OF MEETING'
`
`Development of T-Cell Acute Lymphoblastic Leukemia (T-ALL) in Two Subjects
`in a Gene Transfer Clinical Trial for X-SCID
`
`February 10, 2003
`
`The Recombinant DNA Advisory Committee (RAC) was convened for its 89th meeting at 8:30 am on
`February 10, 2003, at the National Institutes of Health (NIH), Building 31C, Conference Room 6,
`Bethesda, MD. Dr. Theodore Friedmann (Chair) presided. In accordance with Public I ew 92-463, the
`meeting was open to the public from 8:30 a.m. until 5:00 pm. The following individuals were present for
`all or part of this meeting.
`
`Committee Members
`
`W. Emmett Barkley, Howard Hughes Medical Institute
`Martha C. Bohn, Northwestern University Medical School
`Baruch A. Brody, Baylor College of Medicine (via teleconference)
`James F. Childress, University of Virginia
`Neal A. DeLuca, University of Pittsburgh
`Theodore Friedmann, University of California, San Diego
`Thomas D. Gelehrter, University of Michigan Medical School
`Larry G. Johnson, University of North Carolina, Chapel Hill
`Philip R. Johnson, Jr., Columbus Children's Hospital
`Terry Kwan, TK Associates
`Bernard Lo, University of California, San Francisco
`Madison Powers, Georgetown University
`David Sidransky, Johns Hopkins University School of Medicine (via teleconference)
`Robert D. Simari, Mayo Clinic and Foundation
`Diane W. Wara, University of California, San Francisco
`
`Office of Biotechnology Activities (OBA) Director
`Amy P. Patterson, Office of the Director (OD), NIH
`
`Executive Secretary
`Stephen M. Rose, 00
`
`Ad Hoc Reviewers/Speakers
`Peter D. Aplan, National Cancer Institute (NCI), NIH
`Alain Fischer, Hôpital Necker-Enfants Malades (via teleconference)
`Dale E. Hammerschmidt, University of Minnesota
`David M. Harlan, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH,
`Biological Response Modifiers Advisory Committee, Food and Drug Administration
`Carl H. June, University of Pennsylvania
`Ilan R. Kirsch, National Cancer Institute, NIH
`Warren J. Leonard, National Heart, Lung, and Blood Institute (NHLBI), NIH
`Harry Malech, National Institute of Allergy and Infectious Diseases (NIAD). NIH
`Gregory H. Reaman, Children's National Medical Center
`Naomi Rosenberg, Tufts University School of Medicine
`
`1 The Recombinant DNA Advisory Committee is advisory to the National Institutes of Health (NIH', aid its
`recommendations should not be considered as final or accepted. The Office of B otechno ogy Activities should be
`consulted for NIH policy on specific issues.
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`Minutes of tie
`
`lit ant DNA Advisory Committee - 2/10/03
`
`Daniel R. Salomon The Scripps Rasearch Institute, Biological Response Modifiers Advisory Committee,
`ood and Drug AdrLunistrtion
`Ricardo U. Sorei tn, Louisiana State University
`Christof von Kalle University of Cincinnati/Cincinnati Children's Hospital Research Foundation
`
`Nonvoting Agency/Liaison Representative
`Sally L. McCamrnon, U.S. Department of Health and Human Services
`
`NIH Staff Members
`Rima Adler, NHLFfl
`Richard A. Anderson, Nat onal Institute of General Medical Sciences, NIH
`Catherine [3arnarc,, OD
`Sebastian [3renre Laboratory of F lost Defenses (LHD), Division of Intramural Research, NIAID
`Sandra H. Brdge., NIAID
`J. Scott Cairns, Ni/ ID
`Fabio Candotti, National Human Genome Research Institute (NHGRI), NIH
`Sarah Carr, OD
`,Jan Casadei, NCI
`Javier Chinen, NHGRI
`Uimook Choi, NIA!)
`Elaine Cclher, Naticnal Center for Research Resources (NCRR), NIH
`Utpal Dave, Nd
`Camilla Day, Cente. for SSentific Review, NIH
`Jordana Deleon LI
`Suksee Deravn BAlD
`Cindy Dunbar, NI 1, UI
`Kelly T. Fenningtcn OD
`<agnew Gebreyenus, NICDK
`Suzanne Goodwin, OD
`Kailash Gupta, NIAID
`Laurie Harris, OD
`Beverly Hay, NHGF-U
`Anthony F-Iayward NCRR
`i>eirnan Hematti NI ILBI
`Valerie H jrt, Offco of the General CoL.nsel, NIH
`Robert Jarnbou, CL
`Llizaeth Kang, NILDK
`Richard Knazek I JCRR
`Ken Kurarnoto, Nr IL 131
`Ching Juh Lai, NI;, [)
`Bob Lanman, OD
`André Larochelle, NHLBI
`Susan Leibenhaut, FDA
`Ke Liu, NI-1
`Allan LocK, Nationa Institute of Ch.Id Health and Human Development, NIH
`Sharon A Mavrouk kis, NCI
`Cherjl McDonali (JD
`U. Rita Misra NOl
`Richard A. Morgan NCI
`Alan Moshelt, Nato ial Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH
`Marina O'Reilly, Cl',
`Roland A Owens, HDDK
`Alexander Rako's:y, OD
`Nicholas estifo, NCI
`Minerva Rojo, Focarty International Center, NIH
`Steven A Rosenbeig NCI
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`Minutes of the Recombinant DNA Advisory Committee - 2110/03
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`Gene Rosenthal, OD
`Michael Schmidt, National Institute of Dental and Craniofacial Research, NIH
`Thomas Shih, OD
`Allan Shipp, OD
`Lana Skirboll, OD
`Danilo A. Tagle, National Institute of Neurological Disorders and Stroke, NIH
`Tina Thomas, OD
`John Tisdale, NIDDK
`Chuck Trimmer, OD
`Giselle White, OD
`Kouhei Yamashita, LHD
`
`Others
`A list of the 157 other attendees appears in Attachment II.
`
`Welcome and Opening Remarks: Review of Purpose and Objectives/Drs. F'ir'dmann,
`Patterson, and Rose
`
`Dr. Friedmann, RAC Chair, called the meeting to order at 8:30 am. on February 10, 2C
`t. Notice of this
`meeting under the NIH Guidelines for Research Involving Recornb1rart DNA Mo'ecu!c NIH Guidelines)
`was published in the Federal Register on February 5, 2003 (68 FR 590h) The RAC meeting focused on
`issues surrounding the development of T-cell acute lymphoblastic leukemia (T-AL) in ',vlo research
`participants in a gene transfer clinical trial for X-linked severe combiied immune deficiency (SCID) being
`conducted in France and the RAC's recommendations as a result of these two events.
`
`Dr. Patterson explained that the NIH convened this special meeting of the RAC to review and discuss a
`second case of leukemia in a gene transfer clinical trial for X-SCID. The goal of the meeting is to work
`ts enrolled in
`toward a common understanding of the two events and what they mean for the particip
`this trial and their families, participants in other similar trials, and potential participants "c ritemplating
`enrollment in future gene transfer trials. On behalf of the Office of Biotechnology Actvi us (OBA) and the
`RAC, Dr. Patterson expressed gratitude to Dr. Alain Fischer and his colleagues for thei penness in
`sharing information about the results. Their integrity in this regard has nllowed the infcrr:tion to be of
`benefit not only to participants in their trial but also to all individuals enrclled in similar trils throughout the
`world. The X-SCID trial has otherwise had very promising outcomes demonstrating efficacy in the
`children enrolled. Because there have been significant benefits in the triaL it is critically important to reach
`an understanding of the significance of the adverse events so that the potential risks anc: tee potential
`benefits of such trials can be determined.
`
`Dr. Friedmann stated that this is a watershed event for the field of human gene transfer the first-ever
`combination of a major clinical therapeutic response in a gene transfer study with an ohious treatment-
`related SAE, a situation indicative of the maturing of the field. He indicated that the RAC s overarching
`goal is to work through public interactions with investigators to help design scientifcally useful studies, to
`identify potential risks, and to incorporate safeguards to maximize the safety of human jene transfer
`research. Today's meeting, he noted, is the result of cooperation among groups nvotvccl with SOlD or
`similar studies and oversight agencies, all of whom feel an urgent need to determine the mechanisms
`responsible for the leukemia, to improve the technology, and to devise more effective 6nd safe
`approaches to future studies. Dr. Friedmann also expressed gratitude to Dr. Fischer and his colleagues
`for the quality of the studies they have done to understand the events; for the rapid, open, and complete
`information sharing they undertook; and for their foresight in archiving samples, which has made possible
`the characterization of this SAE.
`
`Case Presentation and Molecular Analyses/Dr. von Kalle (and Dr. Fischer via teleconference)
`
`Dr. von Kalle summarized the characteristics of X-linked SOlD as an inm.unodef:ciency caused by a
`genetic deficiency of the gamma chain that is common to a family of interleukin (1L) receptors The
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`Minutes of the Recornni ant DNA Advisor! Committee - 2/10/03
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`deficiency blocks T cell differentiation, likely at a common lymphoid progenitor or early T-cell progenitor
`stage, so that patients do not develop functional T-cell or B-cell immunity. Currently, available therapy
`includes replacement of the immune function with allotranspianted bone marrow, either from a human
`leukocyte antigen (I ILA) identical family donor or from a haploidentical family donor. The success rate of
`stem-cell transplantation has increased over the years, but there are still significant problems, especially
`for patients who have existing infcctons and other serious clinical problems. The option of transplantation
`s also limited o aenor availability.
`
`The gene transfer fnr X-linked SCID uses a retroviral vector expressing the human common gamma chain
`(gamma-C) compl:rilentarj deoxyribonucleic acid (cDNA), which integrates randomly into chromosomes.
`Expression of the ti .insgene reconstitutes gamma-C chain expression, which results in the restoration of
`cell surface expresnion of a family of interleukin receptors necessary for T cell development. It has been
`shown in animal models, as well as in the current clinical trial, that gamma-c reconstitution results in
`functional T cell development, as evidenced by generation of a polyclonal T-cell receptor repertoire. Bone
`marrow derived stam cells (e.g., 0D34+) transduced with the retroviral vectors were reinfused into the
`participants without any chemotherapy or other conditioning treatment. About 2 to 3 months after
`reinfusion, T-cef recovery occurred. Because gamma-c expression is required for T cell growth, nearly
`100 percent of the i cells in the peripheral blood contained the vector, and there was about one copy of
`the retrcviral vect a per cell. The transduced cells had normal functional characteristics, and the thymus
`showed the presence of recent thymic immigrants (indicating normal thymic function).
`
`From a total of 11 participants in the Paris trial, 9 have experienced immune reconstitution. Most
`:articipants in the F ranch trial are young (ranging in age from 1 month to 11 months), many had
`preexisting infections and other ciinical problems symptomatic of immune deficiency, and all exhibited no
`evidence of endocenous gamma-C activity.
`
`Participant 4 received gore transfer at I month of age and showed development of T- and B-cell immune
`responses including a protective immune response to a varicella-zoster virus (VZV) infection that
`occurred 30 month: after gene transfer. At month 34, an abnormal number of a gamma-delta TCR+ T
`cell was present ad rest arch participant 4 was considered to have developed an unusual form of acute
`1-ALL The resot.r Jr participant has a family history of childhood cancer, which could have been a
`contributing factoi Research participant 5 received gene transfer at 3 months of age and showed normal
`I'-cell counts up t
`rcnth 31. HoAever, at month 34, research participant 5 presented with
`splenomegaly, enia:qed mediastinum, and anemia. Further analysis led to a diagnosis of acute T-ALL,
`but in this case the a appeared tc be three T cell clones due to the presence of three independent alpha-
`beta TCR rearranqarnents.
`
`Molecular analyses by LAM-PCR c:etected insertion of the vector in the reverse orientation into the first
`nitron of trio LMO-2 gene n research participant 4. In research participant 5, the integration occurred 3
`Kb upstream of L170-2. The leuk3rnic clones of that participant also showed evidence of a trisomy 10 and
`a Sib- TAL fusion trcn5cript. To calculate the likelihood of insertional activation of Imo-2 in this type of
`experiment, Dr. Jun Kalle considered tne size of the human genome (3 x 109 kb) and length of sequence
`.round IMO-2 thct has been obsareo to activate LMO-2 by translocation (3 x 1 kb), and estimated that
`in 100,000 ntecja tons events would randomly occur in the region of activation. Depending on the dose
`of transduced cell; Jiat ranged from 2 x 10' cellsto 150 x 106 cells, participants may have received 500-
`1000 cells with inr
`rations in the region of activation of LMO-2. There may be other contributing factors
`to leukemia such a, the elfect of the gamma-c transgene on T cell growth and differentiation, clonal
`seeding efficiency and immune tolerance.
`
`Insertion site pattern analysis was done for all participants, and five insertions were detected in the LMO-
`2 region. A second non-leukemic clone was found in research participant 4 with the vector inserted 40
`kb upstrearn of i.11fl-2. TNO insertions were foLnd in the LMO-2 region of a third research participant
`who has no signs ci clonal expans on. More than 100 retroviral vector insertion sites have been
`nequenced from ( 134+ srem cells obtained from research participants, but so far, no others have been
`'lapped to region; )f the :iost genome that would raise concern.
`
`4
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`Minutes of the Recombinant DNA Advisory Committee - 2/10/03
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`
`Additional investigations are concentrating on the mechanisms of LMO-2 dysregulatior und analysis of
`LMO-2 transcripts, retrospective clone tracing, profile of gene expression in 1-ALL clon;, mechanisms
`for screening defects in DNA repair, and additional comprehensive anal jsis to map all i ilegration sites in
`CD34+ stem cells from treated research participants.
`
`Drs. Fischer and von Kalle stated that research participants 4 and 5 are both doing weJi at present, after
`receiving chemotherapy for the T-ALL.
`
`A. RAC Discussion
`
`Dr. DeLuca asked whether, using archival material, it is possible to know the LM-2 trorscriptional
`activity prior to transduction. Dr. von Kalle responded that while LMO-2 is not active in mature T cells,
`LMO-2 is transcribed in the CD34+ cells that were transduced.
`
`Dr. Salomon noted that research participant 4's insertion was in the reverse transcriptional orientation and
`asked about the orientation of the vector in the cells of participant 5. Dr 'ion Kale explaned that the
`insertion is just upstream of the distal promoter and is in forward transcriptional criemalon.
`
`Dr. Kirsch noted that a SIL-TAL rearrangement had been discovered in research participant 5 and
`wondered whether such a rearrangement was found in any other study oarticipants. Di. Fischer
`responded that this rearrangement has been found only in the cells of research participant 5 so far;
`samples are still being analyzed for some of the other participants.
`
`Dr. Noguchi asked whether the researchers believed that preexisting infections in thes' nhildren might
`have had a role in causing the SAE. Dr. Fischer indicated that research participants 4 ;tnc 5 were the
`youngest and also the healthiest at the time of gene transfer. Research participant 4 was fully
`asymptomatic at the age of 1 month, when he was treated, and developed no infections other than
`chicken pox 3 years later. Research participant 5 had skin lesions related to the presei ice of maternal T
`cells, commonly seen in some patients with SOlD, but he did not have any identViable nèctious diseases
`at the time of experimental intervention. All of the other nine research participanis did Have infections,
`some of them serious.
`
`Dr. Sorensen asked for a definition of "residual disease" in this case. Dr. Fischer explaired that 1
`leukemic cell in 1,000, detected in the bone marrow, would probably be considered an indication to
`perform an allogenic hematopoietic stem-cell transplantation, provided an appropriate Honor could be
`lower in
`found. If the number of remaining cells carrying the insertion in the LMO2 site is mud
`frequency, it will be difficult to determine how to proceed, but the participants will be mcr'tored regularly.
`Research participant 4 still has some retrovirus transduced T cell with irsertions in the i.M.J2 site
`following chemotherapy, but these cells lack the chromosomal transiocation seen in tb' T-ALL cells.
`
`Dr. DeLuca asked how the researchers plan to proceed regarding 'participant A' (so dosignated to
`maintain this person's confidentiality) who also had insertions in the LMQ-2 region. Dr. Fischer explained
`that he and his colleagues are monitoring the participants every 3 months even in the absence of clinical
`symptoms. Included in the monitoring are careful immunological investigations of 1-cell ohenotype and
`evaluation of LMO-2 expression by reverse transcriptase polymerase chain reaction (RT-PCR).
`
`Dr. Friedmann asked about the immunological status of research participants 4 and 5 after
`chemotherapy. Dr. Fischer responded that research participant 5 only recently received chemotherapy,
`so no results are known at this time. However, research participant 4 does have gamma c+ transduced I
`cells in his blood, and his T-cell counts are similar to those expectec in a child with prin 'ary leukemia who
`received chemotherapy treatment. Research participant 4 s T-cell reconstitution and his capacity to
`produce I cells are fairly modest. Dr. Sorensen added that research participant 5, who reports to his
`clinic, also has T cells, continues to produce immunoglobulins, and is doing well clinically.
`
`B. Comments and Questions from the Public
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`Minutes of tne RecomL inant DNA Advisory Committee - 2/10/03
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`Dr. Richard P. Junqhans, Beth lsiael Deaconess Medical Center/Harvard Medical School, asked about
`the possibility of a n:cor.dary event bang involved in the development of this SAE. Dr. von Kalle stated
`that it is unikely tl't every insertional event in the proximity of LMO-2 would induce tumorigenesis
`because this type c SAE would have been seen earlier and in other research participants. Studies by Dr
`Baum showed that over e<pression of LMO-2 in transgenic mice was associated with T-cell expansion
`and eventual development of leukemia in a significant number of animals. Further research, however, is
`needed to determine the effect of expression promoted by the viral LTR and the effect of gamma-c
`expression
`
`Pediatric TALL/Dr. Reaman
`
`Dr. Reaman provided a synopsis of T-ALL in childhood, in the context of ALL. ALL is the most common
`pediatric malignancy, representing 25 percent of all cancer diagnoses in individuals younger than 21
`years of age. TIre oak ae of incidence is between 2 and 6 years, and more than 3,000 cases are
`ciagnosad per yo.:rr in the United States, with an incidence rate of 3.7 per 100,000 children. ALL is more
`common in boys th.n in girls and in whites than in African Americans.
`
`There are three major classifications for ALL: immunophenotypic, morphologic, and molecular genetic.
`lmmunophenotypi: classification defines the cell of origin. B cell precursors account for nearly 85% of
`childhood ALL while T cel ALL accounts for approximately 15 percent of ALL cases.
`
`Regarding prognostic factors in childhood ALL, the age at time of diagnosis is extremely important in
`ALL—children YOL nqer than 10 years old but older than 1 year old have a much better outcome in
`response to therapp than Jo adolescents and young adults. Patients presenting with white counts lower
`than 10,030 have an extremely favorable prognosis, and individuals with white counts lower than 50,000,
`currently used as a defirit on of a standard risk group of children with ALL, have a better outcome in
`response to thera;. than Jo those with higher white counts. Early response to therapy is defined as
`either the disappc.a once of peripheral blood blasts during a 7-day pretreatment with steroids or the
`r.resence of less tic n 5 percent leukemic lymphoblasts in the bone marrow following 1 week of multi-
`induction chemotherapy Early response can also be defined by the absence of minimal residual disease
`efter the induction phase of chemotherapy. The other important prognostic factor in childhood ALL is the
`extent of .eukernic burden at the time of diagnosis—children who have mediastinal mass or massive
`hepatospienomegoly have a less favorable outcome. Immunophenotype by itself currently is not
`considered a prognostic factor, taken independently. Cytogenetic nonrandom structural chromosomal
`abnormalities ie g , Philac.elphia chromosome) are another significant indicator of poor prognosis in
`childhocd ALL; Solie suggest little chance of survival without hematopoietic stem-cell transplantation.
`
`I'-ALL more commonly occurs in males and at age greater than 15 years. Many of these children present
`with mediastinal no as, hyoerleukocytosis, white cell counts exceeding 100,000/mm3, massive
`iepatosplenomega:, an increased incidence of central nervous system (CNS) involvement, and an
`increased incidence of CNS relapse. While cytogenetic analysis of T-ALL cells has determined that 25%
`of patients have rai'rnal karyotypos, chromosomal deletions or translocations are frequently observed,
`including a number of trarslocatiors involving T cell receptor genes.
`
`Successful treatment of individue s viti ALL has greatly improved in recent decades. Currently, 75
`Percent to 80 percent of ciildren are cured, defined as long-term complete remission off therapy for
`periods exceedinq P year,,. Mcci of this is the result of the development of risk based specific
`treatments using rognos'ic factors such as age, white count, and early response to therapy. Treatment
`consists of an inbur tion phase involving multiple chemotherapeutic agents, followed by the consolidation
`Phase directed at J.e CNS, and then maintenance therapy for 2-3 years using antimetabolite based
`chemotherapy
`
`A. RAC Discussien
`
`Dr. Noguchi suggecced that Dr. Reaman's comprehensive review seems to indicate that these two cases
`cf T-ALL are atyni:tl comoared to naturally occurring cases of the disease, and he asked what that might
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`Minutes of the Recombinant DNA Advisory Committee - 2/10/03
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`mean for the evaluation of these SAEs. Dr. Reaman responded that, aihough the clinical presentation of
`patients 4 and 5 is atypical, the immunophenotype of these individuals is not particularly atypical.
`
`Dr. von Kalle asked Dr. Reaman for advice on how to evaluate minimal residual disease Dr. Reaman
`stated that evaluating minimal residual disease in childhood ALL is an area of significa it debate within the
`specialty and that no definitive consensus has been reached. Yet preliminary studies here and in Europe
`suggest the following definition: presence of more than 1 ALL cell in 100,000 cells usin I PCIR or more
`than 1 in 10,000 cells using multiparameter flow. The outcome for patients with rnnim I residual disease
`is less positive than for those who show no evidence of it.
`
`Dr. Sorensen asked about residual cells with the insertion that are not malignant and how to predict which
`cell may become proliferative again. Dr. Reaman responded determining which of the remaining residual
`cells are truly clonogenic and will continue to divide is a main challenge of this field.
`
`B. Public Comment
`
`Joanne C. Delenick requested an estimate of the total number of children that have be, 1) placed in
`remission in all studies in the past 45 years. Dr. Reaman responded that, in the oast 1) Ic 12 years more
`than 15,000 patients have been treated in multicenter cooperative trials for childhood I ukemia.
`
`IV. Retroviruses and Insertional Mutagenesis/Dr. N. Rosenberg
`
`Dr. N. Rosenberg reviewed retroviral integration, and the effects on gene expression. There are four
`common mechanisms by which insertional mutagenesis occurs with retioviruses (1) promoter insertion,
`(2) enhancer insertion, which is the most common mechanism and is orentation and positon
`independent, (3) leader insertion, and (4) terminator insertion. Consequences of insertional activation
`include upregulation, deregulation, or loss of expression, or altered or truicated gene rrcucts.
`
`Changes in gene expression may be quite common as result of retroviral insertion, but most such
`changes are of no consequence to the cell. More rarely, the cell acquires a selectve g'cwth or survival
`advantage such as when the proviral integration affects expression of genes for transcr ptbn factors,
`chromatin remodeling proteins, growth factors or apoptosis proteins Assuming that th:ro are 20J proto-
`in 15,000 exposures could result
`oncogenes in a cell and a target size of approximately 1 kilobase, then
`in a tumorigenic event. Model animal systems show that a single integration is usually associated with
`other changes leading to transformation, but the integration event is the primary rate-limiting step.
`
`While the general consensus in the field is that retroviral integration is largely rardom, there are
`conflicting results from different studies. A study of HIV integration by Bushman's group ndicated that a
`disproportionate number of integrations occurred within transcription units, particularly ictive transcription
`units. A different study done with an avian retrovirus indicated that nontran scribe, d regie 's were
`preferred. More experimentation is needed to determine whether these cifferences arc clue to differences
`in the viruses or in methods of analysis. In addition to integration, insertional muagencsis can be
`affected by other viral factors, such as replication, and LTR sequences that deteimine issue specificity
`and pathogenecity. Host factors, such as genetic background, age, arc target cell typo ohio infuence
`tumorigenecity.
`
`Dr. N. Rosenberg noted that integration of the provirus is an inevitable consequence of rotrovirus-based
`gene transfer. Researchers have yet to learn how to target integration and insertional mutagenesis and
`proto-oncogene activation are likely to occur with high numbers of integrations. To minimize the potential
`for insertional mutagenesis, Dr. N. Rosenberg suggested keeping the number of transc:Iuced cells to the
`minimum needed, avoiding the use of LTR enhancer and promoter sequences from hic: hly leukemogenic
`viruses, using a cell-type specific promoter (rather than an LTR), developing better srnll animal models
`to test viral vectors, and considering alternative vector designs such as entivirusbasec ectors or
`inclusion of insulator elements.
`
`UPenn Ex. 2050
`Miltenyi v. UPenn
`IPR2022-00855
`Page 9
`
`

`

`Minutes of Vie Re co ni i a nt DN\ Advisory' Committee —2110/03
`
`A. RAC Discussion
`
`Dr. L. Johnson asked whether there is evidence to show that the use of self-inactivating (SIN) vectors
`decreases the frequency of tumorigenesis in various models. Dr. N. Rosenberg stated that, where the
`potential for replication exists, these vectors do decrease tumorigenesis frequency. The SIN modification
`may be more useful in lentiviral vectors than in vectors derived from MLV since the SIN modification leads
`to problems generafing sLificient vector titer levels.
`
`In response to Cr. Kirsch's question, Dr. N. Rosenberg suggested that the SAEs in the X-SCID study
`cccurred as a result of a random .integration followed by selection—expression of a gene in a particularly
`susceptible cell type
`
`Dr. Wara asked abut the effect of host age at time of infection on integration patterns. Dr. N. Rosenberg
`described a stucy clone comparing avian retrovirus infection in chick embryos to newborn chicks. The
`study investigated the integrations found in tumors that arose after the infection. The tumors had
`integrations into d,fierent genes depending whether the infection occurred in embryos or newborn chicks.
`However, the study did nct analyzc global integration patterns.
`
`Dr. von Kalle asked wheff er evidertce