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`BLUEBIRD EXHIBIT 1004
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`BLUEBIRD EXHIBIT 1004
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`
`THERAPEUTIC HEMOGLOBIN SYNTHESIS IN BETA-THALASSEMIC MICE
`
`EXPRESSING LENTTVIRUS-ENCODED HUM AN BETA-GLOBIN
`
`A Thesis
`
`Presented to the Faculty of the Graduate School
`
`of Cornell University
`
`in Partial Fulfillm ent of the Requirements for the D egree of
`
`Doctor of Philosophy
`
`by
`
`C had M. May
`
`M ay 2001
`
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`
`
`UMI Number 3020182
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`___
`
`®UMI
`
`UMI Microform 3020182
`Copyright 2001 by Bell & Howell Information and Learning Company.
`All rights reserved. This microform edition is protected against
`unauthorized copying under Title 17, United States Code.
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`
`© 2001 Chad M. May
`
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`
`
`THERAPEUTIC HEMOGLOBIN SYNTHESIS IN BETA-THALASSEMIC MICE
`
`EXPRESSING LENTTVIRUS-ENCODED H U M A N BETA-GLOBIN
`
`Chad M. May, Ph.D.
`
`Cornell University 2001
`
`The stable introduction of a functional globin gene in autologous
`
`hem atopoietic stem cells is a potentially pow erful approach to treat (3-
`
`thalassemia. The challenge facing this approach is to stably express high levels
`
`of the hum an p-globin gene in an erythroid-specific, regulated, an d sustained
`
`fashion. Low level expression, position effects and transcriptional silencing have
`
`ham pered until n o w the effectiveness of viral transduction of the h u m an P-globin
`
`gene linked to m inim al regulatory sequences. I show here that the use of
`
`recom binant lentiviruses enables efficient transfer and faithful integration of the
`
`h u m an P-globin gene together w ith large segm ents (3.2 kb) of its locus control
`
`region (LCR). Studies com paring a vector containing a 3.2 kb LCR, term ed TNS9,
`
`to one w ith a m inim al 1.0 kb LCR, term ed RNS1, dem onstrate both a higher
`
`m ean level of h u m an P-globin expression by TNS9, and a higher fraction of cells
`
`expressing h u m an P-globin following vector integration at random sites. In long
`
`term studies in recipient mice engrafted w ith TNS9-transduced bone m arrow
`
`cells, production of lentivirus-encoded P-globin is substantially augm ented,
`
`ow ing to an increase in both the level of globin expression, as show n b y RNA
`
`analysis, and the fraction of red cells expressing h u m an pA, as dem onstrated by
`
`im m unostaining. M urine oCj: hum an pA2 tetram ers account for u p to 13% of total
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`Page 6 of 150
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`
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`hem oglobin in m ature red cells in norm al long-term bone m arrow chim eras.
`
`M ost im portantly, higher levels are obtained in P-thalassemic mice, ranging from
`
`17 to 24% fifteen w eeks after transplant, resulting in a substantial increase in
`
`hem oglobin concentration and hem atocrit levels, w ith a concom itant reduction in
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`reticulocyte counts. Red cell m orphology (anisocytosis and poikilocytosis) is
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`m arkedly corrected. Therapeutic benefits are stable up to 40 w eeks resulting in
`
`reduced extram edullary erythropoiesis in the spleen. My findings dem onstrate
`
`th at high-level, tissue-specific gene expression can be achieved in the progeny of
`
`unselected genetically m odified stem cells. Therefore, a genetic ap p roach could
`
`be successful in treating P-thalassemia. Furtherm ore, these findings provide a
`
`parad ig m for stem cell therapy requiring regulated expression of a tissue-specific
`
`transgene in the progeny of genetically m odified stem cells.
`
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`Page 7 of 150
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`BIOGRAPHICAL SKETCH
`
`The author w as b o m and raised in Fridley, M innesota. H e attended the
`
`U niversity of M innesota in M inneapolis, and received a Bachelors of Science
`
`D egree from the D epartm ent Genetics an d Cell Biology. As a n u n d erg rad u ate
`
`his first experience in scientific research w as w orking as a stu d en t technician in
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`the laboratory of Dr. Bruce R. Blazar, u n d er the supervision of Dr. Paul O rchard,
`
`in the D epartm ent of Pediatric O ncology/H em atology. U pon g raduation he
`
`joined the laboratory of Dr. R. Scott M clvor in the Institute of H u m an G enetics at
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`the U niversity of M innesota, as a senior technician. Dr. M clvor fostered his
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`interest in the area of gene therapy a n d provided him w ith the su p p o rt an d
`
`intellectual guidance necessary to be accepted into to the Im m unology Ph.D.
`
`P rogram a t the C ornell University G raduate School of M edical Sciences, in N ew
`
`York City in 1995. The authors Ph.D. w ork w as com pleted u n d er the guidance
`
`a n d m entorship of Dr. M ichel Sadelain in the D epartm ent of H u m an Genetics at
`
`M em orial Sloan-Kettering Cancer C enter. Dr. Sadelain w as an exceptional
`
`teacher an d continued to foster the author's enthusiasm for scientific research.
`
`Ill
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`Page 8 of 150
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`DEDICATION
`
`Dedicated to Deborah,
`
`m y parents Judith and William,
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`and m y brother Guy.
`
`iv
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`ACKNOWLEDGEMENTS
`
`There are a num ber of people I w ould like to thank w ho have given m e
`
`trem endous support and encouragem ent in m y p u rsu it of this goal. M y first
`
`scientific m entor, Dr. Scott M clvor, gave me the opportunity an d confidence to
`
`seek this degree. M y Ph.D. m entor, Dr. Michel Sadelain, fostered m y
`
`independence as a researcher, m otivated me to perform a t the highest level, an d
`
`shaped m e into a scientist. I w o u ld also like to thank the m em bers m y
`
`comm ittee, Dr. Lucio Luzzatto and Dr. Malcolm M oore, for their guidance and
`
`support throughout this process.
`
`The day to day rigors o f research were m ade m uch easier and enjoyable
`
`by the m em bers of the lab p a st and present. Thanks to those w ho have w orked
`
`together w ith m e on this project, Stefano Rivella a great scientist and friend,
`
`John Callegari, and Cui-w en Tan. A special thanks to M ichael G ong and Renier
`
`Brentjens for your friendship and your often unsolicited advice. Thanks to
`
`Jean-Baptiste Latouche for being Jean-Baptiste Latouche, an d to Millie G allardo
`
`for years of support and assistance. A generous thanks to the p ast and present
`
`m em bers of the lab; Clay Lyddane, Stefan Schnell, John M aher, Steve D ando,
`
`Jakob D upont, G ertrude A tkinson, Eduardo Nerioe, Tanya H enderson, and
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`Laurie Lindsley. All of you have helped me along the way.
`
`I w ould also like to th an k m y parents, m y family, an d m y friends for
`
`their unw avering encouragem ent a n d support. Lastly, I th an k D eborah for
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`m aking this long road a w onderful journey.
`
`v
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`
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`TABLE OF CONTENTS
`
`BIOGRAPHICAL SKETCH
`
`DEDICATION
`
`ACKNOWLEDGEMENTS
`
`TABLE OF CONTENTS
`
`LIST OF TABLES
`
`LIST OF FIGURES
`
`Chapter 1. Introduction
`
`P-Thalassemia
`
`M olecular Pathology
`
`Effects of Excess a-G lobin
`
`Clinical Form s
`
`M edical T herapy
`
`Bone M arrow T ransplantation
`
`W hy gene therapy?
`
`Analysis of H u m an P-Globin Gene Expression
`
`in Transgenic M ice
`
`Retroviral G ene Transfer of the H um an P-globin
`
`Gene: The Early Years
`
`vi
`
`iii
`
`iv
`
`v
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`vi
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`x
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`xi
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`1
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`1
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`3
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`6
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`7
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`10
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`11
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`11
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`13
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`13
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`Identifying and D efining the H um an P-Globin
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`Locus C ontrol Region
`
`The H um an P-Globin Locus C ontrol Region:
`
`Function
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`D N ase I H ypersensitive Sites
`
`5' H ypersensitive Site 1
`
`5' H ypersensitive Site 2
`
`5' H ypersensitive Site 3
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`5' H ypersensitive Site 4
`
`5' H ypersensitive Site 5
`
`H ypersensitive Site Synergy
`
`Proxim al P-Globin E nhancer Elem ents
`
`The H u m an p-Globin Prom oter
`
`R etroviral Gene Transfer of the H um an P-Globin Gene:
`
`1990 to the Present
`
`Lentiviral Vectors
`
`A nim al M odels of Disease
`
`C h ap ter 2. M aterials a n d M ethods
`
`Vector Construction
`
`Vector Production
`
`S outhern Blot Procedure
`
`vii
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`14
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`16
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`18
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`19
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`20
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`24
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`26
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`27
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`28
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`29
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`30
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`33
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`36
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`38
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`40
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`40
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`40
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`41
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`N orthern Blot Analysis
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`H eLa Cell Infection and Viral Transm ission Assay
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`MEL Cell Infection and Screening
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`Q uantification of H um an P-Globin RNA
`
`Bone M arrow T ransplantation
`
`Protein A nalyses
`
`Red Cell M orphology and Hem atologic Studies
`
`Statistics
`
`Chapter 3. G oals and H ypothesis
`
`Goals
`
`H ypothesis
`
`Chapter 4. R esults
`
`Vector C onstruction and A nalysis
`
`Vector Construction
`
`Vector Analysis
`
`In Vitro Expression Analyses
`
`Expression Levels and Tissue Specificity
`
`M onitoring Expression at Individual Integration Sites
`
`In Vivo Studies in Bone M aroow Chim eras
`
`Long-term Vector C opy N um ber and RNA Expression A nalysis
`
`viii
`
`43
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`44
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`44
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`45
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`46
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`48
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`49
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`49
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`50
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`50
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`51
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`53
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`53
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`53
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`55
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`57
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`57
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`58
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`63
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`63
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`Analysis of Vector Silencing O ver Time
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`H um an P-Globin Protein Expression Analyses
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`H em atopoietic Stem Cell Transduction Analyses
`
`In Vivo Expression Analysis in a M ouse M odel of 3-Thalassemia
`
`Short-Term Therapeutic Effect
`
`Long-Term Therapeutic Benefit
`
`C h ap ter 5. D iscussion
`
`C h ap ter 6. A ppendix
`
`C h ap ter 7. References
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`65
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`67
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`72
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`76
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`76
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`79
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`85
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`98
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`107
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`ix
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`LIST OF TABLES
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`Table 1
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`M ouse oc^human (3A2 tetram ers
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`in the p eripheral blood of long-term
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`bone m arro w chim eras.
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`Table 2
`
`Secondary bo n e m arrow transplants.
`
`69
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`74
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`LIST OF FIGURES
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`C hapter 1
`
`Figure 1.01
`
`The distribution of regions w ith high incidences of (3-thalassemia.
`
`Figure 1.02
`
`The hum an (3-like globin gene cluster.
`
`Figure 1.03
`
`M utations in the p-globin gene that lead to P-thalassemia.
`
`Figure 1.04
`
`Effects of excess production of free a-globin chains.
`
`Figure 1.05
`
`H olocom plex m odel of LCR activation.
`
`Figure 1.06
`
`The hum an P-globin prom oter.
`
`xi
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`2
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`4
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`5
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`8-9
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`17
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`31
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`Figure 1.07
`
`HIV-1 derived lentiviral vector system.
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`C hapter 4
`
`Figure 4.01
`
`Structure of recom binant lentiviral vectors.
`
`Figure 4.02
`
`Stability of recom binant lentiviral vectors.
`
`Figure 4.03
`
`Prim er extension to m easure expression in vivo.
`
`Figure 4,04
`
`Increased lineage- and stage-specific hum an p-globin expression in MEL
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`cells transduced w ith the TNS9 vector.
`
`Figure 4.05
`
`Increased proportion of clones expressing detectable h u m an P-globin
`
`w ith the TNS9 vector.
`
`xii
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`37
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`54
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`56
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`59
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`60
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`62
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`
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`Figure 4.06
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`Long-term stability of vector copy num ber and h u m an P-globin RNA
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`levels in th e peripheral blood of bone m arrow chim eras.
`
`Figure 4.07
`
`64
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`66
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`Long-term stability of hum an P-globin RN A expression in TNS9 transduced
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`bone m arrow chim eras w ith no apparent vector silencing.
`
`Figure 4.08
`
`Fraction of hem oglobin tetram ers in the peripheral blood of long-term
`
`bone m arrow chim eras that contain h u m an P-globin polypeptide.
`
`Figure 4.09
`
`Fraction of h u m an P-globin positive m ature red cells in long-term bone
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`m arrow chim eras.
`
`Figure 4.10
`
`Integration of lentiviral vectors into hem atopoietic stem cells.
`
`Figure 4.11
`
`Fraction of hem oglobin tetram ers in the peripheral blood of secondary
`
`transplant recipients that contain hum an P-globin polypeptide.
`
`xni
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`68
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`70-71
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`73
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`75
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`Figure 4.12
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`C orrection of anisocytosis an d poikilocytosis in bone m arrow chim eras
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`reconstituted w ith TN S9-transduced H bbth3/+ bone m arrow cells.
`
`Figure 4.13
`
`A m elioration of hem atological param eters in bone m arrow chim eras
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`reconstituted w ith TN S9-transduced Hbbth3/+ bone m arrow cells.
`
`Figure 4.14
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`77
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`78
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`80
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`Fraction of hem oglobin tetram ers that contain hum an P-globin polypeptide
`
`in m ice transplanted w ith p° heterozygote bone m arrow cells.
`
`Figure 4.15
`
`82
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`Long-term am elioration of hem atological param eters in bone m arrow chim eras
`
`reconstituted w ith TN S9-transduced H bbth3/+ bone m arrow cells.
`
`Figure 4.16
`
`Long-term analyses of hum an P-globin protein expression.
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`Figure 4.17
`
`Reduced splenom egaly in H bbth3/+long-term bone m arrow chim eras
`
`transduced w ith the TNS9 lentiviral vector.
`
`xiv
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`83
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`84
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`
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`C h ap ter 6
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`Figure 6.01
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`Progress in viral encoded P-globin expression in vivo.
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`Figure 6.02
`
`H em oglobin production in norm al an d H bb th3/+ heterozygous chimeras.
`
`Figure 6.03
`
`Potential im provem ents to the TNS9 lentiviral vector.
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`87
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`90
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`94
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`xv
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`
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`Chapter 1
`
`Introduction
`
`1.1
`
`^-Thalassemia
`
`The thalassem ias, a form of congenital anem ia ranging from severe to
`
`m ild, are recognized as the m ost com m on m onogenic disorders in hum ans
`
`(1). D escribed first in 1925 b y Thom as Cooley an d Pearl Lee (2), they are a
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`heterogeneous group of inherited red blood cell diseases resulting in
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`defective synthesis and subsequent im balanced a - o r p-globin chain
`
`production. Expression of the adult a-an d P-globin genes is lim ited to
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`erythroid cells. U nder norm al conditions they are balanced so that equal
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`am ounts of the tw o polypeptides are available to assem ble into the
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`hem oglobin heterotetram ers (c^: P 2). U nder severe conditions the faulty
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`p roduction of hem oglobin in a - or P-thalassemia gives rise to red blood cells
`
`th at cannot hold an adequate supply of oxygen to m aintain norm al life
`
`processes. P-thalassemia, am ong the m ost intensively stu d ied disorders in the
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`w orld, is m ost prevalent throughout countries of the M editerranean, N orth
`
`Africa, the M iddle East, the Indian subcontinent, an d Southeast Asia (Fig.
`
`1.01). Estim ates of gene frequencies range from 3 to 10% in som e areas (3). It
`
`has been suggested th at the high frequency of p-thalassem ia observed is a
`
`result of negative selection, providing heterozygous carriers of the m utant
`
`allele protection against P. falciparum m alaria (4).
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`1
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`Page 21 of 150
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`Figure 1.01. The distribution of regions w ith high incidences of 0-
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`thalassem ia th roughout the M editerranean, Africa, the M iddle East, the
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`Indian subcontinent, an d Southeast Asia. (A dapted from W eatherall, D. J.
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`1994 [3])
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`
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`3
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`Molecular Pathology
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`The h u m an (3-like globin genes are a cluster of five functioning globin
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`genes, in the order of 5'-e-yG-j A-S-(3-3'. These genes lie in a region that is
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`approxim ately 60 kb and are located on the short arm of hum an chrom osom e
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`11 (Fig. 1.02). The genes are expressed distinctly in a developm ental-, stage-,
`
`and tissue-specific manner. The embryonic e-globin gene is expressed in the
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`yolk sac, the fetal Y2- and yA -globin genes are expressed prim arily in the fetal
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`liver, an d the adult 5- and P-globin genes are expressed prim arily in the bone
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`m arrow . M utations in this gene family result in a broad range of genetic
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`diseases. P-thalassemia results from upw ards of 200 different m utations
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`w ithin this P-like globin gene cluster (5), affecting P-globin transcription,
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`m R N A processing, translation, and post-translational stability of the p-globin
`
`polypeptide chain. While m ost are single base pair nucleotide substitutions
`
`or deletions in prom oter, splice-site, CAP-site, and polyadenylation
`
`sequences (Fig. 1.03), some of the m ost inform ative results regarding
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`regulation of the locus arise from large deletions w ithin a 20 kb region
`
`upstream of the globin genes, term ed the locus control region (LCR [Fig.
`
`1.03}). N aturally occurring deletions (Fig. 1.03 [6]) and translocations (7) th at
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`rem ove the locus control region b u t leave the globin genes intact results in
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`the failure to transcriptionally activate the cis-linked globin genes in
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`erythroid cells.
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`4
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`Human
`Chromosome 11
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`H ■ i i M
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`t
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`LCR
`54321
`
`5'HS:
`
`e
`
`8
`
`3'HS
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`L
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`Hispanic deletion
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`10 kb
`
`Figure 1.02. The h um an (3-like globin gene cluster. The h u m an globin genes are
`
`located on the short arm of chrom osom e 11 and are represented b y black boxes.
`
`The location of the 5' D N ase I hypersensitive sites are indicated b y num bered
`
`black arrows. The bracket indicates the extent of the naturally occurring
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`'hispanic' deletion w ithin the loci (6).
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`Page 24 of 150
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`
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`Deletions
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`/M
`/M
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`JM
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`Introntf Exon 1 Intron
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`Exon 2
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`Intron
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`1
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`Exon 3
`
`Intron
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`I
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`5
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`3 '
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`Other Mutations
`»■ Mutations affecting initiation of transcription
`^ Mutations affecting splicing of
`RNA from introns
`| Polyadenylation signal mutation
`a n Mutations affecting initiation of translation
`■ Nonsense mutation
`+ Frame shift mutation
`
`F igure 1.03. The n orm al structure of the P-globin gene and the location of
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`know n m utations th at result in the loss of P-globin expression a n d that
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`subsequently leads to p-thalassemia. The consequence of the deletions is to
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`either abolish the production of P-globin chains or reduce the level of
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`expression. The different m utations that act in this w ay m ay interfere w ith the
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`action of the P-globin gene at the transcriptional level, in the processing of the
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`p rim ary transcript, in the translation of P-globin m essenger RNA, or in the
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`post-translational stability of the P-globin gene product. (A dapted from
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`W eatherall, D. J. 1994 [3])
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`6
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`The Effects of Excess oc-Globin
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`In severe form s of p-thalassemia im balanced a:|3 globin chain synthesis
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`results in excess a-chains, w hich have a resulting deleterious effects on
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`erythroid cell m aturation (Gt phase cell cycle arrest [8]) and survival
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`(apoptosis in the late erythroblast stage [9,10]), the degree of w hich is
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`directly related to the level of a-chain excess. U npaired a-chains are unable
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`to form productive hem oglobin tetram ers and precipitate w ithin developing
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`and m atu re erythrocytes form ing inclusion bodies. These intracellular
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`precipitates signal their rem oval from the hem atopoietic com partm ent, a
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`process th a t dam ages the cells, and shortens their life span, resulting in
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`destruction of erythroid precursors and subsequent ineffective erythropoiesis
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`(10). In addition, a-globin chain degradation products dam age red blood cell
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`m em branes leading to hemolysis and contributing to the anem ia (11,12).
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`Anem ia leads to ineffective erythropoiesis resulting in up to a 10-15-fold
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`expansion of the erythroid bone m arrow com partm ent (13). The anem ia is
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`further w orsened b y hem odilution, caused by the shunting of blood through
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`the expanded m arrow , and by splenom egaly resulting from entrapm ent of
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`abnorm al red cells in the spleen (3). A nother consequence of the m arrow
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`expansion is seen in the characteristic deform ities of the skull and face, as
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`well as osteopenia (a condition of diffuse decrease in bone density) and in
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`defects in bone m ineralization (14,15). Bone changes can lead to repeated
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`Page 26 of 150
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`fractures. The effects o f excess a-globin chain production is sum m arized in
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`7
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`Figure 1.04.
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`Clinical Forms
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`A result of its considerable molecular heterogeneity, the clinical spectrum of
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`P-thalassem ia produced by different m utations is extensive, from transfusion
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`dependency beginning in infancy, to a m ild condition requiring little if any
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`m edical intervention (3). Thalassemia m ajor is the severe form of the disease,
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`presenting w ith transfusion-dependent anem ia, generally in the first year of
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`life. Thalassemia interm edia is the less severe form of the disease, and is
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`highly variable. H eterozygous carriers of m utations are often asym ptom atic
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`and are silent carriers of the P-thalassemic trait. Presenting no obvious
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`phenotype, they are com m only identified by hem atological screening in
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`populations w ith high m utation frequencies. Those that require m edical
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`m anagem ent are the interm ediate and severe forms, w hich generally result
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`from hom ozygous or com pound heterozygous carriers of m u tan t p-globin
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`alleles. In addition, a num ber of com bined genetics factors m ay am eliorate
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`the severity of the disease, such as the persistence of fetal y-globin expression
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`(16), or a n inherited deficit in a-globin production (17). Both of w hich
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`contribute to reducing the a - to non a-globin chain im balance.
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`Page 27 of 150
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`8
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`Figure 1.04. Effects of excess production of free a-globin chains. Excess
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`u n b o u n d a-globin chains precipitate in red-cell precursors causing defective
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`m aturation and ineffective erythropoiesis. Hemolysis, d ue to th e presence of
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`inclusions in the red cells an d dam age to their m em branes by a-globin chains
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`an d their degradation products, also contributes to the anem ia. A nem ia
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`stim ulates the synthesis of erythropoietin, leading to an intense proliferation
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`of the ineffective m arrow . The anem ia is further exacerbated b y
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`splenom egaly resulting from entrapm ent of abnorm al red cells in the spleen.
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`Bone m arrow expansion also results in characteristic deform ities of the skull
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`an d face, severe osteopenia, an d increased iron absorption.
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`Page 28 of 150
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`9
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`Normal Situation:
`Balanced Synthesis
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`p-Thalssemia:
`Excess (unpaired) a-Globin
`
`a
`
`a
`
`1
`
`Adult Hemoglobin
`
`a
`
`I a
`
`i
`
`Normal Erythropoiesis
`
`a
`
`a
`
`I
`
`Anemia\
`/
`
`Splenom egaly
`
`Ineffective
`Erythropoiesis
`
`Erythroid Marrow
`Expansion
`
`/
`\
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`Iron Toxicity
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`Figure 1.04 (continued) Effects of excess production of free a-globin chains.
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`Page 29 of 150
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`10
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`Medical Therapy
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`Early on hem atologists recognized that blood transfusions w ere
`
`necessary to relieve the severity of the anemia, and to suppress the
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`detrim ental consequences of ineffective erythropoiesis. A stan d ard red blood
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`cell transfusion regim en produces significant increases in survival and
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`quality of life for suffers. Despite progress brought by transfusion therapy,
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`progressive transfusion-related iron accum ulation and enhanced
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`gastrointestinal absorption results in iron overload and iron related tissue
`
`dam age. The excess iro n overw helm s the iron carrying capacity of transferrin
`
`(a protein that carries iron in the bloodstream ), resulting in the em ergence of
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`toxic non-transferrin b o u n d iron (18). W ithout iron chelation th erap y a lethal
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`accum ulation of iron occurs by the second decade of life w ith num erous
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`complications, including delayed or absent sexual developm ent, diabetes
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`m ellitus, cirrhosis, hepatocellular carcinoma, cardiac arythm ia, an d
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`congestive heart failure (3). Together, transfusion and iron chelation therapy
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`has contributed to im proved survival of those effected.
`
`Regardless the decreased m orbidity and increased survival in patients
`
`associated w ith im provem ents in transfusion and iron chelation regim ens
`
`during the past two decades, iron chelation is cumbersome an d tim e
`
`consum ing, and adherence to nightly chelation is difficult for m an y patients.
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`The uncomfortable adm inistration, high cost, and complications contribute to
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`variable patient adherence. A num ber of patients opt to discontinue iron
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`Page 30 of 150
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`11
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`chelation because of physical a n d /o r em otional traum a associated w ith the
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`frequent prolonged subcutaneous infusion (Dr. G iardina personal
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`comm unication). Also, patients continue to suffer transfusional iron overload
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`due to inadequate iro n chelation therapy. Furtherm ore, red blood
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`transfusion therapy is associated w ith alloim m unization (19) an d exposure to
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`blood borne viral agents including HIV, hepatitis B, C, an d CM V (20).
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`Bone Marrow Transplantation
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`Bone m arrow transplantation from HLA-identical donors has been
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`perform ed w ith success in children and adults (21,22). In fact, a stu d y of 826
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`(3-thalassemic patients, age 1 through 35, w ho received H LA -m atched bone
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`m arrow transplants, dem onstrated a 72% disease-free survival rate overall
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`(22). W hile a cure w ith bone m arrow transplantation is a p ro v en option, it is
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`available to only a select few that have a com patible sibling or parental donor
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`(approxim ately 40% of thalassemic children [22]). Pending a significant
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`im provem ent in the outcom e of bone m arrow transplantation from a
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`m ism atched or unrelated donor, the pursuit of an alternative treatm ent for
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`patients w ith severe form s of (3-thalassemia is w arranted.
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`Why gene therapy?
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`The accessibility of bone m arrow and the success of allogeneic bone
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`12
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`m arrow transplantation validate the introduction of corrected hem atopoietic
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`stem cells as a m eans to treat p-thalassem ia. The stable gene transfer of a
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`functional P-globin gene in the autologous hem atopoietic stem cells of a
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`p atien t is therefore a potentially p ow erful approach to perm anently correct
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`the disease. It does no t require the identification of a m atching d o n o r an d the
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`p atien t does not ru n the risk of graft versus host disease. W hile h azard s are
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`still inherent in an y bone m arrow transplantation strategy, a gene th erap y
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`approach rem ains a prom ising option.
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`Because P-thalassemia is associated w ith the loss of function of a single
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`gene, it w as one of the earliest disease candidates considered for treatm ent by
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`gene therapy. H ow ever, the prospect of gene therapy using the P-globin gene
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`h as proven to be exceedingly difficult to attain d ue to m any factors. O ne
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`crucial factor is the high levels of P-globin gene expression required for this
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`approach to be therapeutically relevant. Analyses of patients indicate th at
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`synthesis of 15-20% of the norm al am o u n t of P-globin w ould be required for a
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`substantial clinical im provem ent (23), an d therefore it is anticipated th at
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`expression from the transferred h u m an P-globin gene w ould need to reach
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`this sam e level. Furtherm ore, p-globin expression m u st be tightly regulated,
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`confined to a specific lineage and developm ental stage, p-globin expression in
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`other tissues could otherwise be toxic. Thus, for this approach to becom e
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`feasible in hum ans a num ber of hurd les have to be overcome, including
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`efficient transfer of the P-globin gene into prim itive hem atopoietic progenitor
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`Page 32 of 150
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`13
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`cells and identification of sequences required for stable and hig h level P-
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`globin expression.
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`Analysis of Human ft-Globin Gene Expression in Transgenic Mice
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`Early studies in transgenic mice carrying the hum an P-globin gene
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`along w ith varying am ounts of 5' (4300,815,360,122,45 bp) and 3' (1700 and
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`1900 bp) flanking genom ic DNA indicated that a stage specific pattern of P-
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`globin expression can be conferred by proxim al acting regulatory elem ents
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`closely linked to the h u m an P-globin gene (24,25). H ow ever, they
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`dem onstrated th at th e level of hum an P-globin expression per transgene copy
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`w as significantly low er than that observed from the endogenous gene, less
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`th an 1% th at of the m urine P-globin gene. In addition, transgene expression
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`w as n ot observed in all mice, even though they carried one or m ultiple copies
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`o f the transgene. This indicated a susceptibility to chrom atin-m ediated
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`transcriptional repression, a consequence determ ined by the site of transgene
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`integration, called position effects (24,25).
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`Retroviral Gene Transfer of the Human f3-Globin Gene: The Early Years
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`N ot long after the transgenic studies, reports describing the successful
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`transduction of m urine hematopoietic stem cells w ith recom binant
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`oncoretroviral vectors based on a m urine leukem ia virus (MLV), encoding
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`Page 33 of 150
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`14
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`the h u m an p-globin gene w ere published (26,2 7 ,28). U pon transplantation
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`an d engraftm ent of the transduced hem atopoietic progenitor cells, h u m an P-
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`globin w as expressed at detectable levels in an erythroid-spedfic m anner. But
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`like their transgenic counterparts, the levels of expression derived from the
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`retroviral vector encoded h um an p-globin w as m uch too low for a potential
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`therapeutic effect (<1% of endogenous m ouse P-g
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