Hematology, 2000, Vol. 4, pp. 437-455
`Reprints available direct! y from the publishcr
`Photocopying permitted by liccnsc on! y
`
`© 2000 OPA (Overseas Publishers Association) N.V.
`Published by liœnse under
`the Harwood Academie Publishers imprint,
`part of The Gordon and Breach Publishing Group.
`Printed in Malaysia.
`
`Erythropoiesis
`
`Review Article
`Slow and Steady Wins The Race? Progress in the
`Development of Vectors for Gene Therapy of
`{3-Thalassemia and Sickle Cell Disease
`
`JANE M. MCINERNEYa, MICHAEL J. NEMETHb and CHRISTOPHER H. LOWREYa,b,•
`
`"Department of Medicine, Dartmouth Medical Sclwol, Hanover, NH, USA; bDepartment of Pharmacologtj and Toxicologtj, Dartmouth
`Medical Sclwol, Hanover, NH, USA
`
`(Received 9 June 1999; In final form 23 June 1999)
`
`The cloning of the human .8-globin genes more than
`20 years ago led to predictions that ,8-thalassemia and
`sickle cell disease would be among the first mono(cid:173)
`genie diseases to be successfully treated by gene
`replacement therapy. However, despite the world(cid:173)
`wide enrollment of more than 3,000 patients in
`approved gene transfer protocols, none have involved
`therapy for these diseases. This has been due to sev(cid:173)
`era! technical hurdles that need to be overcome before
`gene replacement therapy for ,8-thalassemia and
`sickle cell disease can become practical. These prob(cid:173)
`lems include inefficient transduction of hematopoi(cid:173)
`etic stem cells and an inability to achieve consistent,
`long-term, high-level expression of transferred ,8-like
`globin genes with current gene transfer vectors. In
`this review we highlight the relationships between
`understanding the fondamental mechanisms of .8-
`globin gene locus function and basic vector biology
`and the development of strategies for .8-globin gene
`replacement therapy. Des pite slow initial progress in
`this field, recent advances in a variety of critical areas
`provide hope that clinical trials may not be far away.
`
`Keywords: Gene therapy, ,8-thalassemia, sickle cell disease,
`,8-globin, retrovirus, lentivirus, chroma tin structure
`
`INTRODUCTION
`
`Mutations of the a- and ,B-globin gene loci com(cid:173)
`prise the most prevalent group of inherited single
`gene diseases. It has been estimated that seven
`percent of the world's population are heterozy(cid:173)
`gous carriers of clinically significant mutations
`affecting the expression of the globin genes or
`the function of their gene products [1]. Mutations
`within the genes of the .B-globin locus give rise
`to ,B-thalassemia and the ,B-hemoglobinopathies,
`including sickle cell disease (SCD). While the
`high prevalence of these mutations appears to
`have arisen because they lessen the morbidity
`of malarial infection in the heterozygous state
`(reviewed in [2]), they continue to result in
`early death and chronic debilitating disease in
`the homozygous state [1]. The underlying patho(cid:173)
`physiologies of these diseases in volve destruction
`of erythrocytes due to abnormal polymerization
`
`*Corresponding au thor. Fax: (603) 650-1129. E-mail: c.lowrey@dartmouth.edu
`
`437
`
`SKI Exhibit 2057
`Page 1 of 19
`
`

`

`438
`
`J. M. MCINERNEY et al.
`
`of hemoglobin molecules containing the sickle (3-
`chain (sickle cell disease) and o:-chain imbalance
`((3-thalassemia) [3,4]. Because erythrocytes are
`derived from the hematopoietic stem cell (HSC),
`transfer of a functional human adult ((3) or fetal (-y)
`globin gene to a patient's stem cells could cure or
`lessen the severity of these diseases. -y-globin gene
`transfer is particularly attractive for seo since the
`-y-globin protein does not participate in the sick(cid:173)
`ling reaction and thus confers greater protection
`than the (3-globin protein [5]. A major rationale
`for using (3-thalassemia and seo as models for
`the development of gene therapy strategies is
`that less than full reconstitution of normal (3-
`or -y-globin gene expression is likely to pro(cid:173)
`duce significant clinical benefit as co-inheritance
`of mutations that confer hereditary persistence
`of fetal hemoglobin (HPFH) have an attenuated
`phenotype [6-8]. A second rationale for (3-globin
`replacement therapy is that significant clinical
`benefits have been achieved using the pharma(cid:173)
`cologie agents 5-azacytidine, hydroxyurea and
`sodium butyrate as inducers of -y-globin synthe(cid:173)
`sis (reviewed in [9,10]). Gene replacement therapy
`offers significant advantages over these phar(cid:173)
`macologie strategies in that a single treatment
`could permanently restore (3-globin (or -y-globin)
`protein production and both known and poten(cid:173)
`tial side-effects of long-term drug therapy could
`be avoided [9]. Successful gene transfer therapy
`would also offer a potentially safer,less expensive
`and more widely applicable curative therapy than
`allogeneic bone marrow transplantation [11,12].
`The (3-globin genes were among the first human
`genes to be cloned [13-15]. This led to specula(cid:173)
`tion that genetic diseases involving these genes
`would be among the first diseases to be treated
`using gene therapy. However, ten years after the
`approval of the first human gene transfer proto(cid:173)
`col [16] and after the treatment of more than three
`thousand patients worldwide, no (3-thalassemia
`or seo patient has been treated in an approved
`gene therapy trial [16,17]. This disappointing lack
`of progress has largely been due to several tech(cid:173)
`nical hurdles which must be overcome before
`
`gene therapy can be applied to these diseases.
`These challenges include developing more effi(cid:173)
`cient methods for the stable transfer of genetic
`material into the genomic ONA of hematopoietic
`stem cells and achieving long-term, high-level
`expression from integrated {3- or -y-globin genes.
`So far, most gene therapy approaches to the
`hemoglobinopathies have utilized retroviral vec(cid:173)
`tors. This gene transfer system has several advan(cid:173)
`tages including the ability to efficiently transduce
`a variety of cell types, the ability to stahly inte(cid:173)
`grate the therapeutic gene into host genomic
`ONA, a size capacity able to accommodate (3-
`globin regulatory and coding sequences and a
`proven clinical safety record [18,19]. Overall,
`retroviral vectors have been the most commonly
`used vector for gene transfer protocols in gen(cid:173)
`eral and for protocols targeting bone marrow
`stem cells in particular [16,17]. Unfortunately,
`retroviral vectors also have shortcomings includ(cid:173)
`ing relatively low level transduction efficiency
`of hematopoietic stem cells. Recent studies have
`shown that this is partly due to low level expres(cid:173)
`sion of appropriate viral receptors on the sur(cid:173)
`face of hematopoietic stem cells [20]. eommonly
`used retroviral vectors, which are based on the
`Moloney Murine Leukemia Virus (MML V) and
`related members of the same retroviral species,
`are also unable to efficiently integrate into the
`genomic ONA of non-cycling target cells. Because
`of these problems, expression of transferred genes
`in human peripheral blood cells remains too
`low to achieve therapeutic benefit [21,22]. New
`strategies designed to overcome these problems
`include up-regulation of retroviral receptors on
`hematopoietic stem cells [23], induction of stem
`cell cycling with hematopoietic growth factors
`and drugs [24,25], pseudo-typing of retroviral
`vector envelope proteins [26], and the recent use
`of lentiviral vectors, which are retroviruses which
`do not require cell cycling for stable integration
`into hematopoietic stem cells [27]. Another novel
`strategy involves the transfer, not of the (3- or -y(cid:173)
`globin genes themselves, but of genes coding for
`regula tory proteins which up-regulate expression
`
`SKI Exhibit 2057
`Page 2 of 19
`
`

`

`GENE THERAPY IN HEMOGLOBINOP A THY
`
`439
`
`of the ')'-globin genes. The stage selector protein
`described by Jane and colleagues is an example
`of such a protein which could participate in the
`reactivation of the fetal globin genes [28,29).
`The second major obstacle to successful gene
`therapy for the ;3-hemoglobinopathies is the
`inability to obtain consistent long-term, high(cid:173)
`level expression of integrated therapeutic genes.
`The importance of this concept was emphasized
`in the report of the Panel to Assess the NIH
`Investment in Research on Gene Therapy which
`noted that " ... very little researclz effort is focused
`on
`the meclzanisms that govem maintenance or
`shutoff of gene expression following gene delivery
`in gene therapy experiments" [30]. The report
`urged the NIH ". . . to give high priority to
`basic researc/z to elucidate lww recipient cells and
`particularly stem cells, lwzdle and express foreign
`DNA sequences." [30]. This concem is especially
`relevant to gene therapy for 3-thalassemia and the
`hemoglobinopathies where !ife-long expression
`from the integrated therapeutic gene is a goal.
`Clearly, the normal J-globin genes are capa(cid:173)
`ble of long-term, high-level expression. This has
`provided a major rationale for studying normal 3-
`globin locus structure and function to define the
`mechanisms by which appropriate expression of
`the endogenous genes is achieved. By incorpo(cid:173)
`rating globin gene regulatory elements (such as
`promoters and enhancers) into gene therapy vec(cid:173)
`tors, it has been hoped that similar expression
`of transferred genes could be attained. How(cid:173)
`ever, after twenty years of investigation into the
`regulation of J-globin gene expression and the
`application of the results of this research to the
`development of ;J-globin gene transfer vectors,
`expression levels adequate to begin human trials
`have yet to be achieved.
`In this review we focus on how understanding
`the basic science of gene regulation in general,
`and of the 8-globin locus in particular, has driven
`the development of 8-globin gene replacement
`vectors. We begin by discussing the role chro(cid:173)
`matin structure plays in the regulation of genes
`integrated into genomic DNA, as understanding
`
`this process appears to be important to devel(cid:173)
`oping vectors capable of high-level expression.
`We then review current information on the reg(cid:173)
`ulation of the genes of the /3-globin locus. Next
`we discuss the development of /3-globin gene
`transfer vectors. Because retroviruses have been
`the principle mode! for the development of gene
`replacement strategies for the /3-globin related
`diseases, we have primarily focused on this sys(cid:173)
`tem. Finally, we discuss how novel insights into
`the chroma tin structure of the 8-globin locus are
`leading to the development of a new generation of
`retroviral vectors specifically designed to address
`the problems of inconsistent and low-level gene
`expression.
`
`CHROMA TIN STRUCTURE AND GENE
`EXPRESSION
`
`Within the nucleus of eukaryotic cells genomic
`DNA is packaged by specifie proteins in a highly
`structured fashion [31]. This combination of pro(cid:173)
`teins and nuclear DNA is termed chromatin.
`The structure of chromatin is highly dynamic
`and plays an important role in the regulation
`of normal gene expression (reviewed in [32])
`and expression resulting from gene therapy vec(cid:173)
`tors integrated into nuclear chromatin [33]. The
`first leve! of packaging involves the wrapping of
`DNA about disc-shaped, multi-protein structures
`ca lied nucleosomes. The nucleosomes themselves
`are comprised of four related proteins termed his(cid:173)
`tones. Each nucleosome packages approximately
`200 bp of DNA which is wrapped twice around
`the structure. Regions of DNA which contain
`actively expressed genes (such as the p-globin
`locus in erythroid cells) are loosely packaged and
`the nucleosomes of the region assume a beads(cid:173)
`on-a-string arrangement. This is associated with
`the formation of enzymatically modified histone
`proteins which become hyperacetylated. Regions
`of DNA which do not contain actively expressed
`genes (such as the p-globin gene locus in non(cid:173)
`erythroid cells) are much more compacted. The
`
`SKI Exhibit 2057
`Page 3 of 19
`
`

`

`440
`
`J. M. MCINERNEY et al.
`
`structure regardless of their site of integration
`within the genome.
`
`REGULATION OF HUMAN ,6-GLOBIN
`GENE EXPRESSION
`
`Perhaps because the human ,B-globin genes were
`among the first mammalian genes to be cloned,
`the human ,B-globin gene locus has served as a
`premiere model for studying the regulation of
`complex mammalian loci. As shown in Figure 1,
`the genes of the locus are arranged in their order
`of developmental expression, from the embryonic
`e-globin gene to the fetal -y-globin genes and the
`adult 8-and ,B-globin genes [2]. Early studies of
`the locus identified promoters and both intronic
`and downstream enhancers of ,B-globin locus
`gene expression (Figure lB). In earl y gene transfer
`experiments, these elements were able to direct
`erythroid specifie expression of the genes but
`expression levels in both transgenic mice and
`tissue culture celllines were very low and highly
`variable [38,39]. Clearly, important elements for
`gene regulation were missing.
`
`nucleosomes in these regions are thought to form
`tightly condensed helical arrays called solenoids
`or 30 nanometer fibers [31]. Inactive areas of
`chromatin are also characterized by DNA methy-
`lation and hypoacetylation of histone proteins
`(reviewed in [34]). Higher levels of packag(cid:173)
`ing also occur in inactive areas but are much
`less well defined. Experimentall~r, differences in
`chromatin structure are often assessed by their
`sensitivity to digestion by nucleases, which are
`enzymes capable of digesting DNA. DNase I is
`a nuclease commonly used in chromatin studies.
`Tightly compacted, inactive areas of chromatin
`are highly resistant to DNase I digestion, while
`transcriptionally active areas of chromatin are
`more sensitive. Transcriptional regulatory ele(cid:173)
`ments such as promoters and enhancers are
`often hypersensitive to nuclease digestion due
`to the displacement or disruption of nucleosomes
`within these areas exposing DNA to the nucle(cid:173)
`ase. These regions of locally altered chromatin
`structure are termed nuclease or DNase I hyper(cid:173)
`sensitive sites or "HSs".
`Chromatin structure is relevant to the devel(cid:173)
`opment of gene transfer vectors because the
`expression of a stably integrated therapeutic gene
`is likely to depend on the surrounding chroma tin
`structure. If the gene is integrated into a com(cid:173)
`pacted, transcriptionally-inactive region of the
`genome it is unlikely to be expressed. This phe(cid:173)
`nomenon is termed "position-dependent expres(cid:173)
`sion" as expression is dependent on the site of
`integration. Position-independent expression or,
`stated in a different way, consistent expression
`is an important goal in the development of gene
`transfer vectors. Furthermore, it has been shown
`that integrated genes which initially express at
`high levels may be subject to transcriptional
`silencing over time [35-37]. This is thought to
`result from a closing down of the chroma tin struc(cid:173)
`ture around the integrated gene. As is discussed
`below, it would be advantageous to be able to
`include elements within gene therapy vectors
`which are able to independently open and main(cid:173)
`tain surrounding domains of active chromatin
`
`100~[3
`~·GLOBIN
`
`:
`
`CHAIN
`SYNTHESIS
`('l'o)
`
`E
`
`EMBAVO
`
`FETlJS
`
`1
`
`1
`BIRTH
`
`~
`ADUL T
`
`6 MONTHS
`
`A
`
`8
`
`• LCR Ill
`5 4 3 2 1
`..
`
`Erythrold spaclllc
`DNasal HSs
`
`E
`
`-10kb
`Chromosome 11 p15.5
`
`FIGURE 1 The_ human .6-&lobin gene locus. A) Expression
`IS developmentally
`regulated.
`the _.6-g!obm genes
`of
`B) Orgamzahon of the locus. Globin genes are depicted
`as black boxes, gene promoters as white boxes and local
`enhanc~rs as di~monds. The .6-globin locus control region
`(LCR) 1s compnsed of five domains of altered chromatin
`strucrure termed DNase 1 HSs. Four of these HSs are erythtoid
`specifie.
`
`SKI Exhibit 2057
`Page 4 of 19
`
`

`

`GENE THERAPY IN HEMOGLOBINOP ATHY
`
`441
`
`The description of the ,8-globin locus control
`in 1987 apparently identified
`region (LCR)
`these critical elements (Figure lB). The LCR is
`comprised of five DNase 1 HSs, four of which
`(5' HS 1-4) are erythroid specifie and are
`able to direct consistent, high-level, position(cid:173)
`independent expression of linked globin genes in
`transgenic mice [40,41]. The functional activities
`of the LCR are primarily mediated by specifie
`core sequences found near the centers of each
`HS domain [42-52]. Each of the 200-400 bp HS
`core elements contain evolutionarily conserved
`binding sites for factors primarily expressed in
`hematopoietic cells, such as NF-E2, GATA-1,
`SSP, and EKLF, as well as more ubiquitous
`factors such as Sp1, YY1, and CDP (reviewed
`in [53]). On a chromatin structural level these
`nuclease sensitive regions are characterized by
`highly specifie nucleosome positioning including
`regions where nucleosomes are disrupted or
`displaced [54,55]. The active elements of the
`LCR HSs closely correspond to the regions
`of nucleosome displacement [47,48,52,54,55]. In
`transgenic mice containing multiple copies of
`linked transgenes and individual LCR elements,
`HS2, HS3, and HS4 are each able to confer
`position independent expression [56]. However,
`only when all of these LCR elements are
`present as full-length, or smaller "mini" or
`"micro" LCRs is near-normal expression of the
`transgene consistently obtained [41,51,57]. In
`single copy transgenic mice HS3 is uniquely able
`to confer position-independent expression [58]. A
`well characterized, classical enhancer element is
`present within the core region of HS2 [59,60]. This
`enhancer activity requires binding sites for the
`hematopoietic transcription factor NF-E2 [52,61].
`The other LCR HSs do not contain significant
`enhancer activity. While relatively little research
`has been performed on HSs 1 and 5, HS1 may
`be important for consistent expression and for
`mediating interactions between the LCR and
`the ,8-globin promoter [62] and, as discussed
`below, HS5 may function as a chroma tin structure
`insulator [63].
`
`Despite intense investigation the mechanisms
`by which the elements of the LCR are able to
`influence expression of the genes of the human ,8-
`globin locus remain unclear. Most current models
`propose the formation of a holocomplex in which
`the core elements of the LCR functionally inter act.
`This holocomplex is then envisioned to form a
`three dimensionallooping structure that is able
`to interact with the promoters of the globin genes
`(reviewed in [53,64]).
`
`DEVELOPMENT OF ,8-GLOBIN GENE
`TRANSFER VECTORS
`
`Retroviral Transfer Vectors
`
`Since the development of the first gene transfer
`trial more than ten years aga, retroviral vec(cid:173)
`tors have been the most frequently used method
`for stable integration of genes into target cell
`genomes [17]. Retroviruses are enveloped RNA
`viruses which enter cells through interactions
`with specifie receptors on the cell surface [65].
`The presence of these receptors on target cells
`is an important determinant of the virus's abil(cid:173)
`ity to transduce a specifie cell type. The infecting
`virus particle carries a single-strand RNA genome
`which is converted into a double-strand DNA
`provirus by the virally-encoded reverse transcrip(cid:173)
`tase within the infected cell. The viral integrase,
`in cooperation with cellular proteins, then medi(cid:173)
`ates incorporation of the DNA provirus into the
`genomic DNA of the target cell [66].
`The retroviral system has been adapted to
`serve as a safe and effective method for stahly
`transferring potentially therapeutic genes into
`the genomic DNA of target cells (Figure 2).
`The retroviral gene transfer vector is commonly
`constructed within the context of a bacterial
`plasmid. The vector contains the viral long
`terminal repeats (LTRs) and packaging signal ('lj;)
`as well as the gene or genes to be transferred.
`Because the retroviral structural genes gag, pol
`and env are not contained within the vector, it
`is unable to direct the formation of infectious
`
`SKI Exhibit 2057
`Page 5 of 19
`
`

`

`442
`
`J. M. MCINERNEY et al.
`
`(..._5_' -L-T-R'lf_T_a_bz_~_~_Sj_,~_fl_J--3-' _L_T_,R) ~
`
`Retroviral Vector
`(in bacterial plasmid)
`
`Packaging Cell
`
`Target Cell
`
`FIGURE 2 Retroviral gene transfer. A retroviral vector
`containing
`the gene
`to be
`transferred
`(transgene)
`is
`constructed in the context of a bacterial plasmid. The plasmid
`is stably introduced into a packaging cellline (step 1) where
`transcription of the retroviral sequences produces the single
`stranded RNA viral genome (step 2). The packaging cellline
`contains the retroviral structural genes (gag, pol and env)
`necessary for producing infectious virions (step 3) which
`then bind to a specifie cell surface receptor on the target
`cel!. Following uptake into the target cel!, the viral genome
`undergoes reverse transcription by the viral polymerase
`resulting in a double stranded DNA copy of the viral genome
`(step 4) which is then stably integrated into the genomic DNA
`of the target cell (step 5). The transgene is then transcribed,
`ultimately producing the protein product of the transferred
`gene (step 6).
`
`vmons in cells other than specially designed
`"packaging" cell lines which have been stably
`transduced with these genes [67]. This allows the
`structural genes of the virus to be replaced by
`the gene or genes to be transferred as well as
`their associated regulatory elements. Following
`transfection of the retroviral vector into the
`packaging cell an RNA copy of the vector is
`
`produced which is then packaged into functional
`viral particles. These viruses are then used to
`transfer the gene or genes of interest to the
`target cells where they are incorporated into the
`target cell genome. Infection of the target cell is
`mediated by binding to specifie receptors on the
`cell surface. Since the genes for the viral structural
`proteins are not packaged, the virus particles can
`infect a single cell, but subsequent production of
`virions by the target cell can not occur. Once
`the vector RNA is inside the target cell it is
`converted to double stranded DNA in the process
`of reverse transcription. This is mediated by the
`viral polymerase gene (pol) product which is
`packaged within the viral particle. The DNA form
`of the vector is then incorporated into the host
`genomic DNA. Transcription of the integrated
`viral sequences then leads to the production of
`the protein coded for by the transferred gene.
`While there are many positive aspects to using
`retroviruses for transferring ,8-globin genes into
`bone marrow stem cells, there are also disad(cid:173)
`vantages. One is that marrow stem cells have
`relatively low levels of viral receptors on their
`surfaces [20]. Strategies to overcome this problem
`include induction of receptor expression on the
`stem cells. A second disadvantage is an inabil(cid:173)
`ity of retroviruses of the Murine Sarcoma and
`Leukemia Virus species (i.e. MMLV) to integrate
`proviral DNA into the genomic DNA of non(cid:173)
`cycling cells [68]. Hematopoietic growth factors
`and the drug 5-fluorouracil have been used to
`induce stem cell cycling and increase transduc(cid:173)
`tion efficiency [24,25]. An alternative approach
`has been to use other viral-based gene transfer
`vectors. The most promising of these include vec(cid:173)
`tors based on adeno-associated viruses (AA V)
`and lentiviruses. Recombinant AA V vectors car(cid:173)
`rying the human 1'-globin gene have been used
`to mediate stable gene transfer with high-level
`expression in K562 human erythroid tissue cul(cid:173)
`ture cells [69-71]. Lentiviruses are also retro(cid:173)
`viruses but comprise a separate genus from the
`MMLV -related retroviruses. They are structurally
`distinct in that their genome and structure are
`
`SKI Exhibit 2057
`Page 6 of 19
`
`

`

`5' LTR
`
`P-GlobinGene
`
`3' LTR
`
`ljl
`
`ljl
`
`'::;;:J
`
`·v~ HS Core
`
`Intron 2 Deletion
`Mutagcnesis of poly A/splicc sites
`'\1
`
`ljl
`
`Intron 2 Deletion
`'V
`'=:fi%>' 1
`
`ljl
`
`11
`
`HS2 • HS3- HS4 ---
`
`A
`
`B
`
`c
`
`D
`
`E
`
`much more complex. Functionally, they exhibit
`the ability to infect non-cycling cells such as qui(cid:173)
`escent bane marrow stem cells [27]. While it has
`been difficult to develop effective packaging cell
`lines for these vectors, significant progress has
`recently been made in this area [72]. To date no
`published reports have demonstrated the appli(cid:173)
`cation of these vectors to globin gene replacement
`therapy.
`
`First Generation Retroviral Gene Transfer
`Vectors
`
`Early attempts to develop retroviral gene ther(cid:173)
`apy vectors for the hemoglobinopathies utilized
`the human ,8-globin genes in combination with
`their proximal regulatory elements [73-78]. An
`example of these vectors is shawn in Figure 3B.
`These initial vectors were hindered by law titer
`and generally very law ,8-globin gene expres(cid:173)
`sion. Subsequent studies demonstrated that the
`law titer was due, in part, to sequences within
`the second intron and within other untrans(cid:173)
`lated sequences S' and 3' of the ,8-globin gene
`[78-80]. These sequences appeared to cause
`proviral instability and law titer by acting as
`polyadenylation signais or cryptic splice sites
`[81]. Higher titers were achieved with vectors
`containing the ,8-globin eDNA than with those
`containing the genomic sequence [78-80]. How(cid:173)
`ever, it was also shawn that sequences within
`the second intron contribute to high-level gene
`expression [43,78,79]. Therefore, to reduce the
`problem of genetic instability while retaining the
`intronic elements, ,8-globin retroviral vectors are
`now commonly designed so that the gene is in
`reverse orientation relative to viral transcription
`(Figure 3A vs. 3B) [73,75,82]. This reduces vec(cid:173)
`tor rearrangement caused by splicing of the viral
`RNA genome because potential splice sites do
`not have their characteristic sequence in this ori(cid:173)
`entation and consequently are not recognized by
`the RNA splicing machinery. Following integra(cid:173)
`tion of the double stranded provirus, the reversed
`
`GENE THERAPY IN HEMOGLOBINOPA THY
`
`443
`
`Retroviral Construct
`
`References
`
`[78, 79]
`
`[73 -79, 83]
`
`[60, 87, 90]
`
`[81, 89, 91, 92]
`
`[33,80]
`
`FIGURE 3 Evolution of retroviral ,B-globin gene transfer
`vectors. A) Retroviral vector containing the ,B-globin gene
`in the "sense" orientation relative to the viral genome. B) The
`orientation of the ,6-globin gene is reversed to decrease the
`incidence of splicing of the viral sequences during the RNA
`stage of viral production. C) lndividual LCR HS cores are
`added to the vector in an effort to increase ,6-globin expression.
`D) Multiple LCR HS cores are added to the vector, potential
`splice sites are mutated and an inhibitory portion of intron
`2 is deleted. E) Different orientations of LCR HS cores are
`evaluated to determine their ability to improve ,8-globin
`expression.
`
`gene is capable of normal expression on the oppo(cid:173)
`site strand.
`With these earl y vectors, ,8- globin expression in
`mouse erythroleukemia (MEL) cells was highly
`varied, averaging between 2 and 28% percent
`of endogenous mouse ,8-major gene expression
`[73-75,78,79]. These results strongly suggested
`that the site of integration of the retroviral vector
`has a profound effect on expression of the ,8-
`globin gene. In experiments involving gene trans(cid:173)
`fer to murine bane marrow stem cells expression
`was tissue-specifie and less varied, but reached
`at most one percent of mouse ,8-major expres(cid:173)
`sion [76,77,83]. ,8- globin vectors thatincorporated
`the 3' ,8-globin enhancer did not display consis(cid:173)
`tent improvement in overall expression levels or
`position-independent expression [77,79,84-86].
`
`SKI Exhibit 2057
`Page 7 of 19
`
`

`

`444
`
`J. M. MCINERNEY et al.
`
`From these early experiments it was evident
`that retroviral vectors incorporating the pro(cid:173)
`moters and enhancers of the ,B-globin genes
`were insufficient to direct high-level position(cid:173)
`independent expression of ,B-globin genes and
`that additional or alternative gene regula tory ele(cid:173)
`ments would be required to achieve high-level
`position-independent expression using retroviral
`gene therapy vectors.
`
`Second Generation Retroviral Vectors:
`Incorporation of LCR Elements
`
`The ,B-globin LCR is generally thought to be
`required for normal expression of the globin
`genes in human hematopoietic cells and in
`transgenic models of ,B-globin gene expression.
`The discovery of the LCR and the characterization
`of its functional properties led to the expectation
`that the inclusion of these elements in ,B-globin
`gene transfer vectors would finally provide
`high-level position-independent expression in
`erythroid cells. Because the packaging capacity
`of retroviral vectors is limited to approximately
`8-9 kb, the entire LCR region was too large for
`inclusion in ,B-globin vectors [67]. However, as
`noted above, experiments in transgenic mice had
`delineated functional HS cores of only a few
`hundred base pairs in size that contained most of
`the relevant LCR activity and conferred high-level
`position-independent expression. In numerous
`studies these elements were incorporated into a
`second generation of ,B-globin retroviral vectors
`(Figure 3C-3E).
`One of the first studies to utilize this strategy
`was reported by Novak et al. ,B-globin retroviral
`vectors were constructed so that each contained
`one of the HS core elements (Figure 3C) [87].
`These retroviral vectors were then used to trans(cid:173)
`duce MEL cells. An encouraging result from
`these experiments was that HS2, HS3, and HS4
`increased average ,B-globin expression by approx(cid:173)
`imately 20-fold, 4-fold, and 6-fold respectively.
`The combination of HS3 and HS4 increased
`
`expression 3-fold. However, the retroviruses in
`this study continued to show low producer cell
`titers in the range of 104 to 105 cfu/ml, typical
`of similarly designed vectors. Vectors containing
`HS2 and HS3-HS4 also demonstrated frequent
`genetic rearrangement. All vectors tested contin(cid:173)
`ued to show highly variable expression indicating
`that the inclusion of HS cores did not produce
`position independent expression.
`Previous studies in transgenic mice and tissue
`culture cell lines, as well as the retroviral study
`of Novak et al. above, indicated strong enhancer
`activity within HS2 [42,44,59,88]. In an attempt
`to circumvent the problem of genetic instability
`associated with the use of HS2 in retroviral
`vectors, Chang et al. reduced the size of HS2 to
`a 36 bp element containing tandem NF-E2/ AP-l
`binding si tes [ 60,61]. Incorpora ti on ofthis element
`into a ,B-globin vector increased expression two(cid:173)
`fold. It also resulted in reduced vector instability
`compared to vectors containing either a 732
`or 412 bp HS2 core element. However, vector
`rearrangement was still observed approximately
`fifty percent of the time and viral titer remained
`low- in the range of 104 cfu/ml. The use of
`multiple copies of the 36 bp HS2 enhancer
`element led to increased vector rearrangement.
`Attempts to overcome the problems of low ti ter
`and genetic rearrangement included studies by
`the Gelinas and Leboulch laboratories. In the first
`of these studies a 374 bp deletion within intron
`2 of the ,B-globin gene improved the titer of a ,B(cid:173)
`globin vector 10-fold without adversely affecting
`gene expression [89]. Leboulch et al. attempted
`to correct these problems in ,B-globin vectors
`which incorporated LCR elements by perform(cid:173)
`ing sequence analysis of the human ,B-globin
`gene and both S' and 3' untranslated regions
`[81]. This led to the identification of poten(cid:173)
`tial polyadenylation signais and splice sites. In
`this study, all ,B-globin LCR vectors containing
`the wild-type ,B-globin sequences were unsta(cid:173)
`ble and characterized by low titer regardless
`of the LCR derivatives used. Targeted muta(cid:173)
`genesis of putative splice sites within intron 2
`
`SKI Exhibit 2057
`Page 8 of 19
`
`

`

`GENE THERAPY IN HEMOGLOBINOP A THY
`
`445
`
`and the 3' flanking region of the ,13-globin gene
`and deletion of a 372 bp region within intron 2
`(Figure 3D) promoted the stability and increased
`titer 10-fold of an HS2-,13-globin vector. This vec(cid:173)
`tor was also stable in murine bone marrow cells.
`These mutations also allowed stable transmission
`of vectors with multiple LCR elements. Incor(cid:173)
`poration of HS2 increased expression 14-f