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`BLUEBIRD EXHIBIT 1032
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`Page 1 of 547
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`BLUEBIRD EXHIBIT 1032
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`

`

`
`BoAAUS ALESic oh.
`le
`
`MSK.P-050
`Patent Application
`
`VECTOR ENCODING HUMAN GLOBIN GENE AND
`USE THEREOF IN TREATMENT OF HEMOGLOBINOPATHIES
`
`Statement Conceming Government Funding
`
`This application was supported by funds provided under NHLBIgrant No.
`
`HL57612. The United States government mayhavecertain rights in the invention.
`
`Statement Concerning Related Applications
`This application claims the benefit of US Provisional Application No. 60/301,861
`filed June 29, 2001 and US Provisional Application No. 60/302,852 filed July 2, 2001, both of
`
`which are incorporated herein by reference.
`
`Background of the Invention
`This application relates to a vector comprising a mammalian, and particularly a
`
`human globin gene andto the use thereof in treatment of hemoglobinopathies, including a- and
`
`f-thalessemia and sickle-cell disease.
`Currenttreatment modalities for B-thalassemias consist of either red blood cell
`
`transfusionplusiron chelation (which extends survival but is cumbersome, expensive and an
`imperfect therapy), or allogeneic bone marrow transplant (whichcarries a lethalrisk and is not
`available to the majority of patients). Thus, there is a substantial need for improved therapeutic
`approaches. The present invention provides a genetic correction in autologous hematopoictic
`stemcells, thus using gene therapy to provide a less-risky and more effective long-term
`treatment.
`
`While gene therapy has been proposed for many years, a significant challenge
`facing efforts to develop genc therapy vectors is the ability to produce therapeutically useful
`levels of a desired protein or peptide. The present invention provides a vector whichis capable
`
`of providing therapeutically meaningful levels of human globin for sustained periods of time.
`
`-l-
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`SRAEES ce AL Pc
`
`MSK.P-050
`Patent Application
`
`This ability arises from the ability to transmit large genomic regulatory sequences that control
`
`expression of the therapeutic gene.
`
`Summary of the Invention
`
`In accordance with the invention, a recombinant lentiviral vector is provided
`
`comprising:
`
`(a)
`
`(b)
`
`a region comprising a functional globin gene; and
`
`large portions of the B-globin locus control regions which include
`
`large portions of DNaseI hypersensitive sites HS2, HS3 and HS4. The regions may be the
`
`complete site or somelesser site which provides the same functionality as the specific sequences
`
`set forth below. This vector provides expression of B-globin when introduced into a mammal,
`
`for example a human,
`
`in vivo. Optionally, the vector further comprises a region encoding a
`
`dihydrofolate reductase.
`
`By incorporation of different globin genes, the vector of the invention may be
`
`used in treatment of hemoglobinopathies, including a- and B-thalessemia and sickle-cell disease.
`
`For cxample, hcmatopoictic progenitor or stem cells may be transformed ex vivo and then
`
`restored to the patient. Selection processes may be used to increase the percentage of
`
`transformed cells in the retumed population. For example, a selection marker which makes
`
`transformed cells more drug resistant than un-transformed cells allows selection by treatment of
`
`the cells with the corresponding drug. Selection and/or enrichment mayalso be carried out im
`
`vivo, for example using methotrexate or similar antifolates to select for cells rendered resistant by
`
`the expression from the vector of a dihydrofolate reductase (DHFR).
`
`Brief Description of the Drawings
`
`Fig. 1 shows the genomicstructure of a recombinant onco-retroviral vector in
`
`accordance with the invention.
`
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`

`SLO AE
`
`MSK.P-050
`Patent Application
`
`Fig. 2 shows the genomicstructure of recombinant onco-retroviral vector within
`
`the scope of the invention.
`Fig. 3 shows experimental results demonstrating increased mean B-globin
`
`expression in transduced MELcells.
`Fig. 4 showsthe average vector copy numberin peripheral blood cells, measured
`periodically for 24 weeks, which confirms showedhighly efficient gene transfer in cells
`
`transduced with the vector of the invention.
`
`Figs. 5A and B show human B-globin expression per endogenousallele 12 days
`
`and 22 weeksafter introduction ofcells transduced with the vector of the invention.
`
`Fig. 6 shows haematocrit level, red blood cell count, reticulocyte count and
`haemoglobinlevel fifteen weeks after transplantation with unselected TNS9-transduced Hbb***
`bone marrow.
`
`Detailed Description of the Invention
`
`Inafirst aspect of the present invention, a recombinantlentirviral vector is
`
`provided comprising:
`
`(a)
`
`(b)
`
`a region comprising a functional globin gene; and
`
`large portions of the B-globin locus control regions, which include
`
`DNase I hypersensitive sites HS2, HS3 and HS4.
`
`Asusedin the specification and claims hereof, the term “recombinantlentiviral
`
`vector” refers to anartificially created polynucleotide vector assembled froma lentiviral-vector
`
`and a plurality of additional segments as a result of human intervention and manipulation.
`
`The term “functional globin gene”refers to a nucleotide sequence the expression
`
`of which leads to a globin that does not produce a hemoglobinopathy phenotype, and whichis
`
`effective to provide therapeutic benefits to an individual with a defective globin gene. The
`
`functional globin gene may encode a wild-type globin appropriate for a mammalian individual to
`
`be treated, or it may be a mutant form of globin, preferably one which provides for superior
`
`-3-
`
`Page 4 of 547
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`

`

`ALALE ae Da ee
`
`MSK.P-050
`Patent Application
`
`properties, for example superior oxygen transport properties. The functional globin gene
`
`includes both exonsand introns, as well as globin promoters and splice doners/acceptors.
`
`Suitably, the globin gene may encode «-globin, B-globin, or y-globin. B-globin promoters may
`
`be sued with each of the globin genes.
`
`The recombinant vectors of the invention also include large portions of the locus
`
`control region (LCR) which include DNase I hypersensitive sites HS2, HS3 and HS4. In prior
`
`studies, smaller nucleotide fragments spanning the core portions of HS2, HS3 and HS4 have
`
`been utilized. Sadelain et al. Proc. Nat'l Acad. Sci. (USA)92: 6728-6732 (1995); Lebouichet al.,
`
`EMBO J. 13: 3065-3076 (1994). The term “large portions” refers to portions of the locus control
`
`region which encompasslarger portions of the hypersensitive sitcs as opposed to previously
`
`tested fragments including only the core elements. The regions may be the complete site or some
`
`lesser site which provides the same functionality as the specific sequences set forth below.
`
`In
`
`preferred embodiments of the invention, the large portions of the locus control regions are
`
`assembled from multiple fragments, each spanning one of the DNase I hypersensitive sites.
`
`In
`
`addition, the locus control region has two introduced GATA-1 bindingsites at the junction
`
`between HS3 and HS4. While not intending to be boundby any specific mechanism,it is
`
`believed that the incorporation of these transcription factor binding sites enhances the
`
`effectiveness of the vector.
`
`The genomic structure of one embodiment of the vector of the invention (TNS9)
`
`is shown in Fig. 1. TNS9 incorporates human §-globin gene (from position -618 to +2484) that
`includes an extended promoter sequence and a 3'-enhancer element. Optionally, a portion of 3'
`U3 region of the lentiviral backbone can be deleted for increased safety.
`In Fig. 1, the exons and
`
`introns of the human B-globin gene are represented by filled and open boxes, The locations are
`
`indicated for the splice donor (SD), splice acceptor (SA), packaging region (1p), rev-response
`
`element (RRE), human B-globin promoter (P) and 3'-B-globin enhancer (E). Thus, in the vector
`
`TNS9, a functional B-globin gene, which includes both the exons and introns of the gene and the
`
`relevant control sequences from the human B-globin locus. These are combined with the large
`
`-4-
`
`Page 5 of 547
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`Page 5 of 547
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`

`

`SLUG) GLE he 2 EPic
`
`MSK.P-050
`Patent Application
`
`fragments of the locus control region. The 3.2 kb LCR assembled into dTNS9 consists of an 840
`bp HS2 fragment (SnaBI-BstXT), a 1308 bp HS3 fragment (HindII-BamHI) and a 1069 bp HS4
`
`fragment (BamHI-BanI)).
`
`In a further aspect of the invention, the B-globin gene coding sequence can be
`
`exchanged and replaced with either the gammaglobin gene(for sickle cell disease) or the alpha
`globin gene (for alpha-thalassemias). In one strategy, a Ncol-Pst I fragment of the B-globin gene
`
`is replaced with the corresponding Ncol-HindIII fragment of the gammaglobin geneorthe
`
`Ncol-PstI fragment of the human alpha globin gene. These fragments start at the translational
`
`start of each globin gene (spanning the Ncolsite) and end pasttheir respective polyadenylation
`
`signals. In the second strategy, chimeric genes can be generated by only swapping the coding
`
`sequenceof each oneofthe three exons of these genes. Thus, for the gammaglobin gene, the
`result is a vector that comprises the beta globin promoter, the beta globin 5' untranslated region,
`
`the gamma exon 1 coding region, the gammaintron 1 the gammaexon2,the beta intron 2, the
`
`gammaexon3, and the beta 3' untranslated region. Thusall the elements of the TNS9 vector
`
`remain in place (promoter, enhancers, 5' and 3' untranslated regions, the LCR elements, the 2
`
`additional GATA-1 binding sites and the introns of the beta globin gene (at least intron 2, which
`
`
`
`is most important). Inathird strategy, the codon usage within exon 3 of the gamma globin gene
`
`can be modified so that its sequence will resemble as much as possible that of the beta globin
`
`gene. The reasonfor testing this is that the beta globin gene is always the best expressed.
`
`Additional elements may be included in the vectors of the invention to facilitate
`
`utilization of the vector in therapy. For cxample, the vector may include selectable markers, to
`
`confirm the expression of the vector or to provide a basis for selection of transformed cells over
`
`untransformed cells, or control markers which allow targeted disruption of transformed cells, and
`
`thus the sclective removalof such cells should termination of therapy become necessary.
`
`In a further specific embodiment, the vector of the invention includes the mouse
`
`PGK promotor and human dihydrofolate reductase (DHFR) cDNAasatranscriptional unit.
`
`Mutant forms of DHFR which increase the capacity of the DHFR to confer resistance to drugs
`
`-5-
`
`Page 6 of 547
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`Page 6 of 547
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`

`

`tee ap enon on aYa gen oe '
`“yyy oq)
`ALSTOE PUISLac
`
`MSK.P-050
`Patent Application
`
`such as methotrexate are suitably used. For example, single and double mutants of DHFR with
`mutations at amino acids 22 and 31 as described in commonly assigned PCT Publication No.
`WO97/33988, which is incorporated herein by reference, may be advantageously utilized.
`Fig. 2 shows the genomicstructure of specific vector within the scope of the
`invention. The vector includes a deleted LTR, from -456 to -9 of HIV LTR and the PGK
`promoter (530 bp) from the murine phosphoglycerate kinase | gene. It also includes a DHFR-
`encoding region encoding human DHFRwiths/f mutation at amino acid 22. The locus control
`region and the B-globin region are the same as in TSN9. This vector is designated dTNS9-PD.
`This incorporation of DHFRinto this vector provides transformed cells with a methotrexate-
`resistant phenotype. As a result, methotrexate, and other antifolates can be used, both in
`vitro and in vivo as a selectionto tool to enhance levels of the functional hemoglobin. When
`hematopoietic stem cells were transformed using dTNS9-PD and reintroduced to mice that were
`then treated with NMVBPR-P (0.5 mg/dose) and TMTX (0.5 mg dose) for five days, observed
`levels of expressed human f-globin were muchhigher in mice transduced with dTNS9-PD
`vectors after treatment with TMX and NMBPR-Pforselection oftransduced cells.
`
`The vectors ofthe invention are used in therapy for treatment of individuals
`suffering from hemoglobinopathies.
`In one embodimentof the invention, hematopoietic
`progenitor or stem cells are transformed ex vivo andthenrestored to the patient. As usedin the
`specification andclaims hereof, the term “hematopoietic progenitor sand stem cells”
`encompasses hematopoietic cells and non-hematopoietic stem cells, e.g., embryonic stem cells,
`hematopoietic stem cell precursors, or any ofthe latter generated by nuclear transfer from a
`somatic cell. It is know in the art that efficient genes transfer into human embryonic stem cells
`
`can be achieved using lentiviral vectors.
`Sclection processes maybe usedto incrcase the percentage of transformedcells in
`the returned population. For example, a selection marker which makes transformed cells more
`drug resistant than un-transformed cells allowsselection by treatment ofthe cells with the
`
`Page 7 of 547
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`Page 7 of 547
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`

`

`LADSER Pa?
`
`MSK.P-050
`Patent Application
`
`corresponding drug. When DIIFRis used asa selection marker, it can be used for enrichment of
`transducedcells in vitro, or for in vivo selection to maintain the effectiveness of the vector.
`
`The invention will now be further described with reference to the following non-
`
`limiting examples.
`
`Example 1
`
`To produce vector TNS9, the human B-globin gene was subcloned from MB6L
`(Sadelain et al. Proc. Nat'l Acad. Sci. (USA)92: 6728-6732 (1995)) into lentiviral vector
`
`pHR’LacZ (Zuffery ct al., Nature 15: 871-875 (1997)) replacing the CMV-LacZ sequence.
`pHR’eGFPwasconstructed by replacing LacZ with the eGFP sequence (Clontech). Viral stocks
`were generated bytriple transfection of the recombinant vectors pCMVAR8.9 (Zuffrey et al.) and
`pMD.G in 293T cells as previously described in Dull, et al., J. Virol. 72: 8463-8471 (1998). The
`pseudotyped virions were concentrated byultracentrifugationm resuspendedandtitrated as
`described in Gallardoet al., Blood 90: 952-957 (1997). For comparison, RSN1 was used which
`
`has a similar structure, except that the LCR contains only the core portion of HS2, HS3 and HS4.
`Northern blot analysis showed full length RNAtranscripts, indicating that the recombinant
`lentiviral genomoesare stable. Southern blot analysis on genomic DNA from transduced cells,
`digested once in each long terminal repeat (LTR)results in a single band correspondingto the
`expected size for the vector, indicating that the proviral structure is not rearranged.
`
`Example 2
`
`To investigate the tissue specificity, stage specificity and expression level of the vector-
`
`encoded human B-globin gene, we transduced RNS1 and TNS9 into MELcells, lymphoid Jurkat
`cells and myeloid HL-60 cclls. Ccll-free viral supernatant was used to infect C88 MELcells in
`the presence of polybrene (8 pg mi'). Transduced MELcells were subcloned bylimiting
`dilution, and screened by PCRfor transduction” using primers that anneal in the human f-globin
`
`promoter sequence (BPS, 5' -GTCTAAGTGATGACAGCCGTACCTG-3)) and in HS2 (C2A,5'-
`
`-7-
`
`Page 8 of 547
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`Page 8 of 547
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`

`

`SCE LER ES meow TPT AL®
`
`MSK.P-050
`Patent Application
`
`TCAGCCTAGAGT GATGACTCC TATCTG-3'). Vector copy number and integrationsite
`analysis was determined by Southern blot analysis’. Transduced MELcells were induced to
`maturation by 5-day culture in 5 mM N,N’- hexamethylene bisacetamide (HMBA,Sigma).
`To induce B-globin transcription, transduced MEL cell pools were differentiated using
`hexamethylene bisacetamide HMBA). Human B-globin (6) and mouse B-globin transcripts
`were measured by quantitative primer extension. After normalization to vector copy number and
`to endogenous B-globin cxpressionper allele, human B-globin levels were 14.2 + 4.7% for RNS1
`and 71.3 + 2.3% for TNS9 in pooled MELcells (Fig. 2a). MEL, Jurkat and HL-60 cells were
`
`transduced with RNS1, TNS9 or control GFP recombinantlentivirus. Human p-globin RNA
`
`expression in HMBA induced MELcells (grey bars) was measured by quantitative primer
`
`extension and normalized to mouse B-globin RNA expression per locus. Expression was then
`
`normalized to the vector copy number determined by Southern blot. No human B-globin RNA
`
`expression was detected in non-induccd MEL (black bars), Jurkat (white bars) or HL-60 cells
`
`(hatched bars), indicating that globin expression was erythroid- and differentiation-specific. No
`
`human B-globin expression was detected in non-induced MEL, Jurkat and HL-60cells (Fig. 3),
`
`indicating that human B-globin expression was appropriately regulated in termsof tissue
`
`specificity and state of differentiation. We generated a panel of MELcell clones that carried a
`
`single copy of either vector to distinguish whether the increased expression obtained in HMBA-
`treated Melcells transduced with TNS9 rather than RNS1 wasthe result of an increase in B*
`
`expression per cell or of an increase in the fraction of cells expressing human B-globin.
`
`Transduced MELcells were subcloned by limiting dilution immediately after transduction,
`
`avoiding any bias towards favourable chromosomalintegration sites as produced by drug
`
`selection’. The proportion of clones expressing human (-globin varied significantly between the
`
`two vectors. One out of ten RNS1 positive clones yiclded measurable human B-globin
`
`transcripts, in contrast to [2 out of 12 for TNS9 also expressed higher levels of human B-globin
`
`than did those bearing RNS1 (P < 0.01, Fisher’s exact test). Cells bearing TNS9 also expressed
`
`higher levels of human B-globin than did those bearing RNS1 (P < 0.01, Wilcoxon rank sum
`
`-8-
`
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`

`

`GLSLeeea a
`
`MSK.P-050
`Patent Application
`
`test). These findings established that both the level and probability of expression at random
`integration sites was increased with the TNS9 vector.
`
`Example 3
`Quantification of human B-globin mRNA
`Total RNA wasextracted from MEL,Jurkat and HL-60cells, or mouse spleen and blood
`using TRIzol. Quantitative primer cxtension assays were done using the Primer Extension
`System-AMVReverse Transcriptase kit (Promega) with [?P] dATP end-labelled primers
`specific for retroviral-derived human $-globin (5' -CAGTAACGGCAGACTTCTCCTC-3’) and
`mouse B-globin (5' -TGATGTCTGTTTCTGGGGTT GTG -3'), with predicted extension
`products of 90 bp and 53 bp,respectively. The probes yield products ofidentical length for Bp",
`B™", B* and B'. Primers were annealed to 4g of RNA and reactions were run according to
`manufacturer’s protocols. Radioactive bands were quantitated by phosphorimager analysis
`(BioRad). RNAisolated from A85.68 mice”wasusedas positive control. After correction for
`primer labelling, the human to mouse RNA signal was 29 + 1% per gene copy in repeated
`experiments (n > 8), in agrecment with previous findings based on RT-PCR”. Values measured
`in bone marrow chimaeras that were obtained in separate runs were standardized to the value
`obtained in the A85.68 RNA sample. In Figs. 2 and 3c, d, human B-globin expression18
`expressed per vector copy and normalized to the endogenoustranscripts (accounting for two
`endogenousalleles). In Fig. 3b, humantranscripts are reported as the fraction of total B-globin
`RNA(Huf / Hu® + MufB)to reflect absolute contribution of vector-encoded transcripts.
`
`Example 4
`To investigate the function of the vectors in vivo, we transduced and transplanted murine
`bone marrow cells without any selection in syngeneic,lethally irradiated recipient mice. Donor
`bone marrow wasflushed from the femursof 8- to 16-week-old male CS7BL/6 or Hbb®"™*
`(Jackson Laboratories) that had beeninjected intravenously (i.v.) 6 days earlier with 5-flurouracil
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`(5-FU, Pharmacia; 150 mg kg’ body weight). Bone marrow cells were resuspended in serum-
`free medium, and supplemented with IL-1a (10 ng mf’), IL-3 (100 U m1’), IL-6 (150 U m1’), Kit
`ligand (10 ng ml) (Genzyme), B-mercaptoethanol (0.5 mM;Sigma), _-glutamine (200 mM),
`penicillin (100 TU mI!) and streptomycin (100 pg m"), and cultured for 18 h. Recipient mice (11-
`to 14-week-old C57/BL6 or Hbb"** mice ) were irradiated with 10.5 Gy (split dose 2 x 5.25 Gy)
`on the day oftransplantation. Bone marrow cells were pelleted and resuspended in serum-free
`medium containing concentrated lentiviral supernatant, and supplemented with polybrene (8g
`mf’), ,-glutamine (200 mM), penicillin ( 100 TU mI”) and streptomycin (100 pg ml"), and
`cultured for 6 h. Transduced bone marrowcells (1 x 10° or 5 x 10°) were then i.v. injected into
`
`eachof the irradiated female recipients to cstablish short-term and long-term bone marrow
`
`chimaeras, respectively.
`In short-term studies, spleens were removed 12 d after transplantation to extract total
`RNAand genomic DNA. To monitor long-term chimaeras, two orthree capillary tubes of blood
`were collected every 4-6 weeks, from which genomic DNA,total RNA and haemoglobin were
`extracted. To examine vector function reliably in long-term animals, erythroid cell populations
`were purified from spleen. Single-cell suspensions were incubated with rat anti-mouse TER-119
`monoclonal antibody (PharMingen). Sheep anti-Rat IgG dynabeads (M-450, DynalInc.) Were
`added to the antibody-coated spleen cells and purified as recommended by the manufacturer.
`Vector copy number, integration pattern and chimaerism were determined by Southern blot
`analysis. The fraction of donor DNArelative to recipient was determined by stripping and
`reprobingthe blot with a [*P] dCTP-labelled probe specific for the Y chromosomeand
`normalizing to an endogenous mouse band. Radioactive bands were quantitated by
`phosphorimager analysis. Sera from five randomly selected long-term bone marrow chimaeras
`(30 weeksafter transplantation) tested negative for HIV-1 gag by RT-PCR usingthe Amplicor
`
`HIV-1 monitor kit (Roche).
`Vector copy number and human f-globin RNAtranscripts were measured during a 24-
`week period in mice transplanted with RNS1 (n = 8) or1'NS9 (n = 10) transduced bone marrow.
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`a, Vector copy number wasassessed by southern blot analysis of genomic DNA isolated from
`peripheral blood at weeks 6, 10, 16 and 24. The average vector copy numberin peripheral blood
`cells, measured periodically for 24 weeks (Fig. 4), showed highly efficient gene transfer with
`both vectors (1.8 + 0.6 and 0.8 + 0.6 average vector copies per cell for B-globin transcript levels
`in the 10-20% range during the same time period. To assess transcriptionalactivity per vector
`copy, steady-state RNA transcripts and vector copy number were quantified in pooled CFU-S,,
`and in erythroid TER-119+ spleen cells. Twelve days after transplantation, human B-globin
`expression per endogenousallele, (Fig. 5a). Twenty weekslater these values were 0.5 = 0.1%
`(significantly lower than on day 12, P = 0.02) and 15.8 + 0.9% respectively (Fig. 5b). These
`findings establishedthat the larger LCR fragments increased globin expressionin vivo and,
`furthermore, suggested that TNS9 is moreresistant to transcriptionalsilencing than is RNS1.
`The levels of TNS9-encoded human f-globin could be produced. Haemoglobintetramers
`incorporating vector-encoded human 6* and endogenous murine a-globin (designated Hbb™)
`were quantitated in peripheral bloodred cell lysates after cellulose acetate gel fractionation.
`Hbb™levels accounting for up to 13% of total haemoglobin were found 24 weeksafter
`transplantation (Fig. 3e, Table 1 in Supplementary Information). In the sameassays, transgenic
`mice bearing one copy of a 230-kb yeastartificial chromosome (YAC) encompassingthe entire
`human B-globin like gene cluster”’ showed 14% oftheir total haemoglobin incorporating human
`B*. No haemoglobin tetramers containing human B* were measurble in any ofthe mice bearing
`RNSI (table 1 in Supplementary Information). The proportion of mature peripheral blood red
`cells expressing human B* was elevate in most TNS9 bone marrow chimaeras, as shownby dual
`staining of human $4“ and TER-119. In contrast, chimaeras engrafted with RNS1-transduced
`bone marrow showedhighly variable fractions of weakly staining B“ -positive erythrocytes.
`Normalized to the fractionof circulating 8B-positive mature red cells, the levels of haemoglobin
`containing lentivirus-encoded B* were on average 64% of those obtained in the YACtransgenic
`
`mice.
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`Example 5
`To ascertain that true HSCs were transduced, we carried out secondary transplants using
`
`marrow from primary recipients collected 24 weeksafter transplantation. The TNS9 and RNS1
`vectors were readily detectedin all secondary recipients 13 weeks after transplantation. Human
`B-globin expression was maintainedin all recipients of TNS9-transduced marrow. The
`successful transduction of HSCs was confirmed by integration site analyses. Southern blot
`analysis was performed on genomic DNAisolated from bone marrow, spleen and thymus of
`secondary bone marrow transplant recipients collected 13 weeksafter transplant (one
`representative RNS1, and two representative TNS9 secondary transplantrecipients are shown).
`Two endogenous bandsare found in the genomic DNA of C57BL/6 (B6) mice.
`
`Example 6
`In view ofthe high levels of cxpression, we tested the extent to which the TNS9 vector
`could correct the phenotype of thalassaemic cells using P° -thalassaemic heterozygote micethat
`lack a copy oftheir bI and b2 B-globin genes (Hbb"**)’'. These heterozygotes have a clinical
`phenotype similar to humanthalassaemia intermedia and exhibit chronic anaemia (haematocrit
`28-30%, haemoglobin 8-9 g dl") and anomalies in red cell size (anisocytosis) and shape
`(poikilocytosis). Fifteen weeksafter transplantation with unselected TNS9-transduced Hbb""
`bone marrow,the haematocrit level, red blood cell count, reticulocyte count and haemoglobin
`
`level were markedly improvedin five out offive recipient mice (Fig. 6). Anisocytosis and
`poikilocytosis were markedly reduced in the peripheral blood smears of chimaeras bearing the
`TNS9 vector. Control mice transplanted with Hbb™* bone marrowcells transduced with a
`vector encoding enhanced greenfluorescent protein (eGFP)all remained severely anaemic (n = 5,
`Fig. 6) and maintained their abnormalred cell morphology. Theseresults establish that levels of
`circulating haemoglobin obtained with TNS9 were indeed therapeutically relevant.
`The combined effect of the high efficiency of gene transfer and the absence of vector
`
`rearrangements afforded by the recombinantlentivirus carrying the B-globin gene and LCR
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`configuration adopted in TNS9 yielded levels of human B* expression in the therapeutic range.
`The higher expression obtained with TNS9 compared with RNS| was associated with a higher
`fraction of permissive integration sites in MELcells and a higher fraction ofhuman *-
`containing red blood cells in bone marrow chimaeras. RNS1, which carries a weaker enhancer,
`silenced over time whereas TNS9retained undiminishedtranscriptional activity over the same
`
`time period and in secondary transplant recipients.
`Higherlevels of murine «,: human 6, tetramers were obtained in peripheral blood
`samples from recipients of TNS9-transduced Hbb"?* bone marrow (21 + 3% oftotal
`haemoglobin, n = 5, than with Hbb** bone marrow (6 + 4%, n + 10). The two groups showed
`comparable peripheral blood vector copy numbers and levels of human B-globin RNA (0.8 + 0.2
`compared with 0.8 + 0.6, and 16.8 + 6% compared with 10.8 + 7%, respectively). This
`observation is consistent with a competitive advantage of murine f-globin over human B-globin
`in associating with murinc «-globin”. In thalassaemicpatients, added human B-chain synthesis
`would improve the «:$ chain imbalance and thus increase red cell survival and ameliorate the
`ineffective erythropiesis in these patients. In patients with sickle cell disease, transduced B*
`chains are expected to have an advantage over the BS chains produced by both endogenous genes
`in competingfor the available «-chains”*. Given that patients with S/B-thalassaemia whose HbA
`represents 10-30% oftheir total haemoglobin are very mildly affected'”*, the clinical benefit of
`such an intervention would