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`SCOREPlaceholder Sheetfor IFW Content
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`Application Number: 12433412
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`BLUEBIRD EXHIBIT 1033
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`BLUEBIRD EXHIBIT 1033
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`DocCode — SCORE
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`SCOREPlaceholder Sheetfor IFW Content
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`Application Number: 12433412
`
`Document Date: 04/30/2009
`
`The presence of this form in the IFW record indicates that the following document type was
`received in electronic format on the date identified above. This content is stored in the SCORE
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`PTO/SB/05 (08-08)
`Approved for use through 06/30/2010. OMB 0651-0032
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`S48S8DV(51890)
`UTILITY
`
`
`PATENT APPLICATION [Firstinventor___|MichelSadelain__|
`VECTOR ENCODING HUMAN GLOBIN GENE
`TRANSMITTAL
`AND USE THEREOF IN TREATMENT OF
`
`(ONLY FOR NEW NONPROVISIONAL APPLICATIONS UNDER
`HEMOGLOBINOPATHIES
`37 CFR 1.53(B))
`Express Mail Label No.
`
`Commissionerfor Patents
`APPLICATION ELEMENTS ADDRESS TO:_P.O. Box 1450
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`See MPEP chapier 600 concerning utility patent application contents.
`Alexandria, VA 22313-1450
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`Both the claims and abstract must start on a new page
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`Ifa CONTINUING APPLICATION, check appropriate box, and supply the requisite information below and in the first sentence of the
`specification following the title, or in an Application Data Sheet under 37 CFR 1.76:
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`[| Continuation X|Divisional [| Continuation-in-part (CIP) of prior application No.: 10/1 88,221
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`
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`Prior application information: Examiner
`Maria Marvich
`Art Unit:
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`PTO/SB/14 (07-07)
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`Title of Invention|eMoGLOBINOPATHIES
`
`cat
`Application Data Sheet 37 CFR 1.76
`Application Number
`VECTOR ENCODING HUMAN GLOBIN GENE AND USE THEREOF IN TREATMENT OF
`
`The application data sheetis part of the provisional or nonprovisional application for which it is being submitted. The following form contains the
`bibliographic data arrangedin a format specified by the United States Patent and Trademark Office as outlined in 37 CFR 1.76.
`This document may be completed electronically and submitted to the Office in electronic format using the Electronic Filing System (EFS) or the
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`Secrecy Order 37 CFR 5.2
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`[_]
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`Applicant Information:
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`Applicant 1
`Applicant Authority @/nventor|()Legal Representative under 35 U.S.C. 117 CParty ofInterest under 35 U.S.C. 118
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`Prefix} Given Name Family Name Middle Name
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`
`Sadelain
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`State/Province
`Country of Residence i
`SicershipmieTORT[ET
`Mailing Address of Applicant.
`
`401 East 89th Street
`Address 1
`
`
`Address 2
`
`Apt. SK
`
`Applicant2
`
`
`
`Applicant Authority (@)Inventor|()Legal Representative under 35 U.S.C. 117 OpPatty of Interest under 35 U.S.C. 118
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`Stefano Rivella
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`736 West End Avenue
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`Postal Code
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`
`May
`Mr.
`Chad
`() Active US Military Service
`Residence Information (Select One) () US Residency
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`City|New York Country Of Residencei|US
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`PTO/SB/14 (07-07)
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`Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid OMB control number.
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`Attorney Docket Number|64836DIV(51590)
`Application Data Sheet 37 CFR 1.76
`—
`Application Number
`VECTOR ENCODING HUMAN GLOBIN GENE AND USE THEREOF IN TREATMENT OF
`HEMOGLOBINOPATHIES
`
`Title of Invention
`
`Mailing Address of Applicant:
`Address 1
`220 Avenue A
`
`
`
`Address 2
`Apt. 3C
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`New York
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`State/Province
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`NY
`
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`Applicant4
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`Mr.
`Joseph
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`Bertino
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`Enter either Customer Number or complete the CorrespondenceInformation section below.
`For further information see 37 CFR 1.33(a).
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`65488
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`
`
`Email Address
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`Lascenrat
`
`Application Information:
`VECTOR ENCODING HUMAN GLOBIN GENE AND USE THEREOF IN TREATMENT OF
`Title of the Invention
`HEMOGLOBINOPATHIES
`Attorney Docket Number] 64836DIVi51500) Small Entity Status Claimed
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`Nonprovisional
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`PTO/SB/14 (07-07)
`Approved for use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
`Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid OMB control number.
`
`Attorney Docket Number|64836DIV(51590)
`Application Data Sheet 37 CFR 1.76
`Application Number
`VECTOR ENCODING HUMAN GLOBIN GENE AND USE THEREOF IN TREATMENT OF
`
`Title of Invention|GeMmoGLOBINOPATHIES
`eighteen monthsafterfiling.
`
`Publication Information:
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`[_] Request Early Publication (Fee required at time of Request 37 CFR 1.219)
`
`Request Not to Publish. | hereby requestthat the attached application not be published under 35 U.S.
`C. 122(b} and certify that the invention disclosed in the attached application has not and will not be the subject of
`an application filed in another country, or under a multilateral international agreement, that requires publication at
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`Representative information should be provided for all practitioners having a power of attorney in the application. Providing
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`Customer Number 65488
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`Prior Application Status|Pending
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`Division of
`10188221
`2002-07-01
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`10188221
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`non provisional of
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`60301861
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`2001-06-29
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`Application Number
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`Continuity Type
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`Prior Application Number
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`Filing Date (YYYY-MM-DD}
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`10188221
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`non provisional of
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`60302852
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`2001-07-02
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`PTO/SB/14 (07-07)
`Approved for use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
`Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid OMB control number.
`
`
`Attorney Docket Number|64836DIV(51590)
`Application Data Sheet 37 CFR 1.76
`Application Number
`VECTOR ENCODING HUMAN GLOBIN GENE AND USE THEREOF IN TREATMENT OF
`
`Title of Invention|GeMmoGLOBINOPATHIES
`
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`Assignee1
`If the Assignee is an Organization check here.
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`Organization Name
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`Memorial Sloan-Kettering Cancer Center
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`1275 York Avenue
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`CFR 1.4(d) for the form of the signature.
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`|Signature[Peter C. Lauro/ Date (YYYY-MM-DD)| 2009-04-30
`
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`

`VECTOR ENCODING HUMAN GLOBIN GENE AND
`USE THEREOF IN TREATMENT OF HEMOGLOBINOPATHIES
`
`MSE.P-050
`Patent Application
`
`Statement Concerning Government Funding
`- This application was supported by funds provided under NHLBIgrant No.
`HLS7612. The United States government may havecertain rights in the invention.
`
`statement Concerning Related Applications
`
`This application claims the benefit of US Provisional Application No. 60/301,861
`
`flied June 29, 2001 and US Provisional Application No. 60/302,852 filed July 2, 2001, both of
`
`which are incorporated hereinby reference.
`
` Backeround of the Invention
`This application relates to a vector comprising a mammalian, and particularly a
`humanglobm gene and to the use thereof in treatment of hemoglobinopathies, including a-and
`R-thalessernia and sickle-cell disease.
`|
`Current treatment modalities for B-thalassemias consist of either red blood cell
`transfision plus iron chelation (which extends survival but is cumbersome, expensive and an
`unperfect therapy), or allogeneic bone marrowtransplant (which carries a lethal risk 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 hematopoietic
`stem cells, 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
`facingefforts to develop gene therapy vectors is the ability to produce therapeutically useful
`levels of a desired protein or peptide. The present invention provides a vector which is capable
`
`of providing therapeutically meaningful levels of human globin forsustained periods of time.
`
`-1-
`
`Page 9 of 248
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`

`

`This ability arises from the ability to transmit large genomic regulatory sequences that control
`
`M3K.P-050
`Patent Application
`
`expression of the therapeutic gene.
`
`Summary of the Invention -
`
`In accordance with the invention, a recombinant lentiviral vector is provided
`
`comprising:
`
`a region comprising a functional.globin gene; and
`(a)
`large portions of the 8-giobin jocus control regions which include
`{bo}
`large portions of DNase I hypersensitive sites HS2, HS3 and HS4. The regions may be the
`complete site or some lesser site which provides the same functionality as the specific sequences
`set forth below. This vector provides expression of B-globin when introduced inte a mammal,
`for example a human,
`in vivo. Optionally, the vector further comprises a region encoding a
`cihydrofolate reductase,
`Byincorporation of different globin genes, the vector of the invention may be
`used in treatment of hemoglobinopathies, including «- and B-thalessemia and sickle-cell disease.
`
`For example, hematopoietic progenitor or stem cells may be transformed ex vivo and then
`restored to the patient. Selection processes may be usedto increase the percentage of
`transformed cells in the returned population. For example, a selection marker which makes
`
`transformed ceils more drug resistant than un-transformed cells allows selection by treatment of
`the cells with the corresponding drug. Selection and/or enrichment may also be carried outin
`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 genomic structure of a recombinant onco-retroviral vector in
`
`accordance with the invention.
`
`Page 10 of 248
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`

`

`MSE.P-050
`Patent Application
`
`Fig. 2 showsthe genomic structure of recombinant onco-retroviral vector within
`
`the scope of the invention.
`
`Fig. 3 shows experimental results demonstrating increased mean §-globin
`
`expression in transduced MELcells.
`Fig, 4 shows the average vector copy number in peripheral blood cells, measured
`periodically for 24 weeks, which confirms showed highly efficient gene transfer in celis
`transduced with the vector ofthe invention.
`|
`|
`Figs. 5A and B show buman $-globin expression per endogenousallele 12 days
`
`ané 22 weeks after introduction of cells transduced with the vector of the invention.
`Fig. 6 shows haematocrit level, red blood cel! count, reticulocyte count and
`haemoglobin level fifteen weeks after transplantation with unselected TNS9-transduced Hbb™”
`
`bone marrow.
`
`Detailed Description of the Invention
`in a first aspect of the present Invention, a recombinant lentirviral vector is |
`provided comprising:
`
`(a)
`
`(b)
`
`ategion comprising a functional globin gene, and
`
`large portions cf the B-globin locus control regions, which mclude
`
`DNase ft hypersensitive sites HS2, HS3 and HS4.
`As used in the specification and claims hereof, the term “recombinantlentiviral
`vector”refers to an artificially created polynucleotide vector assembled from a 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
`ofwhich leads to a globin that does not produce a hemoglobinopathy phenotype, and which is
`effective to provide therapeutic benefits to an individual with a defective globin gene. The
`
`functional globin gené may encode a wild-type globin appropriate for a mammalian individuaito
`
`be treated, or it may be a mutant formof globin, preferably one which provides for superior
`
`~3-
`
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`MSK._P-050
`Patent Application
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`properties, for example superior oxygen transport properties. The fimctional globin gene
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`includes both exons and introns, as well as globin promoters and splice doners/acceptors.
`
`Suitably, the globin gene may encode a-globin, B-globm, or y-globin. B-globin promoters may’
`
`.
`be sued with each of the globin genes.
`The recombinant vectors of the invention also mclude large portions of the locus
`control region (LCR) which include DNase I hypersensitive sites H$2, HS3 and HS4. In prior
`
`studies, smaller nucleotide fragments spanning the core portions of HS2, HS3 and HS4 have
`been utilized. Sadelain et al. Proe. Nat'l Acad. Sci. (USA)92: 6728-6732 (1995); Lebouich et al.
`EMBO J. 13: 3065-3076 (1994). The term, “large portions” refers to portions of the locus contro}
`region which encompasslarger portions of the hypersensitive sites as opposedto previously
`tested fragments including only the core elements, The regions may be the completesite 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,
`addition, the locus control region has two introduced GATA-1 binding sites at the junction
`between HS3 and HS4. While not intending to be bound by any specific mechanism,it is
`believed that the incorporation ofthese transcription factor binding sites enhances the
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`In
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`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 6-globin gene are representedby filled and open boxes, The locations are
`indicated for the splice donor (SD), splice acceptor (SA), packaging region (ir), rev-response
`element (RRE), human 6-globin promoter (P) and 3'-B-globin enhancer (E). Thus, in the vector
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`TNS9, a functional 6-globin gene, which includes both the exons and introns of the gene and the
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`relevant control sequences from the human B-globin locus. These are combined with the large
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`_4.
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`MSKE.P-050 —
`Patent Application
`
`fragments of the locus control region. The 3.2 kb LCR assembled into dTNS9 consists of an 840
`bp HS? fragment (SnaBlL-BstXD, a 1308 bp HS3 fragment (HindIl-BamiD and a 1069 bp HS4
`fragment (BamHI-Banlt}.
`
`In a further aspect of the invention, the B-globin gene coding sequence can be
`exchanged and replaced with either the gamma globin gene(for sickle cell disease) or the alpha
`globin gene (for alpha-thalassemias).
`In one strategy, a Ncol-Pst I fragmentof the 6-globin gene
`
`is replaced with the corresponding Ncol-HindI] fragment of the gamma globin geneor the
`_ Neol-Pstl fragment of the human alpha globin gene. These fragmentsstart at the translational
`start of each globin gene (spanning the Ncolsite) and end past their respective polyadenylation
`signals. In the second strategy, chimeric genes can be generated by only swapping the coding
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`sequence of each one ofthe three exons of these genes. Thus, for the gammaglobin gene, the
`result is a vectorthat comprises the beta globin promoter, the beta globin 5’ untranslated region,
`the garoma exon 1 coding region, the gamma intron 1 the gamma exon 2, the beta intron 2, the
`gamma exon 3, and thebeta 3' untranslated region. Thus all the elements of the TNS9 vector
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`remain in place (promoter, enhancers, 5’ and 3' untranslated regions, the LCR elements, the 2
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`additional GATA-1 binding sifes and the introns of the beta globin gene(at least intron 2, which
`is most important).
`In a third strategy, the codon usage within exon 3 of the gamma globin gene
`can be modified so that its sequence will résemble as muchas possible that ofthe beta globin
`gene. The reason for testing this is that the beta globin geneis always the best expressed.
`Additional elements may be included in the vectors of the invention to facilitate
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`utilization of the vector in therapy. For example, the vector may include selectable markers, to
`confirm the expression of the vector or to provide a basis for selection of transformed cells over
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`untransformedcells, or control markers which allow targeted disruption of transformed cells, and
`thusthe selective removal of such cells should termination of therapy become necessary.
`Ina further specific embodiment, the vector of the invention Includes the mouse
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`PGK promotor and human dihydrofolate reductase (DHFR) ¢DNA as a transcriptional unit.
`Mutant forms of DHFR which increase the capacity of the DHFR to confer resistance to drugs
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`MSK.P-050
`Patent Application
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`such as methotrexate are suitabiy used. For example, single and double mutants of DHFR with
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`tnutations at amino acids 22 and 31 as described in commonly assigned PCT Publication No.
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`WO 97/33988, which is incorporated herein by reference, may be advantageouslyutilized.
`
`Fig. 2 shows the genomic structure of specific vector within the scope of the
`
`invention. The vector includes a deleted LTR, from -456 to -9 of HIV LTR and the POK.
`promoter (530 bp) from the murine phosphoglycerate kinase 1 gene. It also includes a DHFR-
`encoding region encoding human DHFR with s/f mutation at amino acid 22. The locus control
`region and the B-globin region are the same as in TSN9. This vectoris designated €TNS9-PD.
`This incorporation of DHFR into this vector provides transformed cells with a methotrexate-
`
`resistant phenotype. As a result, methotrexate, and other antifolates can be used, both in
`vitro and tn vivo as a selection to 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 NMBPR-P (0.5 mg/dose) and TMTX (0.5 mg dose) for five days, observed
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`levels of expressed human B-globin were much higher in mice transduced with dTNS9-PD
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`vectors after treatment with TMTX and NMBPR-Pforselection of transduced celis.
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`The vectors of the invention are used in therapy for treatmentof individuals
`suffering from hemoglobinopathies. In one embodimentofthe invention, hematopoietic
`
`progenitor or stem cells are transformed ex vive and then restored to the patient. As usedin the
`specification and claims hereof, the term “hematopoietic progenitor sand stem cells”
`encompasses hematopoietic cells and non-hematopoietic stem cells, e.g., embryonic stem cells,
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`hematopoietic stern cell precursors, or amy of the latter generated by nuclear transfer from a
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`somatic cell. It is know tn theart that efficient genes transfer into human embryonic stem cells
`can be achieved using lentiviral vectors.
`| —
`- Selection processes may be used to increase the percentage of transformed cells in
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`the returned population. For example, a selection marker which makes transformed. cells more
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`drug resistant than un-transformed cells allows selection by treatment of the cells with the
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`corresponding drug. When DHFRis used as a selection marker, it can be used for enrichment of
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`transduced cells in vitro, or for in vive selection to maintain the effectiveness of the vector.
`The invention will now be further described with reference to the following non-
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`MSE.P-050
`Patent Application
`
`limiting examples.
`
`Example J
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`To produce vector TNS9, the human 8-globin gene was subcloned from MB6L
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`(Sadelain et al. Proc. Natl Acad. Sci. (USA}92: 6728-6732 (1995)) into lentiviral vector
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`pHR’LacZ (Zuffery et al, Nature 15: 871-875 (1997)) replacing the CMV-LacZ sequence.
`pHR’eGFP was constracted by replacing LacZ with the eGFP sequence (Clontech). Viral stocks
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`were generated by triple transfection of the recombinant vectors pCMVARS.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 by ultracentrifugationm resuspended andtitrated as
`described in Gallardo et al., Blood 90: 952-957 (1997), For comparison, RSNIL was used which
`has a similar structure, except that the LCR contains on