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WO2025151744A1 - Globine améliorée et autres vecteurs lentiviraux destinés à une thérapie génique - Google Patents

Globine améliorée et autres vecteurs lentiviraux destinés à une thérapie génique

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Publication number
WO2025151744A1
WO2025151744A1 PCT/US2025/011122 US2025011122W WO2025151744A1 WO 2025151744 A1 WO2025151744 A1 WO 2025151744A1 US 2025011122 W US2025011122 W US 2025011122W WO 2025151744 A1 WO2025151744 A1 WO 2025151744A1
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cells
globin
seq
nucleotides
plasmid
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Frank PARK
Andrew WILBER
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Southern Illinois University System
Southern Illinois University Edwardsville
University of Tennessee Research Foundation
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Southern Illinois University System
Southern Illinois University Edwardsville
University of Tennessee Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present subject matter relates to recombinant lentiviral transfer plasmids for producing lentiviral vectors with increased vector titer and performance.
  • These transfer plasmids are constructed so that a central polypurine tract (cPPT) is centered between the lentiviral long terminal repeats (LTRs) and include a 6x stop codon sequence block downstream of the 3’ LTR and woodchuck post-transcriptional regulatory element (WPRE).
  • cPPT central polypurine tract
  • LTRs lentiviral long terminal repeats
  • WPRE woodchuck post-transcriptional regulatory element
  • Transduced cells, pharmaceutical compositions and methods of treating or ameliorating hemoglobinopathies with these lentiviral vectors are also provided. Additional transfer plasmids, cells, and compositions are provided that are useful for delivering any gene of interest to a cell, as well as plasmids for production of lentiviral vectors for treating other erythroid-specific diseases or disorders such as anemia and cancer.
  • BACKGROUND [0002] The severe hemoglobin disorders are prevalent genetic diseases transmitted through autosomal recessive mode of inheritance [1].
  • SCA sickle cell anemia
  • HbS ⁇ - chain of hemoglobin S
  • lentiviral vectors are the only integrating vector system that is available and is a requirement for effective ex vivo gene delivery methodologies. All other currently available viral vector systems are largely episomal and do not integrate. Therefore, proliferating target cells will lose vector genomes over time and result in a loss of the therapeutic gene and its effect. [0005] Another major benefit of the lentiviral vector system is a more expansive packaging capacity compared to the predecessor MMLV-based vector system. This enables the incorporation of larger gene expression cassettes, such as the inclusion of the ⁇ -globin genomic sequences and enhancer regions as previously described [15, 16].
  • All other vectors have designed their cPPT-containing lentiviral vectors using the pre-existing location either 5’ or 3’ to the rev-responsive element (RRE) and 5’ to any of the expression cassettes.
  • the newly configured design, with the repositioned cPPT and with the stop codons in all reading frames following the 3’ LTR, and external WPRE has a positive impact on gene transfer and vector- encoded ⁇ -globin production.
  • SUMMARY OF THE DISCLOSURE [0007] The present disclosure provides recombinant lentiviral vectors for gene therapy based on new and improved recombinant lentiviral transfer plasmids.
  • the recombinant lentiviral transfer plasmids comprise (a) a globin expression cassette which comprises sequences for an artificial ⁇ -globin locus control region (LCR), a central polypurine tract (cPPT), a first promoter, a globin gene functional to ameliorate a hemoglobinopathy, and a 3’ erythroid-specific enhancer, with these sequences being operably linked within the globin expression cassette to enable expression of the globin gene in a mammal when present in a cell; (b) a lentiviral vector expression cassette which comprises a second promoter, a 5’ LTR, lentiviral vector packaging sequences, the globin expression cassette, a 3’ LTR, and posttranscriptional regulatory elements, wherein the globin expression cassette is oriented in an antisense direction on the sense strand of the lentiviral vector expression cassette, wherein the cPPT is positioned approximately midway between the 5’ and 3’ LTRs of
  • the cPPT sequence is positioned no more than about 250 nucleotides upstream or downstream of the midpoint of the lentiviral vector cassette.
  • the midpoint of the cPPT sequence is positioned from about 10% to about 20% upstream or downstream of the midpoint between the first nucleotide of the 5’ LTR and the last nucleotide of the 3’ LTR.
  • the LCR is no larger than about 4 kb.
  • the LCR comprises HS2, HS3 and HS4. In embodiments of any of the foregoing or following subject matter, the said LCR consists essentially of HS2, HS3 and HS4.
  • the first promoter is a ⁇ -globin promoter.
  • the globin gene is selected from the group consisting of a ⁇ -globin gene, a ⁇ -globin gene, and a ⁇ -globin gene. In some of these embodiments, the globin gene is human ⁇ -globin gene.
  • human ⁇ -globin gene is a wild- type human ⁇ -globin gene or a mutant human ⁇ - globin gene.
  • the mutant human ⁇ -globin gene is the human ⁇ - globin gene T87Q mutant.
  • the 6x stop codon sequence block comprises the nucleotide sequence of GGATAAGACAGGACCTGGATGAGCAAGGCAATAGGATAGGCAAGGCATAGGATAA GACAGGCTATGGATAAGACAGGCCAGGATGAGACAGG (SEQ ID NO: 14).
  • the 3’ LTR comprises an insulator.
  • the lentiviral vector expression cassette is TAT-independent and self-inactivating (SIN).
  • the plasmid is SRT1, SRT2, SRT3, SRT4, SRT5, SRT8 or SRT11.
  • the globin transfer plasmid is SRT9.
  • Another aspect of the disclosure provides isolated lentiviral particles comprising the lentiviral vector encoded in any of the lentiviral transfer plasmids of the disclosure, as well as pharmaceutical compositions for transducing cells comprising an effective amount of the lentiviral particles of the disclosure in admixture with a pharmaceutically acceptable carrier.
  • a further aspect of the disclosure relates to hematopoietic cells transduced ex vivo with the lentiviral particles of the disclosure or a pharmaceutical composition of the disclosure.
  • the cells are selected from the group consisting of hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and hemogenic endothelium cells.
  • the hematopoietic stem cells are CD34 + hematopoietic stem cells.
  • the transduce cells are provided as a pharmaceutical composition for treating a hemoglobinopathy which comprises an effective amount of these cells and a pharmaceutically acceptable carrier.
  • the disclosure provides methods of treating or ameliorating a hemoglobinopathy in a subject which comprise administering an effective amount of the transduced cells of the disclosure or a pharmaceutical composition comprising those cells to the subject to thereby treat or ameliorate said hemoglobinopathy.
  • the cells are autologous, allogeneic, syngeneic or xenogeneic.
  • the methods further comprise (a) harvesting bone marrow cells from a subject; (b) selecting CD34+ cells from said bone marrow cells; and (c) expanding and/or conditioning said CD34+ cells to thereby produce the said hematopoietic cells.
  • the hemoglobinopathy is selected from the group consisting of hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, ⁇ -thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemia, and hemoglobin H disease.
  • the globin gene is selected from the group consisting of a ⁇ -globin gene, a ⁇ -globin gene, and a ⁇ -globin gene.
  • the globin gene is human ⁇ -globin gene.
  • human ⁇ -globin gene is a wild- type human ⁇ -globin gene or a mutant human ⁇ - globin gene.
  • the mutant human ⁇ -globin gene is the human ⁇ -globin gene T87Q mutant.
  • the heterologous gene encodes a transcription factor such as GATA1, KLF1, RUNX1, GATA2, HOXB2 or TAL1; a chromatin remodeler such as ATRX; a histone methyltransferase such as ASH1L; a chromatin assembly protein such as codanin1; a protein involved in vesicle formation such as SEC23B; a heme biosynthetic pathway enzyme such as ALAS-E, porphobilinogen deaminase, delta aminolevulinate dehydratase, ferrochelatase or CPO; or another enzyme such as PKLR, glutathione peroxidase, 15-lipoxygenase or carbonic anhydrase I.
  • a transcription factor such as GATA1, KLF1, RUNX1, GATA2, HOXB2 or TAL1
  • ATRX histone methyltransferase
  • ASH1L a chromatin assembly protein
  • codanin1 a
  • the LCR comprises an HS2 region consisting of the 853 nucleotides of SEQ ID NO: 1, an HS3 region consisting of the 999 nucleotides of SEQ ID NO: 4 and an HS4 region consisting of the 539 nucleotides of SEQ ID NO: 5.
  • any of these LCRS are present in an expression cassette operably linked to an erythroid-specific promoter and a heterologous gene, wherein said artificial LCR comprises an HS3 region consisting of the 999 nucleotides of SEQ ID NO: 4 or an HS4 region consisting of the 539 nucleotides of SEQ ID NO: 5.
  • the LCR of the expression cassette comprises an HS2 region consisting of the 853 nucleotides of SEQ ID NO: 1, an HS3 region consisting of the 999 nucleotides of SEQ ID NO: 4 and an HS4 region consisting of the 539 nucleotides of SEQ ID NO: 5.
  • the erythroid- specific promoter of the expression cassette is a ⁇ -globin promoter.
  • the expression cassette further comprises a 3’-erythroid-specific enhancer operably linked to the heterologous gene.
  • Functional titers of viral products were determined by quantitative PCR (qPCR) detection of lentiviral GAG sequences in genomic DNA isolated from transduced cells and are reported as transducing units (TU/mL). * P ⁇ 0.05 significant difference in viral titer between SRT2 versus SRT1.
  • Figure 4 Transduction of Human Erythroleukemia K562 Cells with Lentiviral Vectors Encoding for Erythroid-specific Expression of Green Fluorescence Protein (GFP).
  • GFP Green Fluorescence Protein
  • This table illustrates some of the differences between the SRT transfer plasmids relative to TNS9.3.55, including the vector length (which is the size of the integrated vector from the beginning of the 5’LTR to the end of the 3’ LTR), the length of the ⁇ -globin promoter, the lengths of the individual HS2, HS3, HS4 elements and the overall LCR length; all values are in base pairs.
  • the cPPT element the location is given relative to position 1 of the 5’ LTR.
  • the positions of HS2, HS3 and HS4 elements are the location of those sequences in GenBank sequence NG_052895.1.
  • nucleic acid molecule or “nucleic acid” is intended to include DNA molecules, RNA molecules (e.g., mRNA, shRNA, siRNA, microRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • RNA molecules e.g., mRNA, shRNA, siRNA, microRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g., mRNA, shRNA, siRNA, microRNA
  • the nucleic acid molecules of the disclosure may be single-, double-, or triple-stranded.
  • a nucleic acid molecule of the present disclosure may be isolated using sequence information provided herein and well known molecular biological techniques (e.g., as described in Sambrook et al., Eds., MOLECULAR CLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).
  • a “vector” refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide or nucleic acid, and which can be used to mediate delivery of the polynucleotide or nucleic acid to a cell, either in vitro or in vivo
  • illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles, such as viruses.
  • the polynucleotide or nucleic acid to be delivered sometimes referred to as a “target polynucleotide” or “transgene,” may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest).
  • Vector thus includes a biological entity, such as a lentivirus or other virus, used for the delivery of genes into an organism or introduction of foreign genes into cells.
  • Transfection or “transduction” as used herein, are terms referring to a process for the introduction of an exogenous polynucleotide, nucleic acid or vector into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant nucleic acids or virus, respectively, to introduce the exogenous polynucleotide to the host cell.
  • Transfer of a polynucleotide into a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot; and measurement of DNA and RNA by heterologous hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays.
  • Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as direct viral infection or non-viral plasmid transfection using methods such as lipids, electroporation, as well as other non-viral gene delivery applications into eukaryotic cells.
  • a “transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription. [0076] “Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner.
  • a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if those sequences or the promoter promotes transcription of the coding sequence.
  • Operably linked sequences or promoters are generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
  • “Heterologous” means derived from a genotypically distinct entity or location from the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a polynucleotide or protein coding sequence removed from its native location in a genome, and which is operably linked to (or otherwise associated with) at least one sequence element with which it is not normally associated (e.g., when cloned into a plasmid or vector), is a heterologous gene or coding sequence with respect to those sequence elements.
  • any sequence element, such as a promoter or a protein coding sequence, that is removed from its native sequence location and linked to a different, non-native sequence location is a heterologous element.
  • exogenous when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means.
  • An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell.
  • an exogenous nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.
  • “Transgenic” is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence.
  • the host cells are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.
  • the term “expression cassette” refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.
  • the expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid region.
  • the expression cassette portion can include a gene to be transcribed and elements that control the expression of the gene (e.g., a promoter, enhancer polyadenylation sequences and the like).
  • lentiviral vector refers, in context, to either the nucleic acid packaged within a lentiviral particle or to the particle itself. Lentiviral vector, LV vector and LVV are also used interchangeably herein with “lentiviral particle” or “lentiviral vector particle.”
  • the “ ⁇ -globin locus control region” or “LCR” was identified over 20 years ago and is a long-range, cis-acting regulatory region required for expression of globin genes in erythroid cells.
  • the ⁇ -globin LCR has five DNase I hypersensitive sites (HS): HS1, HS2, HS3, HS4 and HS5.
  • LCRs of the ⁇ -globin genes have been published, e.g., human [36, 37], mouse [38], rabbit [39] and goat [40].
  • the LCRs used in the present disclosure are artificial LCRs derived from the native LCRs.
  • the ⁇ -globin LCRs in the present disclosure confer erythroid specific enhancer activity of gene expression.
  • the term “recombinant” includes reference to a cell or a vector that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention or may have reduced or eliminated expression of a native gene.
  • the term “globin” refers to a family of heme-containing proteins that are involved in the binding and transport of oxygen. Subunits of vertebrate and invertebrate hemoglobins, vertebrate and invertebrate myoglobins or mutants thereof are included by the term globin, whether wild type or mutant.
  • an “effective amount,” “therapeutically-effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.
  • Artificial Locus Control Regions LCRs
  • the LCRs used in the transfer plasmids of the present disclosure are artificial ⁇ -globin LCRs (artificial LCRs are also referred to herein as modified LCRs; these terms are used interchangeably).
  • the transfer plasmids of the disclosure comprise a lentiviral vector cassette having a gene expression cassette inserted therein in the antisense directions (relative to transcription of the lentiviral vector cassette) and are an improvement over previous transfer plasmids for at least two reasons.
  • the lentiviral vector cassette in the transfer plasmids of the disclosure have a cPPT sequence positioned approximately midway between the 5’ and 3’ LTRs in the cassette and, second, the lentiviral vector cassette also includes a 6x stop codon sequence immediately following the 3’ LTR, external WPRE and poly(a) signal of the lentiviral vector cassette.
  • a “lentiviral vector expression cassette” comprises a promoter to control transcription of the lentiviral vector, the 5’ and 3’ LTRs for integration, packaging sequences, posttranscriptional regulatory elements and an antisense-facing (relative to the transcription direction of the lentiviral vector cassette) gene expression cassette located between the LTRs, with the cPPT sequence positioned in the central region of the lentiviral vector in a manner that does not disrupt the function of the other elements in the cassette (in other words, the cPPT sequence could be placed upstream or downstream of the promoter in the gene expression cassette provided it remains approximately centered between the LTRs).
  • the lentiviral transfer plasmid is a globin-specific transfer plasmid. In some embodiments, the lentiviral transfer plasmid is an erythroid-specific transfer plasmid.
  • exemplary lentiviral transfer plasmids are shown in Figs.1, 2 and 8.
  • A. Globin-specific transfer plasmids [00105] In accordance with the disclosure, certain embodiments relate to globin-specific transfer plasmids.
  • the marker gene is green fluorescent protein (GFP), it provides a simple method to assess viral vector transduction.
  • GFP green fluorescent protein
  • the presently disclosed subject matter provides globin expression cassettes which comprise sequences for an artificial ⁇ -globin LCR, a cPPT, a first promoter, a globin gene functional to ameliorate a hemoglobinopathy, and a 3’ erythroid-specific enhancer, wherein these sequences (also termed herein sequence elements) are operably linked within the globin expression cassette to enable expression of the globin gene in a mammal when present in a cell.
  • these sequence elements are present in 5’ to 3’order (for a sense strand) of the ⁇ -globin LCR, cPPT, promoter, globin gene and the 3’ erythroid-specific enhancer.
  • these sequence elements are present in 5’ to 3’order (for a sense strand) of the ⁇ -globin LCR, promoter, cPPT, globin gene and the 3’ erythroid-specific enhancer.
  • the cPPT sequence is within the HS2 region of the LCR.
  • the LCR comprises the HS sites from TNS9, which has the BstXI and SnaBI HS2-spanning nucleotide fragment, the BamHI and HindIII HS3-spanning nucleotide fragment and the BamHI and BanII HS4-spanning nucleotide fragment (see U.S. Patent No.7,541,179).
  • the LCR comprises the HS sites of TNS9.3.55 (which is 6 bp longer than those cloned into SRT1-5 for HS2).
  • the placement of the overall cPPT in the any of the plasmids and vectors of the disclosure can be calculated and, if needed, adjusted to avoid disruption of other nearby sequence elements.
  • the middle of the sequence between the two LTRs corresponds to the midpoint of the integrated genome length.
  • cPPTs are well known in the art and typically range from about 115 to about 118 nucleotides [27 and 41-57]. In some embodiments, the cPPT is 118 nucleotides. In some embodiments, the sequence of the cPPT is as follows: TTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGAC ATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCA AAATTTT (SEQ ID NO: 15). c. ⁇ -globin promoter [00122] In accordance with the present disclosure, the globin expression cassette further comprises a ⁇ -globin promoter.
  • the ⁇ -globin promoter is positioned between the globin gene or functional portion thereof and the ⁇ -globin LCR.
  • the length and the sequence of the ⁇ -globin promoter can vary.
  • the ⁇ -globin promoter is from about 100 bp to about 1600 bp in length, e.g., from about 200 bp to about 700 bp, from about 100 bp to about 200 bp, from about 200 bp to about 300 bp, from about 300 bp to about 400 bp, from about 400 bp to about 500 bp, from about 500 bp to about 600 bp, from about 600 bp to about 700 bp, from about 700 bp to about 800 bp, from about 800 bp to about 900 bp, from about 900 bp to about 1000 bp, from about 1000 bp to about 1100 bp, from about 1100 bp to about 1200 bp, from about 1200 bp, from about 1
  • the ⁇ -globin promoter is a human ⁇ -globin promoter that is about 130 bp, about 613 bp, about 663 bp, about 265 bp, about 316 bp or about 1555 bp, in length. In certain embodiments, the ⁇ -globin promoter is a human ⁇ -globin promoter that is about 663 bp in length. In certain embodiments, the ⁇ -globin promoter is a human ⁇ -globin promoter that is about 316 bp in length. d. Globin Genes [00123] In accordance with the present disclosure, the globin expression cassette comprises a globin gene functional to ameliorate a hemoglobinopathy.
  • the globin gene comprises a non-wild-type (mutated or modified) human ⁇ -globin gene.
  • the human ⁇ -globin gene comprises a human ⁇ - globin gene with a deletion in intron 2 (IVS2).
  • the deletion in IVS2 is about 370 bp.
  • the deletion in IVS2 can eliminate AT-rich (ATR) sequences that comprise a cryptic polyadenylation site responsible for premature termination of the transcription.
  • the human ⁇ -globin gene comprises a human ⁇ -globin gene encoding a threonine to glutamine mutation at codon 87 ( ⁇ T87Q ).
  • a functional portion of a globin gene has at least about 80%, at least about 90%, at least about 95%, at least about 99% or at least about 100% identity to a corresponding wild-type reference polynucleotide sequence.
  • the human ⁇ -globin gene is a human ⁇ -globin gene encoding a threonine to glutamine mutation at codon 87 ( ⁇ T87Q ).
  • the human ⁇ - globin gene encoding a threonine to glutamine mutation at codon 87 ( ⁇ T87Q ) further comprises a deletion in intron 2 (e.g., an about 370 bp deletion).
  • the human ⁇ -globin gene is a non-wild-type human ⁇ -globin gene selected from a human ⁇ -globin gene comprising one or more deletions of intron sequences, a human ⁇ -globin gene encoding at least one anti-sickling amino acid residue, and a human ⁇ - globin gene comprising one or more deletions of intron sequences and encoding at least one anti- sickling amino acid residue.
  • the globin expression cassettes of the disclosure may optionally further comprise a 3’ erythroid-specific enhancers and poly(A) signal sequences operably linked with the LCR, promoter and globin gene to control or regulate expression of the globin gene in a mammal. Additional sequence elements which enhance expression or regulate erythroid-specificity may also be present in the globin expression cassettes of the disclosure.
  • the 3’erythroid-sepecific enhancer is a human ⁇ -globin 3’ enhancer and is positioned downstream of the globin gene.
  • the human ⁇ - globin 3’ enhancer has the nucleotide sequence as found at nucleotides 2270...2885 of SRT2 (Table 7) –in its form as the complement.
  • Polyadenylation (poly(A)) signal sequences ensure proper mRNA transport, processing, and stability and are generally included in the globin expression cassettes of the disclosure. Poly(A) signals are well known in the art and any poly(A) signal can be used. 2.
  • sequence elements can be included in the lentiviral expression cassettes of the disclosure, including but not limited to, a deleted gag gene ( ⁇ gag), RRE and insulators.
  • Useful promoters, packaging signals ( ⁇ ), LTRs, packaging signals, ⁇ gag, and RRE are all known in the art.
  • the promoter is a strong promoter such as a CMV promoter.
  • the LTRs are self-inactivating.
  • the LTRs are TAT-independent.
  • the 3’ LTR comprises an insulator.
  • the insulator is the A1 insulator.
  • the lentiviral vector expression cassettes hereof comprise posttranscriptional regulatory elements that are adjacent to and downstream from the 3’ LTR. These elements are, in 5’ to 3’ order, a woodchuck post-transcriptional regulatory element (WPRE; or an equivalent such element), a poly(A) signal and a sequence block encoding stop codons in all six reading frames (referred to interchangeably herein as “6x stop codons” or “a 6x stop codon sequence block”).
  • WPRE woodchuck post-transcriptional regulatory element
  • 6x stop codons or “a 6x stop codon sequence block”.
  • these three sequence elements need not be contiguous with each other but should be within a few to about 50 bps.
  • repositioning the WPRE outside of the lentiviral vector and/or adding the 6x stop codons improves vector titers.
  • these elements ensure efficient termination and transport of the lentiviral nucleic acid to be packaged into the lentiviral vector that in turn will deliver the globin gene.
  • the WPRE and other equivalent sequences are well known in the art as are poly(A) signals that are useful with the lentiviral vector expression cassettes.
  • the poly(A) signal is the bovine growth hormone poly(adenylation) signal.
  • the 6x stop codons can comprise any short sequence of DNA that provides stop codons, i.e., TAA, TAG and TGA, in all possible reading frames.
  • a 6x stop codon element is GGATAAGACAGGACCTGGATGAGCAAGGCAATAGGATAGGCAAGGCATAGGATAA GACAGGCTATGGATAAGACAGGCCAGGATGAGACAGG (SEQ ID NO: 14).
  • the plasmid backbone can be from any plasmid suitable for replication in bacteria, yeast or other organism, and may, optionally, provide antibiotic resistance genes or gene sequences suitable for use as selectable markers.
  • erythroid-specific transfer plasmids of the disclosure are recombinant lentiviral transfer plasmids comprising: (a) an erythroid-specific expression cassette which comprises sequences for an artificial ⁇ -globin locus control region (LCR), a central polypurine tract (cPPT), a first promoter, a heterologous gene, and a 3’ erythroid-specific enhancer, with these sequences being operably linked within the erythroid-specific expression cassette to enable expression of the heterologous gene in a mammal when present in a cell; (b) a lentiviral vector expression cassette which comprises a second promoter, a 5’ LTR, lentiviral vector packaging sequences, the erythroid-specific expression cassette, a 3’ LTR, and posttranscriptional regulatory elements, wherein the erythroid-specific expression cassette is oriented in an antisense direction on the sense strand of the lentiviral vector cassette, wherein the cPPT is
  • heterologous genes present in the erythroid-specific expression cassette can be any gene whose product is desired for expression in an erythroid cell.
  • the heterologous gene is expressed at a level to provide therapeutic benefit.
  • the heterologous gene is for treatment of a hematological disease or disorder, including anemia, cancer, blood clotting disorders, and the like.
  • Heterologous genes for use herein include but are not limited to, the following oligonucleotides and polynucleotides: • An siRNA, shRNA or other RNA that controls or regulates expression of any gene specific to an erythroid cell.
  • Lentiviral Vector Expression Cassette [00144] The lentiviral vector expression cassette for the erythroid-specific-transfer plasmids are as described above in Section A.2.
  • these expression cassettes comprise at least the following sequence elements: a second promoter (to control expression of the lentiviral vector expression cassette), a 5’ LTR, lentiviral vector packaging sequences, the erythroid-specific expression cassette, a 3’ LTR, and posttranscriptional regulatory elements, wherein the erythroid-specific expression cassette is oriented in an antisense direction on the sense strand of the lentiviral vector cassette, wherein the cPPT is positioned approximately midway between the 5’ and 3’LTRs of the lentiviral vector cassette, and wherein the post transcriptional regulatory elements are adjacent to and downstream of the 3’ LTR and comprise a woodchuck post- transcriptional regulatory element, a polyadenylation signal and a 6x stop codon sequence block, in that order.
  • the LCRs are removed, and the promoters and enhancers for controlling gene expression are tailored as needed based on the gene to be expressed and the tissue-specificity or desired spatial or timing of expression.
  • the cPPT is positioned approximately midway between the 5’ and 3’ LTRs of the lentiviral vector expression cassette- without disrupting other elements present in the gene expression cassette. Any of the above contemplated combination of the remaining sequence elements, lentiviral vector expression cassettes and plasmid backbones are contemplated for use with the cPPT-centered transfer plasmids of the disclosure.
  • cPPT-centered transfer plasmids of the disclosure are recombinant lentiviral transfer plasmids comprising: (a) a gene expression cassette which comprises sequences for, in 5’ to 3’ order, (i) a central polypurine tract (cPPT) and a promoter or (ii) a promoter and a cPPT, and a gene of interest, said sequences operably linked for expression of said gene of interest; (b) a lentiviral vector expression cassette which comprises a second promoter, a 5’ LTR, lentiviral vector packaging sequences, the gene expression cassette, a 3’ LTR, and posttranscriptional regulatory elements, wherein the gene expression cassette is oriented in an antisense direction on the sense strand of the lentiviral vector expression cassette, wherein the cPPT is positioned approximately midway between the 5’ and 3’ LTRs of the lentiviral vector expression cassette, and wherein the post transcriptional regulatory elements are adjacent to and downstream of
  • the gene expression cassettes comprise sequences for a central polypurine tract (cPPT), a first promoter, a gene desired for expression, and, optionally, a 3’ enhancer, with these sequences being operably linked within the gene expression cassette to enable expression of the gene desired for expression.
  • Other sequence elements including a poly(A) signal may be present.
  • the gene is expressed at therapeutic levels to ameliorate a disease or condition for which delivery of the gene leads to a therapeutic benefit. Any of the relevant sequence elements described herein for the globin gene expression cassette are useful in the gene expression cassettes of the disclosure.
  • the cPPT sequence is also positioned as described herein.
  • the genes present in the gene expression cassette can be any gene whose product is desired for expression by a lentiviral vector, i.e., a gene of interest.
  • the gene product is expressed at a level to provide therapeutic benefit.
  • the gene of interest is for treatment of a hematological disease or disorder, including anemia, cancer, blood clotting disorders, and the like.
  • the gene of interest is any of the heterologous genes listed above as well as coagulation factors VIII and IX, and alpha-1- antitrypsin.
  • a “gene” encodes a gene product that is a peptide, protein, oligonucleotide or polynucleotide.
  • the coding sequence may be present as encoded in the native form of the gene product, e.g., with exons and introns, as a spliced form (as found in cDNA), or in some smaller form of the gene (e.g., missing introns, as a splice variant or having smaller introns) provided a functional gene product is produced.
  • the lentiviral vector expression cassette for the cPPT-centered transfer plasmids are as described above in Section A.2. Accordingly, these expression cassettes comprise at least the following sequence elements: a second promoter (to control expression of the lentiviral vector expression cassette), a 5’ LTR, lentiviral vector packaging sequences, the gene expression cassette, a 3’ LTR, and posttranscriptional regulatory elements, wherein the gene expression cassette is oriented in an antisense direction on the sense strand of the lentiviral vector cassette, wherein the cPPT is positioned approximately midway between the 5’ and 3’LTRs of the lentiviral vector cassette, and wherein the post transcriptional regulatory elements are adjacent to and downstream of the 3’ LTR and comprise a woodchuck post-transcriptional regulatory element, a polyadenylation signal and a 6x stop codon sequence block, in that order.
  • Plasmid backbone for the cPPT-centered transfer plasmids are as described above in Section A.3. Hence, the plasmid backbone can be from any plasmid suitable for replication in bacteria, yeast or other organism, and may, optionally, provide antibiotic resistance genes or gene sequences suitable for use as selectable markers. Lentiviral Particles and Production Thereof [00153] Another aspect of the disclosure relates to lentiviral vectors (LVVs) produced from the lentiviral transfer plasmids of the disclosure.
  • the LVVs comprise the lentivirus genome encoded in a lentiviral transfer plasmid of the disclosure which has been encapsulated by envelope glycoproteins.
  • the envelope glycoproteins are supplied in trans by either helper plasmids or by a packaging cell line.
  • the glycoproteins can be from HIV or other lentivirus or can be pseudotyped by using a glycoprotein from another virus such a vesicular stomatitis virus (VSV- G), lymphocytic choriomeningitis virus (LCMV), measles viral (MV), hepatitis C virus (HCV) and the like.
  • VSV- G vesicular stomatitis virus
  • LCMV lymphocytic choriomeningitis virus
  • MV measles viral
  • HCV hepatitis C virus
  • the envelope glycoprotein is VSV-G.
  • transient transfection systems are used with one or more helper plasmids and a lentiviral transfer plasmid of the disclosure.
  • transfection systems are well known in the art and include, but are not limited to, triple and quadruple transfection systems.
  • the triple transfection system uses a transfer plasmid with two helper plasmids.
  • helper plasmids pCMV ⁇ R8.9, pCMV ⁇ R8.74, or similar constructs
  • pMD.G envelope protein-expression
  • helper plasmids pALD-Rev-K, pALD-Gagpol-K, and pALD-VSV-G-K as supplied from the pALD-Lenti System (Aldevron) for transfection into 293T cells.
  • Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer plasmid to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods.
  • the method comprises contacting cells with an LVV of the disclosure and selecting for transduced cells.
  • the cell can be autologous to the subject (i.e., from the subject) or it can be non-autologous (i.e., allogeneic or xenogeneic) to the subject.
  • the LVVs of the disclosure which express a globin gene or other gene in an erythroid- specific manner are particularly useful in the transduction of human hematopoietic progenitor cells or hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like.
  • preferred targets are CD34+ cells.
  • the present disclosure is directed to a method for transducing a human hematopoietic stem cell comprising contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentiviral vectors under conditions to affect the transduction of a human hematopoietic progenitor cell in said population by the vector.
  • the stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject.
  • the human stem cell can be removed from a human, e.g., a human patient, using methods well known to those of skill in the art and transduced as described above.
  • the transduced stem cells are then reintroduced into the same or a different human.
  • the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 ⁇ 10 5 to 50 ⁇ 10 5 transducing units of the viral vector per 10 5 cells.
  • MOI multiplicities of infection
  • the methods involve isolating cell populations, e.g., stem cells or bone marrow cells, from a subject, optionally expanding the cells in tissue culture, and transducing those cells with a lentiviral vector whose presence results in production of a globin gene in the cells. The transduced cells are then returned back to the subject, where, for example, they may provide a population of red blood cells that produce the globin in therapeutically effective levels.
  • cell populations e.g., stem cells or bone marrow cells
  • a population of cells which may be cells from a cell line or from an individual other than the subject, can be used.
  • Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc., in patients undergoing chemotherapy.
  • stem cells can be derived from a number of sources including bone marrow (BM), cord blood (CB) CB, mobilized peripheral blood stem cells (mPBSC), and the like.
  • one aspect of the present disclosure relates to various methods of treating or ameliorating a hemoglobinopathy in a subject with the present LVVs or hematopoietic cells transduced with the present LVVs to stably produce a globin.
  • the method for treating or ameliorating a hemoglobinopathy in a subject comprises administering an effective amount of transduced cells of the disclosure or a pharmaceutical composition comprising such cells to a subject.
  • Such treatments may increase the subject’s hemoglobin levels in an amount sufficient to treat or ameliorate the hemoglobinopathy.
  • such treatments may restore the subject’s ability to produce red blood cells containing normal/functional hemoglobin to and thereby treat or ameliorate the hemoglobinopathy.
  • the transduced cells can be autologous cells, allogeneic cells, syngeneic cells or xenogeneic cells. Selection of the appropriate cells for a subject and methods for harvesting and transducing those cells are known in the art. In any of the embodiments of any of the methods hereof, the hematopoietic cells are obtained from bone marrow of a subject.
  • the hemoglobinopathy for treatment of amelioration is selected from the group consisting of hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, ⁇ - thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemia, and hemoglobin H disease.
  • the globin gene is selected from the group consisting of a ⁇ -globin gene, a ⁇ -globin gene, and a ⁇ -globin gene.
  • the globin gene is human ⁇ - globin gene.
  • the human ⁇ -globin gene is a wild- type human ⁇ -globin gene.
  • the human ⁇ -globin gene is a mutant human ⁇ -globin gene.
  • human ⁇ -globin gene encoding at least one anti-sickling amino acid residue is selected from the group consisting of a human ⁇ -globin gene encoding a threonine to glutamine mutation at codon 87 ( ⁇ T87Q ), a human ⁇ -globin gene encoding a glutamic acid to alanine mutation at codon 22 ( ⁇ E22A ), a human ⁇ - globin gene encoding an asparagine to lysine mutation at codon 80 ( ⁇ N80K ), a human ⁇ -globin gene encoding a glutamic acid to alanine mutation at codon 22 and an asparagine to lysine mutation at codon 80, and a human ⁇ -globin gene encoding a glutamic acid to alanine mutation at codon 22 and a threonine to glutamine mutation at codon 87.
  • the presently disclosed subject matter is for use in gene therapy methods to treat, prevent, or ameliorate a hemoglobinopathy that is selected from the group consisting of: hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, ⁇ -thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemia, and hemoglobin H disease.
  • a hemoglobinopathy is selected from the group consisting of: hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, ⁇ -thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemia, and hemoglobin H disease.
  • the hemoglobinopathy is ⁇ -thalassemia.
  • the hemoglobinopathy is sickle cell anemia.
  • the methods of the disclosure provide a method of treating or ameliorating anemia, cancer, a blood clotting disorder or other disease or disorder associated with erythroid cells in a subject by comprises administering an effective amount of cells transduced with any of the LVVs to a subject to thereby treat or ameliorate the anemia, cancer, a blood clotting disorder or other disease or disorder associated with erythroid cells.
  • the cells are autologous, allogeneic, syngeneic or xenogeneic cells.
  • Pharmaceutical Compositions, Administration and Dosing [00177] The present disclosure further provides pharmaceutical compositions comprising a LV vector of the disclosure, together with a pharmaceutically acceptable carrier, excipient or vehicle.
  • pH buffering agents may be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof.
  • TIS tris(hydroxymethyl)aminomethane
  • TAPS N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid
  • ammonium bicarbonate diethanolamine
  • histidine which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof.
  • pharmaceutically-acceptable salt refers to the salt of the compounds. As used herein a pharmaceutically-acceptable salt retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound.
  • Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts.
  • Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Examples of basic salts include salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and ammonium ions + N(R 3 ) 3 (R 4 ), where R 3 and R 4 independently designate optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl, and more specifically, the organic amines, such as N, N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • alkali metals such as sodium and potassium
  • alkaline earth metals such as calcium and magnesium
  • R 3 and R 4 independently designate optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl
  • the pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.
  • compositions may be formulated for any suitable route and means of administration.
  • Pharmaceutically-acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.
  • An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • Parenteral administration is typically associated with injection at or in communication with the intended site of action, including infraorbital, intraarterial, intracapsular, intracardiac, intracerebroventricular, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.
  • Pharmaceutical compositions of the disclosure may be administered alone or in combination with one or more other therapeutic agents.
  • a combination therapy may include an LV vector of the disclosure combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated.
  • agents include, inter alia, a psychoactive drug, anti-inflammatory or anti-proliferative agent, growth factors, cytokines, an analgesic, a therapeutically-active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates expression of one or more genes, one or more modifiers of signaling pathways and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.
  • pharmaceutically acceptable carrier includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the LV vector may be coated in a material or materials intended to protect it from the action of acids and other natural inactivating conditions to which the LV vector may encounter when administered to a subject by a particular route of administration.
  • a pharmaceutical composition of the disclosure also optionally includes a pharmaceutically acceptable antioxidant.
  • Exemplary pharmaceutically acceptable antioxidants are water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lec
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • Compositions of the disclosure may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Such media and reagents for pharmaceutically active substances are known in the art.
  • compositions of the disclosure may include any conventional media or agent unless any is incompatible with the LV vectors of the disclosure.
  • Supplementary active compounds may further be incorporated into the compositions.
  • Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage.
  • the composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art.
  • isotonic agents e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition.
  • Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as, e.g., sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • acids or bases such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Sterile injectable solutions may be prepared by incorporating an LV vector in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration.
  • Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains dispersion medium and other ingredients, such as those described above.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • An isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • a pharmaceutical composition of the present disclosure may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case-by-case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient.
  • a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure depends on the specific characteristics of the active compound and the particular therapeutic effect(s) to be achieved, taking into consideration the treatment and sensitivity of any individual patient.
  • the dosage range will generally be from about 1 x 10 6 transduced cells to 1 x 10 8 transduced cells per host body weight (in kg). Exemplary doses are greater than or equal to about 9 x 10 6 transduced cells per kg.
  • a selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular transduced cells of the disclosure or the LV vectors of the disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.
  • factors such as pharmacokinetic factors, including the activity of the particular transduced cells of the disclosure or the LV vectors of the disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being
  • transduced cells of the disclosure or the LV vectors of the disclosure may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention or lessening of impairment or disability due to the disease affliction.
  • the transduced cells of the disclosure or the LV vectors of the disclosure or composition of the present disclosure may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and/or mode of administration will vary depending upon the desired results.
  • Routes of administration for transduced cells of the disclosure or the LV vectors of the disclosure and compositions containing such vectors include, e.g., intracerebroventricular, intravenous, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraperitoneal, subcuticular, intraarticular, subcapsular, subarachnoid, epidural and intracisternal magna injection and infusion.
  • the changes made relative to TNS9.3.55, as depicted schematically in Fig.1, include (a) incorporation of an A1 insulator [30] in the 3’LTR; (b) repositioning the cPPT sequence [25] to be approximately centered between the 5’ and 3’ LTRs; (c) use of the ⁇ -globin T87Q variant; (d) addition of 6 stop codons (2 x 3 different frameshifts) at the end of the bovine growth hormone poly(A) sequence; and/or (e) use of alternative backbones, including a Nanoplasmid® (Aldevron), pUC57-Kan (GenScript) and pUC57-Brick-Kan (GenScript).
  • each transfer plasmid and its SEQ ID NO: is provided in in Table 3B, with the nucleotide sequence for each plasmid provided in the accompanying Sequence Listing. Table 8, following Example 14, provides the sequence location for the various sequence elements in SRT2.
  • Table 3A SRT Transfer Plasmid Elements Transfer cPPT A1 ⁇ -globin 6x stop Backbone Plasmid insulator n n Table 3B.
  • the TransIT lentiviral vector system (Mirus, Madison, WI) was used with a total of 21 ⁇ g total plasmid per 10-cm dish and 2.5 ⁇ L per ⁇ g DNA of the TransIT transfection solution as follows: 2.5 ⁇ g pALD-VSV-G-K plasmid, 2.5 ⁇ g pALD-Rev-K plasmid, 6 ⁇ g pALD-Gag-pol plasmid, and 10 ⁇ g transfer plasmid.
  • the plates were placed into an incubator at 5% CO 2 :95% O 2 for 60-72 hours.
  • Genomic DNA isolated from cell line (GA8) harboring a single copy lentiviral vector was serially diluted from 100 ng to 0.1 ng and quantitative PCR performed using primer-probe sets to lentiviral vector ⁇ sequence or the cellular control gene RNaseP to establish standard curves for VCN calculations.
  • the D432-GFP control virus titer was 9.2x10 5 TU/mL based on flow cytometry.
  • the flow cytometry histograms demonstrating GFP expression are shown in Fig.4B with the percentage of GFP+ cells plotted as a function of VCN shown in Fig.4C.
  • Fig.4D shows the VCN as a function of viral particles added to the K562 cells.
  • K562 cells were seeded at 1x10 5 per well in a 12-well plate and transduced with 10, 30 or 100 microliters of SRT1 or SRT2 lentiviral vector in a total volume (2 mL) of complete growth medium supplemented with 100 ⁇ g/mL F108 (Fig.5A). Cultures were grown for 7 days for total RNA isolation, 10 days for genomic DNA isolation, or 14 days for flow cytometry analysis of intercellular ⁇ -globin protein using clone 37-8; AlexaFluor 488 (Santa Cruz Biotechnology) at 1:500 dilution according to manufacturer’s instructions.
  • RNA transcript levels are shown in Fig.5B and the intracellular ⁇ -globin protein expression is shown in Fig.5C for all the transduced cell populations for SRT1 (left panels) and SRT2 (right panels).
  • the average VCN for each transduction is reported below the X-axis with the exception for SRT1 at 100 microliter volume which was not determined (nd).
  • Example 5. MOI Vector Testing A.
  • K562 cells were seeded at 1x10 5 per well in a 12-well plate and transduced with SRT1 or SRT2 lentiviral vector at equivalent MOI (multiplicity of infection) of 0.3, 1, 3, and 10 in a total volume of 2 mL of complete growth medium supplemented with 100 ⁇ g/mL F108 (Fig. 6A). Cultures were grown for 7 days for total RNA isolation, 10 days for genomic DNA isolation, or 14 days for flow cytometry analysis of intercellular ⁇ -globin protein using clone 37- 8; AlexaFluor 488 (Santa Cruz Biotechnology) at 1:500 dilution according to manufacturer’s instructions.
  • SRT1 or SRT2 lentiviral vector at equivalent MOI (multiplicity of infection) of 0.3, 1, 3, and 10 in a total volume of 2 mL of complete growth medium supplemented with 100 ⁇ g/mL F108 (Fig. 6A). Cultures were grown for 7 days for total RNA isolation, 10 days for genomic DNA isolation,
  • RNA transcript levels are shown in Fig.6B and the intracellular ⁇ -globin protein expression is shown in Fig.6C for all the transduced cell populations for SRT1 (left panels) and SRT2 (right panels).
  • the average VCN for each transduction is reported below the X-axis.
  • the log of the percentage of intracellular ⁇ -globin protein expression obtained in Fig.6 is plotted as a function of the log of the vector copy number for cells transduced with SRT1 ( ⁇ ) or SRT2 ( ⁇ ) (Fig.7). Two independent experiments were conducted, and the average of that data is presented in Fig.6 and Fig.7.
  • LTR integrity PCR assay Lentiviral vector preparations for SRT1 and SRT2 were tested on K562 cells to determine stability of vector integration. Viral particles were normalized based on the functional titer and applied to cells at equivalent MOI. Genomic DNA was extracted and used for PCR analysis.
  • PCR primers were obtained from Integrated DNA Technologies (IDT) for the sense: U3(+) 5’- GGAAGGGCTAATTCACTCCC-3’ (SEQ ID NO: 16); and antisense direction: R(-) 5’- GGGTTCCCTAGTTAGCCAGAG-3’ (SEQ ID NO: 17); and U5(-) 5’- CTCTAGTTACCAGAGTCACAC-3’(SEQ ID NO: 18).
  • the primers were used at a concentration of 1 mM, with the genomic DNA and the Platinum Blue PCR supermix (Invitrogen).
  • SRT8 Three additional lentiviral transfer plasmids were designed and synthesized, SRT8, SRT9 and SRT11.
  • SRT8 (1) the HS3 and HS4 LCR regions were reduced in size to 999 and 539 bp, respectively, and (2) the ⁇ -globin promoter was shortened to 316 bp.
  • SRT9 is the same as SRT8 except that the GFP gene was incorporated in frame with the globin gene to enable transduced cells to be readily marked by the detection of GFP fluorescence.
  • SRT11 is the same as SRT8 except that the A1 insulator was incorporated into the 3’ LTR region as in the plasmid constructs of Example 1.
  • SRT8, SRT9 and SRT11 plasmids have the pUC57-Kan backbone and are schematically depicted in Fig.8; their various elements are also summarized in Table 3A.
  • the sizes of these three transfer plasmids and their SEQ ID NO: is provided in in Table 3B, with the nucleotide sequence for each plasmid provided in the accompanying Sequence Listing.
  • the sizes of each HS region in the modified LCRs in all plasmid constructs and the size of the ⁇ -globin promoter are provided in Table 5. Table 5.
  • helper packaging plasmids were supplied from the pALD-Lenti System (Aldevron) to express in trans the accessory and structural proteins, pALD-Rev-K, pALD-Gagpol-K and envelope protein, pALD- VSV-G-K.
  • Aldevron the pALD-Lenti System
  • pALD-Rev-K the accessory and structural proteins
  • pALD-Gagpol-K envelope protein
  • envelope protein pALD- VSV-G-K.
  • K562 cells were seeded at 1x10 5 per well in a 12-well plate and transduced with SRT1, SRT2, SRT8, SRT11 or SRT-CA lentiviral vectors at MOIs of 3 and 10 in a total volume of 2 mL of complete growth medium supplemented with 100 ⁇ g/mL F108 (Fig.11A).
  • SRT-CA is a control vector which is biosimilar to BB305 in terms of the location of the cPPT sequence.
  • RNA transcript levels per vector copy number are shown in Fig.11B and the intracellular ⁇ -globin protein expression normalized to vector copy number is shown in Fig.11C for all the transduced cell populations at each MOI.
  • SRT Vector ⁇ -globin Expression Vector MOI % RNaseP Fold % ⁇ -globin+ % Change Change [0 g. 1, SRT2, SRT8 and SRT-CA at MOIs of 3 or 10, as indicated. Note the increased mean fluorescence intensity (MFI) and % positive cells for SRT8 relative to the other vectors. [00229] Overall, the collective findings demonstrate that SRT8 can be produced at extremely high viral titers with the improved functional capabilities in terms of ⁇ -globin gene expression and transduction efficiency as determined by the ⁇ -globin-positive cells relative to SRT-CA, which is a lentiviral vector with a cPPT positioned in a 5’ site similar to biosimilar vector BB305.
  • SRT-CA which is a lentiviral vector with a cPPT positioned in a 5’ site similar to biosimilar vector BB305.
  • CD34+ Differentiation and Transduction Efficiency Mobilized peripheral blood CD34+ cells from a healthy adult donor were pre- stimulated and transduced with the SRT8, SRT9 or TNS9.3.55 lentiviral vectors at an MOI of 100. Transduced cells (300 – 400) were mixed with semi-solid methylcellulose and cultured for 14 days. Formed colonies were visually inspected using an inverted light microscope and characterized as non-erythroid or erythroid and counted to yield colony-forming units (CFU). Fig.13 shows total CFU by cell type. Colony-forming potential was similar for all transduced populations with nearly equal fractions of non-erythroid and erythroid colonies being observed.
  • SRT9 is identical to SRT8 but includes a GFP marker downstream of the ⁇ -globin coding sequence.
  • the majority of the transduced cells were maintained for 5 days in liquid growth medium that does not support differentiation.
  • about 300-400 cells were mixed with semi-solid methylcellulose, formation of erythroid and non-erythroid colonies were scored after 14 days.
  • Cells in liquid culture were evaluated by flow cytometry for expression of GFP (Fig.14, left panel).
  • Transduced cells were mixed with semi-solid methylcellulose and cultured for 14 days to promote differentiation into non-erythroid and erythroid cells.
  • Genomic DNA was isolated from differentiated cells and quantitative PCR performed to assess average VCN calculated from a standard curve established from a cell line harboring a single lentivirus vector integrant. No vector copies were detected in the genomic DNA from the mock treated cells.
  • VCN for the differentiated cells transduced with TNS9.3.55, SRT8, and SRT9 was 2.0, 3.6, and 1.0, respectively (Fig.15).
  • Example 11 Example 11
  • Hbb + Hba levels of Hbb over total
  • mock (vehicle) treated cells set equal to 1.0. Relative changes in the transcript levels from the lentiviral vector-transduced cells were compared against the mock value.
  • Cells transduced with TNS9.3.55 had 70% higher levels of Hbb, which was similar to SRT9 (60% higher).
  • SRT8 showed a dramatically higher level of Hbb of 180% compared to the mock-treated cells, which was also ⁇ 65% higher than TNS9.3.55 (Fig.16).
  • T87Q globin a Mobilized peripheral blood CD34+ cells from a healthy adult donor were pre- stimulated and transduced with either vehicle (mock) or lentiviral vectors at MOI 100 (TNS9.3.55, SRT8 or SRT9). Transduced cells were mixed with semi-solid methylcellulose and cultured for 14 days to promote differentiation into non-erythroid and erythroid cells. Total RNA was isolated from differentiated cells and quantitative RT-PCR performed to using PCR primers specific to the lentiviral vector-encoded T87Q ⁇ -globin variant as described in provisional application U.S. Ser. No.63/729,416, filed December 8, 2024.
  • the primer pair for detecting the T87Q ⁇ -globin consisted of a forward primer comprising the sequence of TCAAGGGCACCTTTGCCCAG (T87Q sense) (SEQ ID NO: 19) and a reverse primer comprising the sequence of CAGCCCCACTTTCTGATAGGCAC (B1 antisense) (SEQ ID NO: 20).
  • the primer pair for detecting the wild-type ⁇ -globin consisted of a forward primer comprising the sequence of TCAAGGGCACCTTTGCCACA (WT sense) (SEQ ID NO: 21) and a reverse primer comprising the sequence of CAGCCCCACTTTCTGATAGGCAC (B1 antisense) (SEQ ID NO: 20). [00234] The results are shown in Fig.17.
  • Transcript levels were calculated as percentage of the cellular control RPL13A and the values were normalized to VCN. Minimally detectable amplification was measured in the mock (0.003) and TNS9.3.55 (0.005) transduced CD34+ cells. TNS9.3.55 expresses only the wild-type Hbb transcript. For SRT8 and SRT9, there was 0.73 and 0.42, respectively, transcript levels per VCN.
  • Example 13 Integration Site Analysis in Human CD34+ cells [00235] PCR amplification and sequencing of the integrated sites for human CD34+ cells transduced with TNS9.3.55 or SRT8 was performed by Azenta Life Sciences.
  • TNS9.3.55 was found to be inserted at 11.1% of the intergenic regions (inter) within the genomic DNA compared to 8.3% for SRT8.
  • the additional 2.8% integration (or >20% more relative to TNS9.3.55) into intragenic regions (intra) for SRT8 suggests a higher probability to have active promoter activity compared to integrations within the intergenic regions.
  • Lentiviral vector integrations near cancer-causing genes were further analyzed. In cases where 1% or a higher number of integration events were detected, no significant difference was found between TNS9.3.55 versus SRT8 integrations at known cancer-causing genes.
  • Fig.18 provides a table highlighting the major differences between the SRT series of transfer plasmids versus TNS9.3.55.
  • the table includes the vector genome lengths of SRT2 and SRT8 as well as sizes of the ⁇ -globin promoter and individual HS2, HS3, HS4 regions (plus the overall LCR length in each of those plasmids).
  • the table provides the location (and span) of the cPPT element from the beginning position of the integrated vector genome, which for this purpose is arbitrarily designated as the first base of the 5’-LTR.
  • the positions of HS2, HS3 and HS4 are the location of those sequences as found in GenBank sequence NG_052895.1.
  • Table 8 SRT Transfer Plasmid Sequence Elements By Location Note: Nucleotide locations marked with * are on the complement of the sense strand, i.e., the given element as usually presented is 5’ to 3’ on the antisense strand.
  • Pestina T.I., et al., Correction of murine sickle cell disease using gamma-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin. Mol Ther, 2009.17(2): p. 245-52. Puthenveetil, G., et al., Successful correction of the human beta-thalassemia major phenotype using a lentiviral vector. Blood, 2004.104(12): p.3445-53. Uchida, N., et al., Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders.
  • Phage-PGK-GFP-T(WT) (Addgene plasmid # 181736 ; http://n2t.net/addgene:181736 ; RRID:Addgene_181736) pWPT-nlsLacZ (Addgene plasmid # 12261 ; http://n2t.net/addgene:12261 ; RRID:Addgene_12261) pSUSL002R:pTRE3G-dCas9-2xKRAB-p2a-TurboRFP (Addgene plasmid # 209299 ; http://n2t.net/addgene:209299 ; RRID:Addgene_209299) pLenti HsATP

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Abstract

La présente invention se rapporte à des plasmides de transfert lentiviraux recombinants permettant de produire des vecteurs lentiviraux avec un titre de vecteurs et des performances accrus. Lesdits plasmides de transfert sont construits de sorte qu'un tractus polypurine central (cPPT) est centré entre les LTR lentiviraux et comprend un bloc de séquence de codons d'arrêt 6x en aval du 3' LTR. Lorsque les plasmides de transfert comprennent une cassette d'expression de globine, les vecteurs lentiviraux produits à partir de ceux-ci sont utiles pour traiter des hémoglobinopathies par thérapie génique. L'invention concerne également des cellules transduites, des compositions pharmaceutiques et des méthodes de traitement ou d'amélioration d'hémoglobinopathies à l'aide desdits vecteurs lentiviraux. L'invention concerne des plasmides de transfert, des cellules et des compositions supplémentaires qui sont utiles pour administrer n'importe quel gène d'intérêt à une cellule, ainsi que des plasmides pour la production de vecteurs lentiviraux permettant de traiter d'autres maladies ou troubles spécifiques aux érythroïdes tels que l'anémie et le cancer.
PCT/US2025/011122 2024-01-10 2025-01-10 Globine améliorée et autres vecteurs lentiviraux destinés à une thérapie génique Pending WO2025151744A1 (fr)

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