EP4651908A1 - Constructions d'acides nucléiques et leurs utilisations - Google Patents

Constructions d'acides nucléiques et leurs utilisations

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Publication number
EP4651908A1
EP4651908A1 EP24808441.0A EP24808441A EP4651908A1 EP 4651908 A1 EP4651908 A1 EP 4651908A1 EP 24808441 A EP24808441 A EP 24808441A EP 4651908 A1 EP4651908 A1 EP 4651908A1
Authority
EP
European Patent Office
Prior art keywords
vector
cell
seq
human
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24808441.0A
Other languages
German (de)
English (en)
Inventor
Haoquan Wu
Ying DANG
Lingling SU
Qing Ye
Jianjun TAO
Xiaodong Hu
Chao Jiang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kanglin Biotech Hangzhou Co Ltd
Original Assignee
Kanglin Biotech Hangzhou Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US18/383,596 external-priority patent/US20240167058A1/en
Application filed by Kanglin Biotech Hangzhou Co Ltd filed Critical Kanglin Biotech Hangzhou Co Ltd
Publication of EP4651908A1 publication Critical patent/EP4651908A1/fr
Pending legal-status Critical Current

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    • 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
    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • 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
    • 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
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • the present disclosure relates to the field of medical technology, in particular to gene therapy and nucleic acid constructs.
  • Inherited anemias such as thalassemia and sickle cell anemia are rare inherited blood diseases, most commonly in patients of Mediterranean, Middle Eastern, Indian and South Asian descent.
  • Thalassemia is typically derived from the imbalance between the single chains of globin hemoglobin tetramer.
  • the imbalance of red blood cell (RBC) a-globin and p-globin often produces various clinical symptoms, for example, 1) lack of sufficient red blood cells and hemoglobin, resulting in inadequacy of oxygen delivered to the whole body; 2) an increase in the hemolysis rate of red blood cells, leading to an increase in the mortality rate of chronic vascular system damage; and 3) spleen and liver damages caused by extreme load of ferine.
  • Allogeneic hematopoietic stem cell transplantation e.g., allogeneic bone marrow transplantation, peripheral blood hematopoietic stem cell transplantation, or cord blood transplantation
  • Allogeneic hematopoietic stem cell transplantation could be a potential cure for thalassemia.
  • the lack of transplant donors and the risk associated with transplantation limit the widespread use of allogeneic hematopoietic cell transplantation in patients with thalassemia.
  • the disclosure provides a vector comprising: a) a left (5') retroviral LTR; b) human p-globin gene; c) human p-globin gene upstream locus control region (LCR); d) a cis-acting posttranscriptional regulatory element; e) a right (3') retroviral LTR; and f) a SV40 polyadenylation signal and/or SV40 origin.
  • the sequence of the human p-globin gene comprises human p-globin gene exon 1, intron 1, exon 2, intron 2, and exon 3.
  • the sequence of the human p-globin gene is according to Ensembl Database Gene: HBB (ENSG00000244734) Transcript: HBB-201 (ENST00000335295.4).
  • the human p-globin gene comprises a human P-globin promoter.
  • human p-globin promoter is about 250 to about 275 bp (e.g., 268 bp) upstream of exon 1 of the human p-globin promoter.
  • the human P-globin gene comprises a human p-globin 3 ’-enhancer.
  • the human p-globin 3 ’-enhancer is about 850 bp to about 900 bp (e.g., 878 bp) downstream of exon 3 of the human p- globin gene.
  • the human p-globin gene comprises one or more (e.g., 2 or 3) wild-type exons.
  • the human p-globin gene comprises one or more (e.g., 2 or 3) codon-optimized exons.
  • the human p-globin gene comprises one or more wildtype introns.
  • the human p-globin gene comprises a wild-type intron 2.
  • the human p-globin gene comprises one or more truncated introns. In certain embodiments, the human p-globin gene comprises a tmncated intron 2. In some embodiments, the human p-globin gene comprises a wild-type exon 2. In some embodiments, the human p-globin gene comprises an exon 2 that encodes a threonine to glutamine mutation at codon 87 (T87Q). In some embodiments, the human p-globin gene comprises the nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a nucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity thereto, or any combination thereof.
  • the upstream locus control region comprises one or more (e.g., 2 or 3) truncated DNase I hypersensitive sites, HS2, HS3 and HS4 of the LCR.
  • the posttranscriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the SV40 poly adenylation signal and/or SV40 origin is located 3’ of the right (3’) retroviral LTR.
  • the WPRE is wildtype WPRE or a mutated WPRE, e.g., a mutated WPRE described herein.
  • the wildtype WPRE comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • the mutated WPRE comprises the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • the vector is a lentivirus vector.
  • the left (5') retroviral LTR is a lentiviral LTR.
  • the right (3') LTR is a lentivirus LTR.
  • the left (5') and right (3’) retroviral LTRs are lentivirus LTRs.
  • the promoter of the left (5') retroviral LTR is replaced with a heterologous promoter.
  • the right (3') LTR is a self-inactivating (SIN) LTR.
  • the vector further comprises one or more (e.g., 2 or 3) of a Psi packaging sequence ('P+), a central polypurine tract/DNA flap (cPPT/FLAP), or a retroviral export element-rev response element (RRE).
  • 'P+ Psi packaging sequence
  • cPPT/FLAP central polypurine tract/DNA flap
  • RRE retroviral export element-rev response element
  • the vector comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or a nucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • the disclosure features a composition
  • a composition comprising a vector described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides a cell comprising a vector described herein.
  • the cell is a human cell. In some embodiments, the cell is selected from the group consisting of an embryonic stem cell, an adult stem cell, an adult progenitor cell, and a differentiated adult cell. In some embodiments, the cell is a hematopoietic stem cell or a hematopoietic progenitor cell. In some embodiments, the cell is a hematopoietic stem cell or a hematopoietic progenitor cell. In certain embodiments, the source of the stem or progenitor cell is bone marrow, cord blood, placental blood, or peripheral blood. In some embodiments, the cell is transduced with the vector.
  • the disclosure provides a composition comprising a cell described herein and a pharmaceutically acceptable carrier.
  • the disclosure provides a method of treating p-thalassemia, comprising administering to a subject in need thereof an effective amount of a cell described herein, or a cell transduced with a vector described herein, thereby treating p-thalassemia.
  • the method further comprises obtaining a cell from the subject. In some embodiments, the method further comprises transducing the cell with the vector. In some embodiments, the cell is a hematopoietic stem cell or a hematopoietic progenitor cell.
  • the method further comprises administering to the subject an effective amount of busulfan and cyclophosphamide prior to administering the cell transduced with the vector to the subject.
  • busulfan is administered at a dose of 2 to 5 mg/kg/day, e.g., 2.4 to 4.8 mg/kg/day, intravenously.
  • busulfan is administered once every 6 hours.
  • cyclophosphamide is administered at a dose of 30-80 mg/kg/day, e.g., 45-65 mg/kg/day, intravenously.
  • cyclophosphamide is administered 18 to 30 hours, e.g., 24 hours, after busulfan is administered.
  • busulfan is administered for 2-4 days and cyclophosphamide is administered for 1-5 days.
  • the administration of the cell transduced the vector is initiated 24-72 hours after the administration of cyclophoshamide is completed.
  • the disclosure provides a method of pretreating a subject, comprising administering to the subject an effective amount of busulfan and cyclophosphamide prior to administering to the subject a therapy for p-thalassemia.
  • the therapy for p- thalassemia comprises a cell transduced with a vector comprising a human p-globin gene, e.g., a cell described herein, or a cell transduced with a vector described herein, to the subject.
  • busulfan is administered at a dose of 2 to 5 mg/kg/day, e.g., 2.4 to 4.8 mg/kg/day, intravenously. In some embodiments, busulfan is administered once every 6 hours.
  • cyclophosphamide is administered at a dose of 30-80 mg/kg/day, e.g., 45-65 mg/kg/day, intravenously. In some embodiments, cyclophosphamide is administered 18 to 30 hours, e.g., 24 hours, after busulfan is administered. In some embodiments, busulfan is administered for 2-4 days and cyclophosphamide is administered for 1-5 days. In some embodiments, the administration of the cell transduced the vector is initiated 24-72 hours after the administration of cyclophshamide is completed. In some embodiments, the subject is pretreated in accordance with a method described in Examples 9-12.
  • the disclosure provides a formulation comprising the vector described herein, a buffer, a stabilizer, and sodium chloride.
  • the vector is present at a concentration of IxlO 8 TU/mL to IxlO 10 TU/mL. In some embodiments, the vector is present at a concentration of 5xl0 8 TU/mL to 5xl0 9 TU/mL. In some embodiments, the vector is present at a concentration of 5xl0 8 TU/mL to IxlO 9 TU/mL, e.g., 6xl0 8 TU/mL or 6.2* 10 8 TU/mL. In some embodiments, the vector is present at a concentration of IxlO 9 TU/mL to 5xl0 9 TU/mL. In some embodiments, the vector is present at a concentration of 2* 10 9 TU/mL to 3*10 9 TU/mL, e.g., 2.5*10 9 TU/mL or 2.8xl0 9 TU/mL.
  • the buffer is phosphate buffer, sodium citrate, or PIPES. In some embodiments, the buffer is present at a concentration of 10 mM to 50 mM, e.g., 10 mM to 30 mM, 20 mM to 40 mM, 30 mM to 50 mM, or 10 mM to 40 mM. In some embodiments, the buffer is present at a concentration of 10 mM, 20 mM, or 40 mM.
  • the stabilizer comprises a sugar or a polyhydric alcohol, e.g., sucrose, trehalose, sorbitol, inositol, glucose, or dextran.
  • the stabilizer is present at a concentration of 1% to 5%, e.g., 1% to 3%, 2% to 3%, or 1% to 2.5%. In some embodiments, the stabilizer is present at a concentration of 1%, 2%, or 2.5%.
  • sodium chloride is present at a concentration of 50 mM to 200 mM, e.g., 50 mM to 70 mM, 70 mM to 90 mM, 80 mM to 100 mM, 100 mM to 120 mM, 140 mM to 160 mM, lOOmM to 150mM, or 50 mM to 150 mM.
  • sodium chloride is present at a concentration of 50 mM, 60 mM, 75 mM, 80 mM, 90 mM, 110 mM, 140 mM, or 150 mM.
  • the formulation comprises a vector described herein, sodium citrate, sucrose, and sodium chloride.
  • the vector is present at a concentration of 5xl0 8 TU/mL to 5xl0 9 TU/mL
  • sodium citrate is present at a concentration of 20 mM to 40 mM
  • sucrose is present at a concentration of 1% to 2%
  • sodium chloride is present at a concentration of 100 mM to 150 mM.
  • the formulation is any of the formulations described in Examples 13-
  • FIG. 1 depicts the schematic design of exemplary viral vector constmcts.
  • FIG. 2 depicts a diagram of an exemplary lentiviral vector packaging backbone plasmid without insertion of gene of interest.
  • FIG. 3 depicts viral vector packaging efficiency for exemplary viral vector constructs shown in FIG. 1.
  • FIG. 4 depicts the schematic design of additional exemplary viral vector constructs.
  • FIG. 5 is a graph depicting VCN values in PBMCs of recipient mice in the mock (without transduction), LV-TH04 (transduction), and positive (wild-type C57BL/6 cells) groups. Blood samples were collected 4, 6 and 8 weeks after bone marrow transplantation of the recipient mice.
  • FIG. 6 is a graph depicting the chimeric rate (%) of PBMCs of recipient mice in the mock (without transduction), LV-TH04 (transduction), and positive (wild-type C57BL/6 cells) groups. Blood samples were collected 4, 6 and 8 weeks after bone marrow transplantation of the recipient mice.
  • FIG. 7 is a series of graphs depicting the hemoglobin content (HGB (g/L); top left graph), percentage of reticulocytes (RET%; top right graph), hematocrit levels (HCT(%); bottom left graph), and average red blood cell volume (MCV(fL); bottom right graph) of recipient mice in the mock (without transduction), LV-TH04 (transduction), and positive (wild-type C57BL/6 cells) groups. Blood samples were collected 4, 6 and 8 weeks after bone marrow transplantation of the recipient animals.
  • FIG. 8 is a series of chromatograms from HPLC assays using supernatants collected from cells transduced LV-TH04 (top chromatogram) or cells without LV-TH04 (bottom chromatogram). The P- globin and a-globin peaks are indicated.
  • FIG. 9 is a graph depicting the absolute neutrophil count (10 9 /L) after infusion of LV-TH04- transduced hematopoietic stem cells.
  • Subjects PJYU and ZRHA were treated with myeloablative conditioning based on BU/CY; subject FAZH was treated with myeloablative conditioning based on BU.
  • FIG. 10 is a graph depicting platelet count (10 9 /L) after infusion of LV-TH04-transduced hematopoietic stem cells.
  • Subjects PJYU and ZRHA were treated with myeloablative conditioning based on BU/CY; subject FAZH was treated with myeloablative conditioning based on BU.
  • FIG. 11 is a graph depicting the loss (%) of lentiviral particles in the formulations described in Table 15 after being placed at room temperature for 1 day, placed at 4 °C for 3 days, or placed under conditions of freeze-thaw for 3 times.
  • FIG. 12 is a graph depicting the lentiviral titers (TU/mL) of the formulations described in Table 16 after being placed under conditions of freeze-thaw for 3 times, or placed under conditions of freezethaw for 9 times.
  • FIG. 13 is a graph depicting the lentiviral titers (TU/mL) of the formulations described in Table 17 after being placed under conditions of freeze-thaw for 3 times.
  • FIG. 14 is a graph depicting the loss (%) of lentiviral particles in the formulations described in Table 18 after being placed at room temperature for 1 day, placed at 4°C for 3 days, or placed under conditions of freeze-thaw for 3 times.
  • FIG. 15 is a graph depicting the lentiviral titers (TU/mL) of a formulation comprising sodium citrate (20mM), sodium chloride (1 lOrnM), and sucrose (1%), or PBS under control conditions, or after being placed at 4°C for 1 day, placed at 4°C for 3 days, or placed at room temperature for 1 day.
  • Lentiviral vectors have the characteristic of host genome integration and therefor are widely considered desirable gene deliver vectors for various genetic diseases, such as hereditary anemia, caused by the loss expression of a single gene.
  • Autologous hematopoietic stem cell therapy can be a promising curative therapy for severe hereditary anemia.
  • a functional gene encoding a human p-globin peptide chain is introduced into a patient’s hematopoietic stem cells ex vivo by lentiviral vector transduction and the transduced cells are infused back to the patient.
  • the methods described herein may achieve complete cure of thalassemia by a onetime treatment.
  • the human p-globin gene comprise a promoter region, 3 exons and 2 introns, a downstream enhancer region, and an endogenous upstream gene expression control region sequence DNase I hypersensitive sites (HSs).
  • HSs DNase I hypersensitive sites
  • the total length of the gene exceeds 60,000 base pairs (bp), which is difficult to be included in any kind of gene therapy vector. For decades, scientists have worked to develop a relatively small p-globin gene expression framework so that it can be used in gene therapy.
  • the term “about” or “approximately” when referring to a measurable value such as an amount, a temporal duration, and the like, are meant to encompass variations of ⁇ 20% or in some instances ⁇ 10%, or in some instances ⁇ 5%, or in some instances ⁇ 1%, or in some instances ⁇ 0.1% from the specified value, as such variations are appropriate in the context of the disclosure.
  • allogeneic refers to a cell of the same species that differs genetically to the cell in comparison.
  • the term “associated with” or “linked,” when used with respect to two or more moieties, means that the moieties are associated or connected, e.g., physically or chemically, with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • the two or more moieties are covalently or non-covalently attached, coupled, linked, or tethered.
  • an association is through direct covalent chemical bonding.
  • the association is through ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the associated or linked entities remain physically associated.
  • autologous refers to a cell from the same subject.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the term “complementary” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form base pairs, e.g., a duplex, with an oligonucleotide or polynucleotide comprising the second nucleotide sequence.
  • base pairs are formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
  • complementary polynucleotide or oligonucleotide strands can form base pairs in the Watson-Crick manner or in any other manner that allows for the formation of duplexes.
  • complementary as used herein can encompass fully complementary, partially complementary, or substantially complementary. “Fully complementary” refers to the situation in which each nucleotide unit of one polynucleotide or oligonucleotide strand can base-pair with a nucleotide unit of a second polynucleotide or oligonucleotide strand.
  • “Substantially complementary” refers to the situation in which two polynucleotides or oligonucleotide strands can be fully complementary or they may form one or more, but generally not more than 1, 2, 3, 4, or 5 mismatched or non-complimentary base pairs upon hybridization for a duplex, while still retaining the ability to hybridize under the conditions most relevant to their ultimate application.
  • control element refers to an element used for expression of a gene or gene product.
  • exemplary “control elements,” “regulatory control elements,” or “regulatory sequences” include, but are not limited to, promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell.
  • IRS internal ribosome entry sites
  • the term “effective amount” refers to an amount of a compound, formulation, material, or composition, to achieve a particular biological result.
  • the effective amount is a therapeutically effective amount.
  • the effective amount of an agent is that amount sufficient to effect a beneficial or desired result, for example, a clinical result.
  • an effective amount of an agent is, for example, an amount sufficient to achieve treatment of the disorder, as compared to the response obtained without administration of the agent.
  • enhancer refers to a segment of DNA which contains a sequence capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • RNA export element refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) and the hepatitis B virus post-transcriptional regulatory element (HPRE).
  • HAV human immunodeficiency virus
  • RRE human immunodeficiency virus
  • HPRE hepatitis B virus post-transcriptional regulatory element
  • the RNA export element is located within the 3' UTR of a gene and is inserted as one or multiple copies.
  • expression refers to transcription and/or translation of a particular nucleotide sequence.
  • Expression can generally include one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • expression control sequence refers to a polynucleotide sequence that comprises one or more promoters, enhancers, or other transcriptional control elements or combinations thereof that are capable of directing, increasing, regulating, or controlling the transcription or expression of an operatively linked polynucleotide.
  • FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and the central termination sequence (cPPT and CTS) of a retrovirus (e.g., HIV-1 or HIV-2).
  • a retrovirus e.g., HIV-1 or HIV-2
  • CTS central termination at the central termination sequence
  • the central DNA flap may act as a cis-active determinant of retroviral genome nuclear import and/or may increase the titer of the virus.
  • a retroviral or lentiviral vector backbone described herein comprises one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vector.
  • a transfer plasmid can include a FLAP element.
  • a viral vector described herein comprises a FLAP element isolated from HIV-1.
  • HSC hematopoietic stem cell
  • myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages e.g., T-cells, B-cells, NK-cells
  • the term “host cell” refers to a cell transfected, infected, or transduced in vivo, ex vivo, or in vitro with a vector or a polynucleotide.
  • Host cells may include packaging cells, producer cells, and cells infected with viral vectors.
  • target cell is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type.
  • the term “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules (e.g. two DNA molecules and/or two RNA molecules) and/or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, they are considered identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which can to be introduced for optimal alignment of the two sequences.
  • calculation of the percent identity of two sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the length of the reference sequence.
  • isolated means a material (e.g., a polynucleotide, a polypeptide, a cell) that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide,” as used herein, refers to a polynucleotide that has been purified from the sequences that flank it in a naturally -occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • lentiviral vector refers to a viral vector containing a structural or functional element, or a portion thereof, that is primarily derived from a lentivims.
  • lentivims refers to a genus of retroviruses.
  • exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency vims, e.g., HIV type 1 and HIV type 2), bovine immune deficiency vims (BIV), caprine arthritis-encephalitis vims (CAEV), equine infectious anemia vims (EIAV), feline immunodeficiency vims (FIV), simian immunodeficiency vims (SIV), and visna-maedi vims (VMV) vims.
  • HIV human immunodeficiency vims
  • BIV bovine immune deficiency vims
  • CAEV caprine arthritis-encephalitis vims
  • EIAV equine infectious anemia vims
  • FIV feline immunodeficiency vims
  • SIV simian immunodeficiency vims
  • VMV visna-mae
  • LTR long terminal repeat
  • the LTR contains a number of regulatory signals, for example, transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome.
  • the LTR typically includes U3, R and U5 regions and appears at both the 5' and 3' ends of the viral genome.
  • the U3 region contains the enhancer and promoter elements.
  • the U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence.
  • the R (repeat) region is flanked by the U3 and U5 regions. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).
  • nucleic acid cassette refers to a sequence within the vector which can express an RNA, and subsequently a polypeptide.
  • the nucleic acid cassette may contains a gene-of-interest and/or one or more expression control sequences.
  • Vectors may comprise one, two, three, four, five or more nucleic acid cassettes.
  • the nucleic acid cassette can be positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post- translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments.
  • the nucleic acid cassette has its 5’ and 3 ’ ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.
  • the nucleic acid cassette contains a polynucleotide sequence that can be used to treat or prevent a disorder. The cassette can typically be removed and inserted into a plasmid or viral vector as a single unit.
  • operably linked refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
  • “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • the term “of’ means either one, both, or any combination of the alternatives, and is used interchangeably with the term “and/or”, unless context clearly indicates otherwise.
  • packaging cell line refers to a cell line that does not contain a packaging signal, but does stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol, and env) which are necessary for the correct packaging of viral particles.
  • viral structural proteins and replication enzymes e.g., gag, pol, and env
  • the term “packaging signal” or “packaging sequence” refers to a sequence located within a retroviral genome that is required for insertion of the viral RNA into the viral capsid or particle.
  • Several retroviral vectors use the minimal packaging signal (also referred to as the psi
  • the terms “packaging sequence,” “packaging signal,” “psi” and the symbol “T” are used interchangeably to describe the non-coding sequence required for encapsidation of retroviral RNA strands during viral particle formation.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media.
  • the term “prevent” or “prevention” means that a subject (e.g., a human) is less likely to have the disorder, e.g., a myeloma, if the subject receives the antibody molecule.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
  • promoter/enhancer refers to a segment of DNA which contains a sequence capable of providing both promoter and enhancer functions.
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic effect or result.
  • retroviral vector refers to a viral vector containing a structural or functional element, or a portion thereof, that is primarily derived from a retrovirus.
  • R region refers to a region within an LTR beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly A tract.
  • the R region is also defined as being flanked by the U3 and U5 regions. Without wishing to be bound by theory, it is believed that in some embodiments, the R region plays a role during reverse transcription in permitting the transfer of nascent DNA from one end of the genome to the other.
  • retrovirus refers to an RNA vims that reverse transcribes its genomic RNA into DNA and subsequently integrates the DNA into a host genome.
  • retroviruses include, but are not limited to, lentiviruses, oncoretroviruses, and spumaviruses.
  • Exemplary oncoretroviruses include, but are not limited to, feline leukemia virus (FLV), Friend murine leukemia virus, gibbon ape leukemia virus (GaLV), Harvey murine sarcoma virus (HaMuS V), Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMS V), murine mammary tumor virus (MuMTV), murine stem cell virus (MSCV), and Rous sarcoma virus (RSV).
  • the retrovirus is a lentivirus.
  • thalassemia refers to a hereditary disorder characterized by defective production of hemoglobin.
  • the term “thalassemia” encompasses hereditary anemias that occur due to mutations affecting the synthesis of hemoglobin.
  • the term includes any symptomatic anemia resulting from thalassemic conditions such as severe or p-thalassemia, thalassemia major, thalassemia intermedia, a-thalassemias such as hemoglobin H disease.
  • thalassemias include a-thalassemia and p-thalassemia.
  • a-thalassemia is caused by deletion of a gene or genes from the globin chain
  • p-thalassemia is caused by a mutation in the p-globin chain, and can occur in a major or minor form.
  • P-thalassemia children typically are normal at birth, but develop anemia during the first year of life.
  • the mild form of p-thalassemia produces small red blood cells.
  • terapéuticaally effective amount refers to an amount effective to achieve the desired therapeutic effect or result.
  • the term “self-inactivating vector” or “SIN vector” refers to a replicationdefective vector (e.g., a retroviral or lentiviral vector) in which the right 3 ’ LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • the right 3’ LTR U3 region is used as a template for the left 5’ LTR U3 region during viral replication and, thus, the viral transcript cannot be made without a functional U3 enhancerpromoter.
  • the 3’ LTR is modified such that the U5 region is replaced, for example, with a poly(A) sequence.
  • stem cell refers to a cell which is an undifferentiated cell capable of (1) long term self -renewal, or the ability to generate at least one identical copy of the original cell, (2) differentiation at the single cell level into multiple, and in some instance only one, specialized cell type and (3) of in vivo functional regeneration of tissues.
  • the term “subject” is intended to include human and non-human animals.
  • the subject is a human subject, e.g., a human patient having a disorder described herein, or at risk of having a disorder described herein.
  • non-human animals includes mammals and non-mammals, such as non-human primates.
  • the vectors, cells, and compositions described herein are suitable for treating human patients a disorder described herein.
  • Patients having a disorder described herein include, e.g., those who have developed a disorder described herein but are (at least temporarily) asymptomatic, patients who have exhibited a symptom of a disorder described herein, and patients having a disorder related to or associated with a disorder described herein.
  • trans-activation response refers to a genetic element located in the R region of retroviral or lentiviral LTRs. Without wishing to be bound by theory, it is believed that in some embodiments, this element interacts with the retroviral or lentiviral transactivator (tat) genetic element to enhance viral replication. In some embodiments, this element is not required wherein the U3 region of the 5 ’ LTR is replaced by a heterologous promoter.
  • a viral vector described herein comprises a TAR element.
  • the term “treat” or “treatment” means that a subject (e.g., a human) who has a disorder and/or experiences a symptom of a disorder will, in some embodiments, suffer less a severe symptom and/or recover faster when a therapy is administered than if the therapy were never administered.
  • Treatment can, partially or completely, alleviate, ameliorate, relieve, inhibit, or reduce the severity of, and/or reduce incidence, and optionally, delay onset of, one or more manifestations of the effects or symptoms, features, and/or causes of a disorder.
  • treatment is of a subject who does not exhibit certain signs of a disorder, and/or of a subject who exhibits only early signs of a disorder.
  • treatment is of a subject who exhibits one or more established signs of a disorder.
  • treatment is of a subject diagnosed as suffering from a disorder.
  • the term “variant” refers to a polypeptide that is distinguished from a reference polypeptide by the addition, deletion, truncations, and/or substitution of at least one amino acid residue, and that retain a biological activity.
  • a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative, as known in the art.
  • vector refers to a nucleic acid molecule that is used as a vehicle to transfer another nucleic acid molecule into a host cell.
  • the transferred nucleic acid molecule is inserted to the vector nucleic acid molecule.
  • a vector may include a sequence that directs autonomous replication in a cell, or may include a sequence sufficient to allow integration into the host cell genome.
  • Exemplary vectors include, but are not limited to, viral vectors, plasmids (e.g., DNA plasmids orRNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
  • Exemplary viral vectors include, but are not limited to, retroviral vectors (e.g., replication defective retroviral vectors) and lentiviral vectors.
  • viral vector refers to a nucleic acid molecule (e.g., a plasmid) that includes one or more virus-derived nucleic acid elements that facilitate transfer of a nucleic acid molecule into a cell, integration of a nucleic acid molecule into the genome of cell, or delivery of a nucleic acid molecule to a viral particle.
  • a nucleic acid molecule e.g., a plasmid
  • virus-derived nucleic acid elements that facilitate transfer of a nucleic acid molecule into a cell, integration of a nucleic acid molecule into the genome of cell, or delivery of a nucleic acid molecule to a viral particle.
  • the disclosure provides a nucleic acid construct or vector (e.g., a viral vector) comprising a human p-globin gene or a functional fragment thereof.
  • a nucleic acid construct or vector e.g., a viral vector
  • the sequence of the human p-globin gene comprises human p-globin gene exon 1, intron 1, exon 2, intron 2, and exon 3.
  • the sequence of the human p-globin gene is according to Ensemble Database Gene: HBB (ENSG00000244734) Transcript: HBB-201 (ENST00000335295.4).
  • nucleic acid construct or vector comprises a regulatory control element. In some embodiments, nucleic acid construct or vector comprises an expression control sequence. In some embodiments, the nucleic acid construct or vector comprises a promoter or a promoter/enhancer.
  • the human p-globin gene comprises a human p-globin promoter. In some embodiment, the human p-globin gene does not comprise a human p-globin promoter. In some embodiments, human p-globin promoter is about 250 to about 275 bp (e.g., 268 bp) upstream of exon 1 of the human p-globin promoter. In some embodiments, the human p-globin gene comprises a human p-globin 3 ’-enhancer. In certain embodiments, the human p-globin 3 ’-enhancer is about 850 bp to about 900 bp (e.g., 878 bp) downstream of exon 3 of the human p-globin gene.
  • the human p-globin gene comprises one or more (e.g., 2 or 3) wild-type exons. In some embodiments, the human p-globin gene comprises one or more (e.g., 2 or 3) codon-optimized exons. In some embodiments, the human p-globin gene comprises one or more wild-type introns. In certain embodiments, the human p-globin gene comprises a wild-type intron 2. In some embodiments, the human p-globin gene comprises one or more truncated introns. In certain embodiments, the human p- globin gene comprises a truncated intron 2. In some embodiments, the human p-globin gene comprises a wild-type exon 2.
  • the human p-globin gene comprises an exon 2 that encodes a threonine to glutamine mutation at codon 87 (T87Q).
  • the human P-globin gene comprises the nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.
  • the nucleic acid construct or vector comprises a nucleotide sequence that is complementary to the nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.
  • the human p-globin gene encodes a human p-globin variant.
  • the variant may include an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the wild-type human p-globin amino acid sequence.
  • the nucleic acid constmct or vector comprises a retroviral (e.g., lentiviral) LTR.
  • the nucleic acid construct or vector comprises a left (5') retroviral LTR and a right (3') retroviral LTR.
  • the right (3') LTR is a selfinactivating (SIN) LTR.
  • the retroviral LTR is unmodified, e.g., a wild-type retroviral LTR.
  • the retroviral LTR is modified, e.g., comprising one or more subtitutions, insertions, and/or deleteions.
  • the left (5’) retroviral LTR is replaced with a heterologous promoter, e.g., cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, a thymidine kinase promoter, or an Simian Virus 40 (SV40) promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • SV40 Simian Virus 40
  • the right (3’) retroviral LTR is absent.
  • the retroviral LTR is a lentiviral LTR.
  • the nucleic acid constmct or vector comprises a human p-globin gene upstream locus control region (LCR).
  • the upstream locus control region (LCR) comprises one or more (e.g., 2 or 3) tmncated DNase I hypersensitive sites, HS2, HS3 and HS4 of the LCR.
  • the nucleic acid construct or vector comprises a cis-acting posttranscriptional regulatory element.
  • the posttranscriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • the nucleic acid constmct or vector comprises a polyadenylation signal and/or origin.
  • the polyadenylation signal is an SV40 polyadenylation signal.
  • the origina is an S V40 origin. The polyadenylation signal and/or origin can be located 3’ of the right (3’) retroviral LTR.
  • the nucleic acid construct or vector comprises one or more (e.g., two, three, four, or all) of a) a left (5') retroviral LTR; b) human p-globin gene; c) human p-globin gene upstream locus control region (LCR); d) a cis-acting posttranscriptional regulatory element; e) a right (3 ') retroviral LTR; and f) a S V40 polyadenylation signal and/or SV40 origin.
  • the nucleic acid constmct or vector comprises a nucleic acid cassette comprising one or more (e.g., two, three, four, or all) of a) a left (5') retroviral LTR; b) human p- globin gene; c) human p-globin gene upstream locus control region (LCR); d) a cis-acting posttranscriptional regulatory element; e) a right (3') retroviral LTR; and f) a SV40 polyadenylation signal and/or SV40 origin.
  • a nucleic acid cassette comprising one or more (e.g., two, three, four, or all) of a) a left (5') retroviral LTR; b) human p- globin gene; c) human p-globin gene upstream locus control region (LCR); d) a cis-acting posttranscriptional regulatory element; e) a right (3') retroviral LTR;
  • the nucleic acid construct or vector further comprises one or more (e.g., 2 or 3) of a Psi packaging sequence ('P+), a central polypurine tract/DNA flap (cPPT/FLAP), or a retroviral export element-rev response element (RRE).
  • 'P+ Psi packaging sequence
  • cPPT/FLAP central polypurine tract/DNA flap
  • RRE retroviral export element-rev response element
  • the nucleic acid construct or vector comprises the nucleotide sequence SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the nucleic acid construct or vector comprises a nucleotide sequence that is complementary to the nucleotide sequence SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the nucleic acid construcxt or vector further comprises a truncated erythroid cell expression control sequence.
  • the lentiviral nucleic acid construct or vector is an HIV nucleic acid construct or vector.
  • the lentiviral nucleic acid construct or vector may be derived from human immunodeficiency- 1 (HIV-1), human immunodeficiency -2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), and the like.
  • compositions comprising a nucleic acid construct or vector described herein or a cell described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.
  • exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions of the disclosure can comprise, for example, one or more polypeptides, polynucleotides, vectors comprising same, or transduced cells, formulated in pharmaceutically - acceptable or physiologically -acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other therapeutic agents or modalities.
  • the compositions of the disclosure may also be administered in combination with other agents, including, but not limited to, cytokines, growth factors, hormones, small molecules, or other pharmaceutically -active agents.
  • compositions of the disclosure formulation of pharmaceutically - acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including, but not limited to, parenteral, intravenous, and intramuscular administration and formulation.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • 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 the use of surfactants.
  • the prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Pharmaceutically -acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • compositions or formulations described herein can comprises a cell contacted with a combination of any number of polypeptides, polynucleotides, and small molecules, as described herein.
  • compositions that comprise a therapeutically - effective amount of one or more polynucleotides or polypeptides, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents (e.g., pharmaceutically acceptable cell culture medium).
  • the disclosure provides formulations or compositions suitable for the delivery of viral vector systems (e.g., viral-mediated transduction), including, but not limited to, retroviral (e.g., lentiviral) vectors.
  • viral vector systems e.g., viral-mediated transduction
  • retroviral vectors e.g., lentiviral
  • Exemplary formulations for ex vivo delivery include, but are not limited to, the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome formulations (e.g., lipid-mediated transfection).
  • various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome formulations (e.g., lipid-mediated transfection).
  • nucleic acid constructs, vectors, compositions, and cells described herein can be used in methods of treating or preventing thalassemia, e.g., p-thalassemia.
  • the vector is administered by direct injection to a cell, tissue, or organ of a subject in need of gene therapy, e.g., in vivo.
  • the cell is transduced in vitro or ex vivo with a nucleic acid construct or vector described herein, and optionally expanded ex vivo.
  • the transduced cell is then administered to a subject in need of gene therapy.
  • Cells suitable for transduction or administration in the methods described herein include, but are not limited to, stem cells, progenitor cells, and differentiated cells.
  • the transduced cell is a hematopoietic stem cell.
  • the transduced cells are hematopoietic stem and/or progenitor cells, e.g., isolated from bone marrow, umbilical cord blood, or peripheral circulation.
  • the transduced cells are hematopoietic stem cells, e.g., isolated from bone marrow, umbilical cord blood, or peripheral circulation.
  • Hemapoietic stem or pluripotent cells may be identified according to certain phenotypic or genotypic markers, which are known in the art.
  • the disclosure provides a method of treating a disorder.
  • the method comprises administering to a subject (e.g., a human subject) in need thereof an effective amount of a nucleic acid construct or vector described herein, or a cell (e.g., a hematopoietic stem or progenitor cell) transduced with a nucleic acid construct or vector described herein, thereby treating the disorder.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the disorder is a disorder is associated with a defective p-globin gene.
  • the disorder is thalassemia (e.g., p-thalassemia).
  • the method further comprises obtaining a cell (e.g., a hematopoietic stem or pluripotent cell) from the subject.
  • the method further comprises transducing a cell (e.g., a hematopoietic stem or pluripotent cell) from the subject with a nucleic acid construct or vector described herein.
  • the method further further comprises isolating the transduced cell.
  • the method further comprises administering to the subject a second therapeutic agent or modality.
  • the disclosure provides a method of providing a transduced cell.
  • the method comprises administering to a subject (e.g., a human subject) in need thereof a cell (e.g., a hematopoietic stem or progenitor cell) transduced with a nucleic acid constmct or vector described herein.
  • a subject e.g., a human subject
  • a cell e.g., a hematopoietic stem or progenitor cell
  • the disclosure provides a method of treating a hemoglobinopathy.
  • the method comprises administering to a subject (e.g., a human subject) in need thereof a nucleic acid construct or vector described herein, or a cell (e.g., a hematopoietic stem or progenitor cell) transduced with a vector described herein.
  • a subject e.g., a human subject
  • a cell e.g., a hematopoietic stem or progenitor cell
  • the disclosure provides a method of selectively expanding the number erythroid cells.
  • the method comprises administering to a subject (e.g., a human subject) in need thereof a nucleic acid construct or vector described herein, or a cell (e.g., a hematopoietic stem or progenitor cell) transduced with a nucleic construct or vector described herein.
  • a subject e.g., a human subject
  • a cell e.g., a hematopoietic stem or progenitor cell
  • the disclosure provides a method of increasing the proportion of red blood cells or erythrocytes compared to white blood cells or leukocytes in a subject.
  • the method comprises administering a nucleic acid construct or vector described herein, or a cell (e.g., a hematopoietic stem or progenitor cell) transduced with a nucleic acid construct or vector described herein.
  • the transduced cell is administered to the subject intravenously.
  • the transduced cells are administered to the subject at a dose of about IxlO 5 to about IxlO 8 cells, e.g., about IxlO 6 to about IxlO 7 cells, about IxlO 6 to about IxlO 8 cells, about IxlO 7 to about IxlO 8 cells, about IxlO 5 to about IxlO 7 cells, or about IxlO 5 to about IxlO 6 cells.
  • the transduced cells are administered as a single dose. Enumerated Emobidments
  • a vector comprising: a) a left (5') retroviral LTR; b) a human p-globin gene; c) a human p-globin gene upstream locus control region (LCR); d) a cis-acting posttranscriptional regulatory element; e) a right (3') retroviral LTR; and f) a cis-acting element SV40 polyadenylation signal and/or SV40 origin.
  • LCR human p-globin gene upstream locus control region
  • upstream locus control region comprises truncated DNase I hypersensitive sites, HS2, HS3 and HS4.
  • the vector of embodiment 12, wherein the wildtype WPRE comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.
  • mutated WPRE comprises the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.
  • the human p-globin gene comprises one, two, or all of the nucleotide sequences of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identical thereto.
  • the vector of any of embodiments 1-16 which is a lentivirus vector.
  • composition comprising the vector of any of embodiments 1-22 and a pharmaceutically acceptable carrier.
  • composition comprising a cell transduced with the vector of any of embodiments 1-22 and a pharmaceutically acceptable carrier.
  • a method of treating p-thalassemia comprising administering to a subject in need thereof an effective amount of a cell transduced with the vector of any of embodiments 1-22, thereby treating p- thalassemia.
  • a formulation comprising the vector of any of embodiments 1-22, a buffer, a stabilizer, and sodium chloride.
  • the stabilizer comprises a sugar or a polyhydric alcohol, e.g., sucrose, trehalose, sorbitol, inositol, glucose, or dextran.
  • a sugar or a polyhydric alcohol e.g., sucrose, trehalose, sorbitol, inositol, glucose, or dextran.
  • FIG. 1 three vectors, P002, P005, and P006, were designed with woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), SV40 virus polyadenylation signal (SV40 pA signal), and/or SV40 virus replication origin (SV40 ori) incorporated.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • SV40 pA signal SV40 virus polyadenylation signal
  • SV40 ori SV40 virus replication origin
  • the gene expression frames designed in Example 1 were cloned into a lentiviral vector backbone, which was from the third generation lentiviral vector backbone made in-house by Kanglin Biotech (Hangzhou) Co., Ltd., pKL-Kan (SEQ ID NO: 1) (FIG. 2).
  • the P-globin gene expression frame P001 (SEQ ID NO: 2) designed in Example 1 was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and cloned in between the multi-cloning sites Xhol/Kpnl of the lentiviral vector backbone pKL-Kan by the homologous recombination method well-known to the field.
  • the sequence of the resultant construct was confirmed by sequencing and named as pKL-Kan-TH-POOl (SEQ ID NO: 3).
  • the P-globin gene expression frame P002 (SEQ ID NO: 4) with an incorporated WPRE was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and cloned in between LCR and 3' LTR of the lentiviral vector pKL-Kan-TH-POOl by the homologous recombination method.
  • the sequence of the resultant construct was confirmed by sequencing and named as pKL-Kan-TH-P002 (SEQ ID NO: 5).
  • the P-globin gene expression frame P005 (SEQ ID NO: 6) with an incorporated SV40 pA signal plus SV40 ori was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and cloned in between 3' LTR and kan ori of the lentiviral vector pKL-Kan-TH-POOl by the homologous recombination method.
  • the sequence of the resultant construct was confirmed by sequencing and named as pKL-Kan-TH-P005 (SEQ ID NO: 7).
  • the WPRE fragment was amplified by PCR and using pKL-Kan-TH-P002 as a template and cloned in between LCR and S V40 pA signal of the lentiviral vector pKL-Kan-TH-P005 by the homologous recombination method.
  • the sequence of the resultant construct was confirmed by sequencing and named as pKL-Kan-TH-P006 (SEQ ID NO: 8).
  • Example 3 Packaging of lentiviruses containing human P-globin gene
  • the lentiviral vectors of P-globin gene (pKL-Kan-TH-POOl, pKL-Kan-TH-P002, pKL-Kan-TH-P005, or pKL-Kan-TH-P006) constructed in Example 2, envelope plasmid (pKL-Kan-Vsvg; SEQ ID NO: 9), and packaging plasmids (pKL-Kan- Rev (SEQ ID NO: 10) and pKL-Kan-GagPol (SEQ ID NO: 11) were used to co-transfect 293T cells (purchased from ATCC; stock number: CRL-3216) on a 10-cm 2 cell culture dish, by PEI (a cationic polymer)-mediated transient transfection of eukaryotic cells according to manufacturer's instructions.
  • PEI-Max transfection reagent was from Polysciences (Catalog Number: 24765-1).
  • lentiviruses (supernatant of the transfected cells) were harvested, and aliquots were stored at -80°C. Variable volumes of lentivirus was inoculated into human CD4+ T cell line-MT4 cell line (purchased from Shanghai Suoer Biotechnology Co., Ltd.) pre-plated in 96- well cell culture plates.
  • Culture supernatant from the cells transfected with a lentiviral vector containing an EGFP reporter gene (lentivirus packaged with pCCL-sin-EFlcx-WPRE-EGFP using the above-described method) was used as positive control, and initial transfection titers of lentivirus in the harvested supernatant were calculated by quantitative PCR (qPCR) and flow cytometry data based on GFP signal using the well-known method in the field.
  • qPCR quantitative PCR
  • flow cytometry data based on GFP signal using the well-known method in the field.
  • LV probe 5’ -CCTCCAGGTCTGAAGATCAGCGGCCGC-3 ’ (SEQ ID NO: 28) HK Forward primer: 5’-GCTGTCATCTCTTGTGGGCTGT -3’ (SEQ ID NO: 29) HK probe: 5’-CCTGTCATGCCCACACAAATCTCTCC -3’ (SEQ ID NO: 30) HK Reverse primer: 5’-ACTCATGGGAGCTGCTGGTTC -3’ (SEQ ID NO: 31)
  • the LV probe carries 6-FAM fluorescent dye at the 5'-end and TAMRA fluorescent dye at the 3'-end.
  • the HK probe carries CY5 fluorescent dye at the 5'-end and BHQ2 fluorescent dye at the 3'- end.
  • the qPCR program 94°C 5 min; 95°C 10 sec, 60°C 30 sec, 40 cycles.
  • the initial transfection titers of the lentiviruses in the harvested supernatants of the four different P-globin gene lentiviral vectors (pKL-Kan-TH-POOl, pKL-Kan-TH-P002, pKL-Kan-TH- P005, pKL-Kan-TH-P006) are shown in FIG. 3.
  • the data show that the cis-acting WPRE and the sequence of SV40 pA signal combined with SV40 ori all significantly increased the initial transfection titers of the lentiviruses in the harvested supernatants, and these two enhancements can be additive.
  • the P-globin gene lentiviral vectors (pKL-Kan-TH-P005, pKL-Kan-TH-P006) were used to transfect 293T cells cultured on 2 of the 15-cm 2 cell culture dishes using the same protocol as in Example 3, to package lentiviruses. 48 hours after transfection, lentiviruses (supernatant of the transfected cells) were harvested and centrifuged in a table-top bucket centrifuge for 5 min at 4000 rpm and room temperature to remove cell debris, followed by centrifuging at 10000 g, 4°C for 4 hours.
  • RPMI complete culture medium was added to the virus pellet to resuspend the virus particles using a micro sample injector.
  • the virus resuspension was aliquoted and stored at -80°C for future use.
  • Variable volumes of lentivirus resuspension were inoculated into MT4 cell line, and transfection titers of the lentivirus resuspension were calculated by qPCR and flow cytometry data based on GFP signal following the protocol described in Example 3.
  • K562 cells are of the erythroleukemia type, derived from a patient of chronic granulocytic leukemia (acute phase), which can produce a small amount of hemoglobin during the fetal development and bear some potential to differentiate into erythrocytes.
  • K562 cells were purchased from ATCC (stock number CCL-243). Based on the above calculated transfection titers of the lentiviruses containing P-globin gene of the resuspension, the lentiviruses were inoculated into K562 cells pre-plated in 96-well plates at various multiplicity of infection (MOI). Some cells were harvested at day 5, 10, and 13 after transfection and used for the following experiments.
  • MOI multiplicity of infection
  • K562 cells transduced by lentivirus were harvested and washed with PBS. Then the cells were collected after centrifuged at 4200 rpm for 5 min and resuspended in 50 uL QuickExtractTM DNA Extraction Solution (purchased from Lucigen; Catalog Number QE09050). The resuspended cells were lysed in a PCR machine running at the following conditions (Table 1) and total DNAs were isolated.
  • the transduced K562 cells were fixed in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton-XlOO in PBS, and stained with FITC-labeled mouse anti-human [1- globin mAb. Flow cytometry based on FITC signal was used to determine the percentage of K562 cells expressing human P-globin protein as well as the relative signal intensity of the expressed human (3-globin protein.
  • this invention also optimized the expression frame of P-globin gene, including intron sequences and coding sequences.
  • FIG. 4 Based on P006, we designed 6 other vectors (FIG. 4), comprising of, wild-type human P- globin gene sequence (P009), codon-optimized exons with T87Q mutation using method 3 (P011), codon-optimized exons with T87Q mutation using method 4 (P012), coding sequence optimized human P-globin gene with full-length intron 2 and T87Q mutation using method 4 (P015), coding sequence optimized human P-globin gene with full-length intron 2 but no T87Q mutation using method 2 (P019), or coding sequence optimized human P-globin gene with full-length intron 2 and T87Q mutation using method 4 but with WPRE removed (P021).
  • P009 wild-type human P- globin gene sequence
  • P011 codon-optimized exons with T87Q mutation using method 3
  • P012 codon-optimized exons with T87Q mutation using method 4
  • P015 coding sequence optimized human P-glob
  • the wild-type human P-globin gene sequence (SEQ ID NO: 12) was amplified by the well- known PCR method in the field, using genomic DNAs isolated from 293T cells, which was derived from human (purchased from ATCC; stock number CRL-3216), as template.
  • the amplified PCR fragment was cloned in between cPPT/CTS and LCR of pKL-Kan-TH-P006 by the homologous recombination method well-known to the field.
  • the sequence of the resultant vector was confirmed by sequencing and named as pKL-Kan-TH-P009 (SEQ ID NO: 13).
  • the sequence of the full-length intron 2 of human P-globin gene was amplified by PCR using the plasmid DNA of pKL-Kan-TH-P009 as a template and cloned in between exon 2 and exon 3 of pKL-Kan-TH-P012 by homologous recombination.
  • the sequence of the resultant vector was confirmed by sequencing and named as pKL-Kan-TH-POl 5 (SEQ ID NO: 18).
  • the T87Q mutation of pKL-Kan-TH-P015 was changed back to T87 by a site-directed mutagenesis kit (purchased from Vazyme Biotech Co., Ltd.; catalog number C214) and confirmed by sequencing.
  • the new vector was named as pKL-Kan-TH-P019 (SEQ ID NO: 19).
  • pKL-Kan-TH-P015 plasmid DNA As a template, two fragments not including the WPRE sequence were amplified and recombined by homologous recombination. The sequence of the resultant new vector was confirmed by sequencing and named as pKL-Kan-TH-P021 (SEQ ID NO: 20).
  • lentiviruses were packaged in 293T cells for the lentiviral vectors pKL-Kan-TH-P006, pKL-Kan-TH-POl 1, and pKL-Kan-TH-P012 constructed in Example 6, and the lentivirus resuspension was aliquoted and stored at -80°C for future use.
  • the expression of P-globin gene mediated by lentivirus was tested in cultured cells. Based on the calculated transfection titers of the lentiviruses containing P-globin gene of the resuspension, the lentiviruses were inoculated into K562 cells pre-plated in 96-well plates at the multiplicity of infection (MOI) shown in Table 4. Some cells were harvested at day 5, 10, and 15 after transduction and used for the following experiments.
  • MOI multiplicity of infection
  • K562 cells transduced by lentiviruses were harvested and lysed, and total DNAs were isolated.
  • the vector copy number (VCN) of the lentivirus of the transduced K562 cells were calculated by qPCR and flow cytometry data based on GFP signal (Table 4).
  • K562 cells transduced by lentiviruses were harvested.
  • Flow cytometry based on PE signal was used to determine the percentage of K562 cells expressing human P-globin protein as well as the relative signal intensity of the expressed human P-globin protein.
  • lentiviruses were packaged in 293T cells for the lentiviral vectors pKL-Kan-TH-P006, pKL-Kan-TH-P009, pKL-Kan-TH-P012, pKL-Kan-TH-P015, and pKL-Kan-TH-P019 constructed in Example 6, and the lentivirus resuspension was aliquoted and stored at -80°C for future use.
  • the expression of P-globin gene mediated by lentivirus was then tested in cultured cells. Based on the calculated transfection titers of the lentiviruses containing P-globin gene of the resuspension, the lentiviruses were inoculated into K562 cells pre-plated in 96-well plates at the multiplicity of infection (MOI) shown in Table 6. Some cells were harvested at day 5 and day 10 after transduction and used for the following experiments.
  • MOI multiplicity of infection
  • K562 cells transfected by lentiviruses were harvested and lysed, and total DNAs were isolated.
  • the vector copy number (VCN) of the lentivims of the transfected K562 cells were calculated by qPCR and flow cytometry data based on GFP signal (Table 6).
  • K562 cells transfected by lentiviruses were harvested.
  • Flow cytometry based on PE signal was used to determine the percentage of K562 cells expressing human P-globin protein as well as the relative signal intensity of the expressed human P-globin protein.
  • WPRE Woodchuck hepatitis virus Post-transcriptional Regulatory Element
  • SEQ ID NO: 33 a mutated WPRE
  • WPRE Woodchuck hepatitis virus Post-transcriptional Regulatory Element
  • the WPRE when placed in the 3' UTR of the gene of interest, can enhance the expression of the transgene in the early stages of RNA transcription by increasing mRNA levels in the nucleus and cytoplasm. When placed in the upstream of 3' LTR, transcription termination is improved, and therefore, transcript read-through can be significantly reduced.
  • the WPRE sequence was mutated at 6 bases (mut6), including 5 bases in the predicted WHX promoter region and 1 base of the starting codon, so that WHX could not initiate expression of the WPRE sequence, thereby enhancing safety.
  • the mutated version of WPRE was named mWPRE (SEQ ID NO:33), and P0012, after modification, was named TH04.
  • the mWPRE gene was synthesized and inserted between Mini and Kpnl of POO 12 by restriction enzyme cleavage and ligation. The new construct was confirmed by sequencing and named TH04.
  • This Example describes evaluation of the efficacy of the TH04 vector in a mouse model of thalassemia.
  • the thalassemia model Hbbth-4/Hbb+ mice (purchased from Southern Model Organisms) were used to test efficacy of the TH04 vector.
  • the mice were 11 weeks old, with females as donors and males as recipients.
  • the bone marrow hematopoietic stem/progenitor cells were collected and purified from donor mice, transduced with TH04 lentivirus ex vivo, and infused into the recipient mice via the tail vein injection.
  • the therapeutic effect of TH04 lentivirus was assessed by determining the vector copy number, chimeric rate, and changes in thalassaemia-related blood indicators.
  • mice Three groups were tested: the treatment group, the negative control group, and the positive control group.
  • the negative control group G 1
  • purified bone marrow stem cells from female Hbbth4 mice without LV-TH04 transduction were transplanted into male Hbbth4 mice.
  • the treatment group G2
  • purified bone marrow stem cells from female Hbbth4 mice after LV-TH04 transduction were transplanted into male Hbbth4 mice.
  • G3 purified bone marrow stem cells from female C57BL/6 mice were transplanted into male Hbbth4 mice. Table 8 includes details of the experimental groups.
  • Isolation of mouse bone marrow stem cells After euthanizing the donor animals with carbon dioxide, the femur, tibia, and iliac were quickly separated in a biosafety cabinet and immediately transferred to a sterile Petri dish containing DPBS to prevent the bones from drying out. Bone marrows were rinsed with 5 mL DPBS (2% fetal bovine serum)/mouse into a new 50-mL tube with a 70 gm cell strainer, and bone marrow cells suspensions were centrifuged at 500xg for 5 min at room temperature.
  • DPBS 2% fetal bovine serum
  • mice bone marrow stem cells were purified with Lineage Cell Depletion Kit and c-Kit positive sorting kit from MACS according to manufacturer's instructions.
  • the transduced cells for the treatment group, as well as the cells of the negative control group and the positive control group, were continued to be cultured in a 5% CO2 cell culture incubator for two days.
  • Irradiation of recipient animals irradiation and cell transfusion On the day when culturing of bone marrow cells was finished, recipient animals were subjected to myeloablative conditioning with X-ray irradiation (4.5Gy), twice, 3 h apart. The cultured and collected cell suspensions were infused individually by tail vein injection to groups of animals within two hours after the myeloablative conditioning was completed, and the cells were infused at 1E+06 cells/ animal.
  • VCN and chimeric rate in PBMCs of mice 100 pL of whole blood samples were collected by retro-orbital bleeding at 4, 6 and 8 weeks after bone marrow transplantation of recipient animals, and about 5E+05 PBMCs were isolated after density gradient centrifugation and used for chimeric rate and VCN analysis.
  • the PBMC genomic DNAs were extracted by magnetic bead genome extraction kit, and the concentration of each extracted template DNA was determined by microspectrophotometer.
  • the genomic DNA samples were uniformly diluted with ultrapure water to about 50 ng/pL. Determination of chimeric rate and VCN was accomplished with real-time PCR.
  • VCN Mouse MKL3 gene was used as the reference gene, and LTR as the gene detection primer probe for integration of lentiviral vectors into cells.
  • the sequences for primers and probes are shown in Table 9, the reaction system is shown in Table 10, and the reaction procedure is shown in Table 11.
  • VCN determination results of the VCN determination are shown in FIG. 5.
  • the VCN determination in PBMC showed that the VCN of PBMC in the LV-TH04 transduction group was between 1 ⁇ 4, which was as expected.
  • chimeric rate The sample preparation and method of chimeric rate determination were the same as those of VCN determination, except that the LTR primers and probe were replaced with those for the SRY gene, which is a gene unique to the mouse Y chromosome.
  • SRY gene which is a gene unique to the mouse Y chromosome.
  • the SRY sequences are shown in Table 12.
  • the reaction system is the same as Table 10, except that the LTR primers and probe were replaced with those for SRY.
  • the reaction procedure is the same as Table 11.
  • results of the chimeric rate analyses are shown in FIG. 6.
  • the chimeric rate assay of PBMCs showed that the implantations of bone marrow stem cells in the three groups were consistent, indicating that LV-TH04 transduction did not affect the implantation of stem cells.
  • Routine blood tests Whole blood samples of 50 pL were collected by retro-orbital bleeding from recipient animals for complete blood count (CBC) at 4, 6, and 8 weeks after bone marrow transplantation. The results of main indicators related to thalassaemia are shown in FIG. 7. After transduction and infusion, the LV-TH04 transduction group had significantly improved the hemoglobin content, hematocrit, and average red blood cell volume compared with the Mock group, while the levels of reticulocytes was significantly decreased in the TH04 transduction group compared with the Mock group (FIG. 7). The levels of hemoglobin content, reticulocytes, hematocrit, and average red blood cell volume were similar between the LV-TH04 transduction group and the positive control group (FIG. 7). These data demonstrates the high therapeutic efficacy of TH04 in animal models of the thalassaemia, with curative effects achieved.
  • CBC complete blood count
  • Example 11 Clinical data 1 - Expression assay in red blood cells directionally differentiated from CD34+ stem cells isolated from thalassemia major patients after ex vivo transduction with LV-TH04
  • This Example describes the effects of TH04 on red blood cells directionally differentiated from CD34+ stem cells isolated from thalassemia major patients.
  • HPLC assay conditions include: column C4 4.6*250nm: UV 220nm detection; Sample loading, 20uL. Table 13. HPLC assay conditions
  • Example 12 Clinical data 2 - Myeloablative conditioning protocols generally shortened neutrophil engraftment time and platelet recovery time
  • This Example describes the effects of different myeloablative condition protocols on neutrophil engraftment time and platelet recovery time in patients infused with lentivirally transduced hematopoietic stem cells.
  • myeloablative conditioning based on BU/CY:
  • Busulfan was intravenously administered for 2 hours and every 6 hours at a dosage of 2.4-4.8 mg/kg/day. Cyclophosphamide was administered 24 hours after busulfan administration. The dosage of cyclophosphamide was 45 to 65 mg/kg/day intravenously. The duration of administration of busulfan was 2 to 4 days, and the duration of administration of cyclophosphamide was 1 to 5 days. Infusion of the composition began 24-72 hours after cyclophosphamide administration.
  • Myeloablative conditioning based on BU Myeloablative conditioning based on BU:
  • Busulfan was intravenously infused for 2 hours and every 6 hours at a dosage of 3.2 mg/kg/day. The duration of administration of busulfan was 4 days. Reinfusion of the composition began 72 hours after busulfan administration.
  • Platelet levels in PJYU and ZRHA showed a continuous upward trend from days 17 and 11, respectively, and recovered to normal levels (>100* 10 9 /L) on days 48 and 29, respectively.
  • Platelet levels in FAZH fluctuated between 20-40* I () 9 /L for up to 28 days, and only began to rise on day 43 and recovered to normal levels for the first time by day 56 (FIG. 10).
  • Example 13 Determination of stabilizers for pharmaceutical compositions comprising lentiviral vectors
  • This Example describes screening and evaluation of stabilizers for pharmaceutical compositions comprising, e.g., lentiviral vectors for thalassemia gene therapy.
  • the addition of sugars or polyhydric alcohols to the formulation inhibited the inactivation of lentiviral particles caused by microtherm and repeated freeze-thaw processes.
  • the formulation with sucrose exhibited the best performance, demonstrating significant protection compared with the formulation without sugars or poly hydric alcohols.
  • the combination of sodium citrate and sucrose in the formulation resulted in good bioactivity of lentiviral particles after repeated freeze-thaw, regardless of the ratio of sodium citrate and sucrose.
  • formulations comprising varying ratios of sodium citrate and sucrose, under isotonic conditions, effectively inhibited loss of the titers and bioactivity of the lentiviral vector particles.
  • Example 14 Determination of buffers for pharmaceutical compositions comprising lentiviral vectors
  • This Example describes screening and evaluation of buffers for pharmaceutical compositions comprising, e.g., lentiviral vectors for thalassemia gene therapy.
  • Initial screening of buffers was performed by dispersing purified lentiviral vectors for thalassemia gene therapy into the systems shown in Table 18 to form a formulation. Each formulation was tested as follows:
  • Table 18 Components of formulations tested during initial screening of buffers.
  • the sodium citrate buffer exhibited a better effect on stabilizing the biological activity of lentiviral particles when compared with other buffers. No obvious inactivation of viral particles was observed when using the sodium citrate buffer, indicating that viral particles are more stable in formulations with this buffer than in traditional formulations.
  • the formulations tested e.g., comprising the stabilizers and/or buffers described in Examples 13 and 14, provide a number of benefits, including good freeze-thaw stability and storage stability for lentiviral particles.
  • the formulations can effectively prevent loss of the biological activity of viral particles caused by repeated freeze-thaw and storage at low temperature, and the viral particles still exhibit good bioactivity after repeated freeze-thaw and long-term storage.
  • the formulations tested can disperse more active lentiviral particles in solution, with no obvious precipitation and turbidity observed even with 2-3 * 10 9 TU/mL viral particles.

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Abstract

Sont divulgués des vecteurs de thérapie génique utilisés pour transduire efficacement des cellules pour exprimer un gène de β-globine humaine. Est spécifiquement divulgué un vecteur d'expression qui consiste en : une cassette d'expression pour un gène de β-globine, qui consiste en des exons et des introns du gène de la β-globine humaine, ainsi que des éléments à action cis consistant en un ou plusieurs des signaux de polyadénylation WPRE, SV40 et/ou SV40 ori. Les vecteurs d'expression divulgués présentent une efficacité d'encapsulation de vecteur viral considérablement améliorée dans des lignées cellulaires d'encapsidation de vecteur viral, ce qui conduit à une intégration efficace de vecteurs lentiviraux et à un niveau d'expression élevé de gène de β-globine dans des cellules cibles. Sont également divulguées des compositions pharmaceutiques et des méthodes thérapeutiques utilisant de tels vecteurs d'expression.
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FR2777909B1 (fr) 1998-04-24 2002-08-02 Pasteur Institut Utilisation de sequences d'adn de structure triplex pour le tranfert de sequences de nucleotides dans des cellules, vecteurs recombinants contenant ces sequences triplex
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