WO2012061075A2 - Cellules souches/progénitrices hématopoïétiques et macrophages résistants au vih et fonctionnels provenant de cellules souches pluripotentes induites - Google Patents

Cellules souches/progénitrices hématopoïétiques et macrophages résistants au vih et fonctionnels provenant de cellules souches pluripotentes induites Download PDF

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WO2012061075A2
WO2012061075A2 PCT/US2011/057522 US2011057522W WO2012061075A2 WO 2012061075 A2 WO2012061075 A2 WO 2012061075A2 US 2011057522 W US2011057522 W US 2011057522W WO 2012061075 A2 WO2012061075 A2 WO 2012061075A2
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hiv
cell
cells
ipsc
stem cell
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Joseph Anderson
Gerhard Bauer
Jan A. Nolta
Amal Kambal
Gaela Mitchell
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The Regents Of The University Of California
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    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
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Definitions

  • the present technology relates generally to methods and compositions for treating HIV infection.
  • HIV gene and cellular therapies hold enormous potential for not only treating HIV infected individuals but also providing a functional cure.
  • novel therapies need to be developed.
  • Current antiretroviral drugs provide control of HIV replication. After prolonged use, however, these drugs can become toxic, escape mutants can arise, and thus do not provide a cure [1 -4].
  • Advantages of HIV gene therapy using hematopoietic stem cells (HSCs) include the possibility of a one-time treatment, controlled or constitutive anti- HIV gene expression, and long-term viral inhibition upon HSC transplantation [5].
  • HSCs have the capacity for self-renewal and the proliferation potential to differentiate into HIV resistant target cells including CD4 T cells, macrophages, and dendritic cells [6]. By engineering a sufficient quantity of HSCs to express anti-HIV genes, these cells may completely reconstitute the immune system with HIV resistant immune cells.
  • CCR5 inhibitors siRNAs, zinc- finger nucleases, ribozymes, and intrabodies
  • HIV fusion inhibitors HIV fusion inhibitors
  • TRIM5a HIV fusion inhibitors
  • HSCs human hematopoietic stem cells
  • iPSCs express anti-HIV genes for HIV gene therapy.
  • HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5a gene and optionally a TAR decoy.
  • a robust quantity of colony forming CD133+ HSCs were obtained.
  • These cells were further differentiated into functional end stage macrophages which displayed a normal phenotypic profile.
  • the present disclosure provides an isolated induced pluripotent stem cell (iPSC) derived from a stem cell selected from a mesenchymal stem cell or a hematopoietic stem cell, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, a recombinant or mutant anti-HIV polynucleotide or polypeptide.
  • iPSC isolated induced pluripotent stem cell
  • the hematopoietic stem cell is CD34+ or CD34+/CD133+.
  • the mesenchymal stem cell or the hematopoietic stem cell stem cell from which the iPSC is derived comprises the mutant anti-HIV
  • the iPSC expresses one or more markers of OCT4, SOX2, c- MYC, NANOG, TDGF1 or REX1 .
  • Another embodiment of the present disclosure provides an induced pluripotent stem cell (iPSC) transduced with an anti-HIV polynucleotide, wherein an anti-HIV polynucleotide is stably integrated at a location that does not activate an oncogene or disrupt a housekeeping gene.
  • iPSC induced pluripotent stem cell
  • the iPSC is derived from a stem cell selected from a mesenchymal stem cell or a hematopoietic stem cell. In one aspect, the
  • hematopoietic stem cell is CD34+ or CD34+/CD133+.
  • the mesenchymal or hematopoietic stem cell from which the iPSC is derived is transduced with an anti-HIV polynucleotide.
  • the iPSC expresses one or more markers of OCT4, SOX2, c-MYC, NANOG, TDGF1 or REX1 .
  • the recombinant or mutant anti-HIV polynucleotide comprises any one or more of: a polynucleotide encoding an shRNA, siRNA, antisense RNA, decoy or ribozyme or the like that is inhibitory of a gene or nucleic acid element involved in HIV infection, such as chemokine receptor type 5 (CCR5) or TAR; a polynucleotide encoding Tripartite motif-containing protein 5 (Trim5a); a polynucleotide encoding a mutant of a gene or nucleic acid element that is involved in HIV infection, e.g., CCR5 or TAR; or a polynucleotide encoding an antibody that specifically recognizes a protein involved in HIV infection, e.g., CCR5 or a transdominant protein of such a protein.
  • the recombinant or mutant anti-HIV polynucleotide comprises a polynucleotide encoding a CCR5 shRNA; and a polynucleotide encoding Trim5a, which can be on a single vector or separate vectors.
  • the recombinant or mutant anti-HIV polynucleotide comprises a polynucleotide encoding a CCR5 shRNA; and a polynucleotide encoding Trim5a, which can be on a single vector or separate vectors.
  • the recombinant or mutant anti-HIV comprises a polynucleotide encoding a CCR5 shRNA; and a polynucleotide encoding Trim5a, which can be on a single vector or separate vectors.
  • polynucleotide comprises a polynucleotide encoding a CCR5 shRNA, a
  • polynucleotide encoding Trim5a and a polynucleotide encoding a TAR decoy, which can be on a single vector or separate vectors.
  • the present disclosure provides an isolated cell differentiated from the induced pluripotent stem cell (iPSC) of any one of the above embodiments.
  • the isolated cell is a hematopoietic stem cell.
  • the isolated hematopoietic stem cell is CD34+ or CD34+/CD133+ .
  • the differentiated cell is a macrophage. In another aspect, the differentiated cell is a dendritic cell. In some aspects, the isolated cell is a mammalian cell, which can be a human, a simian, a murine, a bovine, an equine, a porcine or an ovine cell and in a particular aspect, is a human cell.
  • Yet another embodiment of the present disclosure provides a substantially homogeneous population of isolated cells of any of the above embodiments.
  • composition comprising a carrier and one or more of: an induced pluripotent stem cell (iPSC), an isolated cell or a population of any of the above embodiments.
  • iPSC induced pluripotent stem cell
  • the carrier is a biocompatible tissue scaffold or carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • the present disclosure provides, in one embodiment, a plurality of isolated induced pluripotent stem cells (iPSC) of any of the above embodiments, wherein at least two of the iPSC in the plurality displays two different HLA types. In some aspects, at least three, or alternatively four, or five, or six, or ten, or twenty, or thirty, or fifty or one hundred, of the iPSC in the plurality displays at least three different HLA types.
  • iPSC isolated induced pluripotent stem cells
  • Also provided is a method for preparing an HIV-resistant pluripotent cell comprising transducing a mesenchymal stem cell or a hematopoietic stem cell with a recombinant or mutant anti-HIV polynucleotide and one or more polynucleotides encoding OCT4, SOX2, KLF4 and c-MYC, thereby preparing the HIV-resistant pluripotent cell.
  • the mutant polynucleotide comprises a mutant CCR5 with reduced CCR5 activity.
  • the mutant CCR5 comprises a CCR5- A32-bp deletion.
  • the hematopoietic stem cell is CD34+ or
  • the present disclosure further in another embodiment, provides a method for preparing an HIV-resistant pluripotent cell, comprising contacting a mesenchymal stem cell or a hematopoietic stem cell with a polynucleotide encoding OCT4, SOX2, KLF4, c-MYC and an anti-HIV polypeptide or nucleic acid.
  • the present disclosure further in another embodiment, provides a method for preparing an HIV-resistant pluripotent cell, comprising transducing a stem cell comprising an anti-HIV nucleic acid with a polynucleotide encoding OCT4, SOX2, KLF4 and c-MYC.
  • the stem cell is a mesenchymal stem cell or a hematopoietic stem cell.
  • the hematopoietic stem cell is CD34+ or CD34+/CD133+. Suitable examples of anti-HIV nucleic acids/polynucleotides are provided above.
  • the anti-HIV polynucleotide is integrated at a location that does not activate an oncogene or disrupt a housekeeping gene.
  • the one or more polynucleotide encoding OCT4, SOX2, KLF4 and c-MYC is omitted and/or substituted by a small molecule.
  • a method for preventing or treating HIV infection in a patient comprising administering to the patient an effective amount of an isolated cell or cell population of any of the above embodiments.
  • the isolated cell or cell population displays a human leukocyte antigen (HLA) type that is the same as the HLA type of the patient.
  • the method further comprises administering to the patient an effective amount of anti-HIV therapy.
  • FIG. 1 shows the generation of anti-HIV iPSCs and end-stage
  • iPSCs were generated by transducing cord blood (CB) CD34+ HSCs with four lentiviral vectors expressing the pluripotency factors OCT4, SOX2, KLF4, and c-MYC either alone (wild-type (WT)) or with an EGFP control vector (EGFP) or a combination anti-HIV vector (anti-HIV).
  • CB transducing cord blood
  • CD34+ HSCs with four lentiviral vectors expressing the pluripotency factors OCT4, SOX2, KLF4, and c-MYC either alone (wild-type (WT)) or with an EGFP control vector (EGFP) or a combination anti-HIV vector (anti-HIV).
  • iPSCs were further co-cultured on OP9 stromal cells where cystic bodies developed.
  • CD133+ HSCs were isolated from the cystic bodies and grown in semi-solid methylcellulose media to form myeloid CFUs. The CFUs were further cultured in media specific for macro
  • EGFP and anti-HIV iPSCs and their differentiated progeny were visualized by both phase and EGFP fluorescence. H9 hESCs and their differentiated progeny were used as controls.
  • B) CB CD34+ cells were used as a positive control and cultured in semi-solid methylcellulose media and macrophage-specific media to derive myeloid CFUs and end-stage macrophages.
  • FIG. 2 shows expression of pluripotency markers by immunofluorescence: iPSCs, WT, EGFP, and anti-HIV were stained with antibodies specific for the pluripotency markers OCT4, SOX2, NANOG, and SSEA4. Cells were visualized for fluorescence. H9 hESCs were used as pluripotency positive controls. Pictures are representative of triplicate experiments.
  • FIG. 3 includes gel pictures showing expression of pluripotency and differentiation genes by RT-PCR:
  • A) Total RNA from undifferentiated iPSCs was extracted and analyzed by reverse transcriptase PCR for the expression of the pluripotency genes OCT4, SOX2, c-MYC, NANOG, TDGF1 , and REX1 .
  • CB CD34+ HSCs were used as a control to detect expression in the "starter cells”.
  • H9 hESCs were used as a pluripotency positive control.
  • D differentiated cells from the cystic bodies which formed in the iPSC/OP9 co- cultures and analyzed by RT-PCR for the expression of genes from all three germ layers including alphafetoprotein (AFP), CK8, brachyury (BRACHY), MSX1 , and PAX6.
  • Undifferentiated (U) cells were used as negative controls for expression.
  • Undifferentiated and differentiated H9 hESCs were used as negative and positive controls, respectively.
  • GAPDH was used as an internal loading control. Experiments were performed in duplicate.
  • FIG. 4 shows detection and proliferation of iPSC derived CD133+ HSCs:
  • A) iPSCs, WT, EGFP, and anti-HIV were differentiated towards the hematopoietic lineage by OP9 co-cultures. On day 9, the cells were analyzed for their expression of CD133 by FACS. Isotype control stained co-culture cells are displayed in the top-left panel.
  • CB CD34+ HSCs were used as a positive control for CD34 and CD133 expression (topmiddle panel). Undifferentiated iPSCs were used as a negative control for CD34 and CD133 expression (top-right panel). Representative FACS plots are displayed from duplicate experiments.
  • CB CD34+ HSCs were used as a positive comparative control.
  • FIG. 5 shows results of henotypic analysis of iPSC derived macrophages: A) Macrophages derived from the WT, EGFP, and anti-HIV iPSCs were stained with antibodies specific for human CD14, CD4, CD68, and CCR5 and analyzed by FACS. CB CD34+ HSC derived macrophages were used as positive controls. Isotype controls are displayed as unshaded histograms. B) EGFP and anti-HIV iPSC derived macrophages were analyzed by FACS for EGFP expression. WT (EGFP negative) cells were used as negative controls and are displayed as unshaded histograms.
  • FIG. 6 shows cytokine secretion of iPSC derived macrophages
  • Macrophages derived from the WT, EGFP, and anti-HIV iPSCs were stimulated with LPS. On days 2, 4, and 7, cell culture supernatants were analyzed for levels of secretion of A) IL-6, B) IL-10, and C) TNFa. CB CD34+ HSC derived macrophages were used as positive comparative controls. Asterisks above the bars indicate statistically significant values as compared to CB HSCs. Experiments were performed in triplicate.
  • FIG. 7A is a chart showing HIV-1 challenge of iPSC derived macrophages: Macrophages derived from the WT ( ⁇ ), EGFP ( ⁇ ), and anti-HIV (A) iPSCs were challenged with an R5-tropic BaL-1 strain of HIV-1 at an MOI of 0.05. On various days post-infection, cell culture supernatants were sampled and analyzed for p24 antigen by ELISA. Experiments were performed in triplicate.
  • FIG. 7B displays similar data as 7A, however, the HIV-1 strain used in the challenge experiments is called 89.6. It is a dual-tropic strain which can utilize either CCR5 or CXCR4 as a coreceptor.
  • FIG. 7C displays the detection of the HIV-1 transcripts, tat (spliced) and pol (unspliced), in uninfected (Ul), WT iPSC, EGFP-alone iPSC, and anti-HIV iPSC derived macrophages which were challenged with either BaL-1 or 89.6 strains of HIV-1 .
  • FIG. 8 illustrates the anti-HIV constructs used in Example 1 .
  • FIG. 9 shows the combination anti-HIV lentiviral vector, in vivo engraftment of transduced cells, and CCR5 down regulation.
  • a third generation lentiviral vector, CCLc-x-PGK-EGFP was utilized to generate the combination anti-HIV construct, (a) human/rhesus macaque TRIM5a isoform was driven under the control of the MNDU3 promoter, and a CCR5 shRNA and a TAR decoy were driven under separate human polymerase-lll U6 small RNA promoter.
  • These three anti-HIV genes were inserted upstream from the EGFP reporter gene,
  • CD34+ HSCs were transduced with the control EGFP-alone or the anti-HIV vector and transplanted into RAG1 pups.
  • Transplanted mice were screened for human CD45 and EGFP expression in the peripheral blood for engraftment of transduced cells, either EGFP-alone or anti-HIV.
  • the peripheral blood of engrafted mice was analyzed or human T cells with antibodies specific for CD3 and CD4 and also for expression of EGFP.
  • Engrafted human cells were analyzed for the expression of CCR5. Bar graphs display averages and standard deviations from eight mice for each cohort
  • NT nontransduced, EGFP-alone, and anti-HIV.
  • FACS plots are representatives for each cohort.
  • FIG. 10 shows marker expression in engraftment of lymphoid organs in transplanted RAG1 mice.
  • RAG1 mice were transplanted with CD34+ HSCs either nontransduced (NT) or transduced with a control EGFP-alone or the anti-HIV lentiviral vector.
  • various lymphoid organs including the (a) thymus, (b) spleen, and (c) bone marrow were analyzed for human cell engraftment.
  • FACS analysis was performed to detect EGFP expression along with total human leukocytes (CD45), T cells (CD3, CD4, and CD8), B cells (CD19), and macrophages (CD14). Data is representative of eight mice for each cohort.
  • FIG. 11 shows detection of human CD4+ cells in HIV-1 infected RAG1 humanized mice.
  • RAG1 mice successfully engrafted with either control EGFP-alone or combination anti-HIV vector transduced cells were infected IV with either an (a) R5-tropic BaL-1 or an (b) X4-tropic NL4-3 strain of HIV-1 .
  • mice were bled and analyzed by FACS for total human CD4+ cell percent. Solid lines represent anti-HIV cell engrafted mice.
  • Dashed lines represent control EGFP-alone cell engrafted mice.
  • infected mice were sacrificed and the spleen was analyzed for CD4+ T cell (CD3+) levels. Bar graphs display averages and standard deviations from eight mice for each cohort. FACS plots are representatives from each cohort.
  • FIG. 12 presents selective survival advantage of anti-HIV gene modified cells in HIV-1 infected RAG1 mice.
  • Mice successfully engrafted with either control EGFP-alone or combination anti-HIV vector transduced cells were infected IV with either an (a) R5-tropic BaL-1 or an (b) X4-tropic NL4-3 strain of HIV-1 .
  • Solid lines represent anti-HIV cell engrafted mice.
  • Dashed lines represent control EGFP-alone cell engrafted mice.
  • FIG. 13 shows detection of in vivo plasma viremia and in vitro HIV-1 challenge of sorted spleen T cells.
  • RAG1 mice successfully engrafted with either control EGFP-alone or combination anti-HIV vector transduced cells were infected IV with either an (a) R5-tropic BaL-1 or an (b) X4-tropic NL4-3 strain of HIV-1 .
  • HIV-1 challenge experiments were performed on human CD3+ T cells, both nontransduced (EGFP-) and anti-HIV vector transduced (EGFP+), with an (c) R5-tropic BaL-1 or an (d) X4- tropic NL4-3 strain of HIV-1 .
  • culture supernatants were collected and analyzed for p24 by antigen ELISA.
  • p24 ELISA samples were performed in triplicate.
  • FIG. 14 includes charts for cytokine expression and karyotypic analysis of anti-HIV vector transduced cells
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
  • compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).
  • composition is also intended to encompass a combination of active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1 -99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
  • polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like
  • alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • pharmaceutically acceptable carrier refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered
  • compositions suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable).
  • semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable).
  • biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.
  • a mammal intends an animal, a mammal or yet further a human patient.
  • a mammal includes but is not limited to a human, a simian, a murine, a bovine, an equine, a porcine or an ovine.
  • oligonucleotide or “polynucleotide” refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any
  • Oligonucleotides are generally at least about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides in length.
  • An oligonucleotide may be used as a primer or as a probe.
  • isolated refers to molecules or biological or cellular materials being substantially free from other materials, e.g., greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98%.
  • isolated refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source and which allow the manipulation of the material to achieve results not achievable where present in its native or natural state, e.g., recombinant replication or manipulation by mutation.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides, e.g., with a purity greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98%.
  • a "recombinant" nucleic acid refers an artificial nucleic acid that is created by combining two or more sequences that would not normally occur together. In one embodiment, it is created through the introduction of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a specific purpose, such as antibiotic resistance.
  • a “recombinant” polypeptide is a polypeptide that is derived from a recombinant nucleic acid.
  • promoter refers to a nucleic acid sequence sufficient to direct transcription of a gene. Also included in the invention are those promoter elements which are sufficient to render promoter dependent gene expression controllable for cell type specific, tissue specific or inducible by external signals or agents.
  • a promoter is an inducible promoter or a discrete promoter.
  • Inducible promoters can be turned on by a chemical or a physical condition such as temperature or light.
  • chemical promoters include, without limitation, alcohol-regulated, tetracycline-regulated, steroid-regulated, metal- regulated and pathogenesis-related promoters.
  • discrete promoters can be found in, for examples, Wolfe et al. Molecular Endocrinology 16(3): 435-49.
  • regulatory element refers to a nucleic acid sequence capable of modulating the transcription of a gene.
  • Non-limiting examples of regulatory element include promoter, enhancer, silencer, poly-adenylation signal, transcription termination sequence. Regulatory element may be present 5' or 3' regions of the native gene, or within an intron.
  • proteins are also disclosed herein with their GenBank Accession Numbers for their human proteins and coding sequences.
  • the proteins are not limited to human-derived proteins having the amino acid sequences represented by the disclosed GenBank Accession numbers, but may have an amino acid sequence derived from other animals, particularly, a warm-blooded animal (e.g., rat, guinea pig, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.).
  • CCR5 or "chemokine (C-C motif) receptor 5" refers to a protein having an amino acid sequence substantially identical to a representative CCR5 sequence of GenBank Accession No. NP_000570.
  • a suitable cDNA encoding CCR5 is provided at GenBank Accession No. NM_000579.
  • Trim5a refers to a protein having an amino acid sequence substantially identical to a representative Trim5a sequence of GenBank Accession No. NP_149023.
  • a suitable cDNA encoding Trim5a is provided at GenBank Accession No. NM_033034.
  • TAR or "HIV trans-activation response element” refers to an RNA element required for the trans-activation of the viral promoter and virus replication.
  • the TAR hairpin acts as a binding site for the Tat protein and this interaction stimulates the activity of the long terminal repeat promoter (Kulinski et al. J. Biol. Chem. 278(40):38892-901 (2003)).
  • a "TAR decoy” is a RNA decoy which the Tat protein binds to instead of the natural TAR protein and thus generates obstacles in the way of HIV replication.
  • a non-limiting example of a TAR decoy is provided in Anderson et al. Mol. Ther.
  • An exemplary nucleotide sequence of the CCR5 shRNA sequence is gagcatgact gacatctact tcaagagagt agatgtcagt catgctc.
  • Alternative CCR5 RNAi for use in this disclosure include a full length coding sequence for Human G-Protein Chemokine Receptor (CCR5) (GenBank Accession No. DM068065) and the following:
  • the sequence below is a polynucleotide encoding a human/rhesus macaque chimeric TRIM5alpha sequence.
  • the first six nucleotides are the Kozak sequence followed by the ATG start codon.
  • the last 39 nucleotides at the end of the sequence (1492-1530) correspond to a hemmaglutinin tag which was put on the end of the protein coding sequence for detection of expression. These 39 nucleotides are followed by the TGA stop codon.
  • the chimeric TRIM5alpha protein was inserted just downstream from the MNDU3 polymerase-ll promoter in the CCLc-MNDU3-x- PGK-EGFP lentiviral vector.
  • Additional Polymerase-ll promoters for use in this invention include a) EF1 -alpha; b) PGK (phosphoglycerate kinase promoter); c) CMV (minimal cytomegalovirus promoter); and d) LTRs from lentiviral and lentiviral vectors.
  • An exemplary HIV TAR decoy sequence is gtcgaccttg caatgatgtc gtaatttgcg tcttactctg ttctcagcga cagccagatc tgagcctggg agctctctgg ctgtcagtaa gctggtacag aaggttgacg aaaattctta ctgagcaaga aa.
  • Additional TAR sequences for use in this invention include: a) cgacttaaaa tcgctagcca gatctgagcc tgggagctct ctggctag or b) gggtctctct ggttagacca gatttgagcct gggagctctc tggctaactag ggaaccc or c) acgaagcttg atcccgtttg ccggtcgatc gcttcga.
  • treating is meant administering a pharmaceutical composition for the purpose of improving the condition of a patient by reducing, alleviating, reversing, or preventing at least one adverse effect or symptom.
  • the term "preventing” is meant identifying a subject (i.e., a patient) having an increased susceptibility to a disease but not yet exhibiting symptoms of the disease, and administering a therapy according to the principles of this disclosure.
  • the preventive therapy is designed to reduce the likelihood that the susceptible subject will later become symptomatic or that the disease will be delay in onset or progress more slowly than it would in the absence of the preventive therapy.
  • a subject may be identified as having an increased likelihood of developing the disease by any appropriate method including, for example, by identifying a family history of the disease or other degenerative brain disorder, or having one or more diagnostic markers indicative of disease or susceptibility to disease.
  • test sample refers to any liquid or solid material containing nucleic acids.
  • a test sample is obtained from a biological source (i.e., a "biological sample”), such as cells in culture or a tissue sample from an animal, most preferably, a human.
  • an effective amount refers to a quantity of a therapeutic composition delivered with sufficient frequency to provide a medical benefit to the patient.
  • an effective amount of a protein is an amount sufficient to treat or ameliorate a symptom of AIDS.
  • a population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.
  • "Substantially homogeneous" describes a population of cells in which more than about 50%, or alternatively more than about 60 %, or alternatively more than 70 %, or alternatively more than 75 %, or alternatively more than 80%, or alternatively more than 85 %, or alternatively more than 90%, or alternatively, more than 95 %, of the cells are of the same or similar phenotype.
  • Phenotype can be determined by a pre-selected cell surface marker or other marker.
  • an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
  • the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.
  • CDR complementarity determining region
  • stem cell defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells.
  • Stem cells include, for example, somatic (adult) and embryonic stem cells.
  • a somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.
  • An embryonic stem cell is a primitive
  • embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation.
  • HES2 also known as ES02
  • H1 also know as WA01
  • embryonic stem cell lines that are recently approved for use in NIH-funded research including CHB-1 , CHB-2, CHB-3, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-1 1 , CHB-12, RUES1 , HUES1 , HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES1 1 , HUES12, HUES13, HUES14,
  • Pluripotent embryonic stem cells can be distinguished from other types of cells by the use of markers including, but not limited to, Oct-4, alkaline phosphatase, CD30, TDGF-1 , GCTM-2, Genesis, Germ cell nuclear factor, SSEA1 , SSEA3, and SSEA4.
  • a “pluripotent cell” broadly refers to stem cells with similar properties to embryonic stem cells with respect to the ability for self-renewal and pluripotentcy (i.e., the ability to differentiate into cells of multiple lineages).
  • Pluripotent cells refer to cells both of embryonic and non-embryonic origin.
  • pluripotent cells includes Induced Pluripotent Stem Cells (iPSCs).
  • iPSCs Induced Pluripotent Stem Cells
  • iPSC induced pluripotent stem cell
  • iPS cell refers to an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell, produced by inducing expression of one or more reprogramming genes or
  • Such stem cell specific genes include, but are not limited to, the family of octamer transcription factors, i.e. Oct-3/4; the family of Sox genes, i.e. Sox1 , Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, i.e. Klf1 , Klf2, Klf4 and Klf5; the family of Myc genes, i.e. c-myc and L-myc; the family of Nanog genes, i.e. OCT4, NANOG and REX1 ; or LIN28. Examples of iPSCs and methods of preparing them are described in Takahashi et al.
  • a "precursor” or “progenitor cell” intends to mean cells that have a capacity to differentiate into a specific type of cell.
  • a progenitor cell may be a stem cell.
  • a progenitor cell may also be more specific than a stem cell.
  • a progenitor cell may be unipotent or multipotent. Compared to adult stem cells, a progenitor cell may be in a later stage of cell differentiation. Examples of progenitor cells include, but are not limited to, satellite cells found in muscles, intermediate progenitor cells formed in the subventricular zone, bone marrow stromal cells, periosteum progenitor cells, pancreatic progenitor cells and angioblasts or endothelial progenitor cells.
  • progenitor cells may also include, but are not limited to, epidermal and dermal cells from neonatal organisms.
  • MSCs Mesenchymal stem cells
  • Mesenchymal stem cells are multipotent stem cells. Mesenchymal stem cells can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells).
  • HSCs Hematopoietic stem cells
  • myeloid monocytes and macrophages
  • neutrophils basophils
  • eosinophils erythrocytes
  • T-cells megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).
  • One embodiment of the present disclosure provides an isolated induced pluripotent stem cell (iPSC) derived from a stem cell selected from a mesenchymal stem cell (MSC) or a hematopoietic stem cell (HSC), comprising a recombinant or mutant anti-HIV polynucleotide or polypeptide.
  • iPSC isolated induced pluripotent stem cell
  • MSC mesenchymal stem cell
  • HSC hematopoietic stem cell
  • MSCs and HSCs can be readily prepared from a patient, a donor or from an established cell line with methods known in the art and identified with known markers.
  • the HSCs can be CD34+ or CD34+/CD133+, which markers are useful in identification and isolation for the HSCs.
  • iPSC can be readily identified with markers expressed on the cell surface.
  • the iPSC expresses one or more markers of OCT4, SOX2, c-MYC, NANOG, TDGF1 or REX1 .
  • the mutant anti-HIV polynucleotide or polypeptide can be introduced into the cell at any time, or alternatively can be a native mutant that has existed in the patient or donor, such as a CCR5 with the A32-bp deletion (Hutter et al. N Engl J Med; 360: 692-8 (2009)), or alternatively can be a mutant generated by a cell manipulation method, such as using a zinc finger nuclease.
  • the recombinant anti-HIV polynucleotide or polypeptide can be introduced into the cell at any time. In one aspect, it is transduced into the MSC or HSC after it is isolated. In another aspect, the anti-HIV polynucleotide or polynucleotide is transduced into the iPSC after the iPSC is derived from the MSC or HSC.
  • the anti-HIV polynucleotide when the anti-HIV polynucleotide is introduced into the MSC, HSC or iPSC, the polynucleotide is stably integrated at a location that does not activate an oncogene or disrupt a housekeeping gene. As such, the anti-HIV polynucleotide does not disrupt the function of a gene which is needed for a cell's normal function. Further, such an integration would not activate an oncogene or a gene that is normally not active in such a cell or a cell differentiated from the cell.
  • Methods of testing whether a polynucleotide is stably integrated at a location that does not activate an oncogene or disrupt a housekeeping gene are known in the art, for example, by PCR or in situ hybridization.
  • the present disclosure provides an isolated cell differentiated from the induced pluripotent stem cell (iPSC) of any one of the above embodiments.
  • the isolated cell is a hematopoietic stem cell.
  • the isolated hematopoietic stem cell is CD34+ or CD34+/CD133+.
  • the iPSCs After the iPSCs is differentiated into HSCs or other types of precursor cells, they can be further induced to differentiate into cells, such as macrophages and dendritic cells, that can be introduced into a patient for providing HIV resistance or therapy. Methods of inducing differentiation of HSCs to macrophages or dendritic cells are known in the art.
  • the present disclosure further contemplates an anti-HIV stem cell bank, which includes a plurality of anti-HIV stem cells, wherein the plurality represent two or more different HLA types.
  • the number of HLA types represented in the stem cell bank is greater than 2, or alternatively greater than 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, or 500.
  • the stem cells in the bank can be iPSC, MSC, HSC or other types of stem cells prepared with methods provided in the present disclosure and are resistant to HIV infection.
  • the bank provides anti-HIV stem cells for patents with different types of HLA.
  • the anti-HIV stem cells can be used for both autologous and allogeneic transplants.
  • An HIV patient can be treated with a matching HLA type of a derived anti-HIV stem cell line.
  • this method can be used as an "off the shelf” therapy where the cells can be shipped anywhere once the specific HLA type is known.
  • the stem cell iPSC, MSC or HSC, includes an anti-HIV polynucleotide or polypeptide which provides HIV resistance to the stem cell.
  • an anti-HIV polynucleotide or polynucleotide can be an inhibitor of a gene involved in HIV infection, or a mutant of a gene involved in HIV infection that competes with the gene.
  • an anti-HIV polynucleotide that inhibits the activity of a gene or nucleic acid element involved in HIV infection, such as CCR5 or TAR, or a polynucleotide encoding such a polynucleotide.
  • a gene or nucleic acid element involved in HIV infection such as CCR5 or TAR
  • a polynucleotide encoding such a polynucleotide include shRNA, siRNA, dsRNA, miRNA, antisense RNA, decoy or ribozyme.
  • CD4 receptor the primary cellular receptor for HIV entry is the CD4 receptor.
  • CXCR4, and CCR5 are necessary co-factors that allow HIV entry when co- expressed with CD4 on a cell surface.
  • CXCR4 or fusin
  • T cells T cells (Feng et al. (1996) Science 10:272(5263):872-7).
  • Co-expression of CXCR4 and CD4 on a cell allow T-tropic HIV isolates to fuse with and infect the cell.
  • HIV gp120 interacts with both CD4 and CXCR4 to adhere to the cell and to effect conformational changes in the gp120/gp41 complex that allow membrane fusion by gp41 .
  • siRNA short interfering RNAs
  • dsRNA double-stranded RNA molecules
  • siRNAi short interfering RNA
  • siRNAi sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by short interfering RNA (siRNA).
  • siRNA includes short hairpin RNAs (shRNAs).
  • a siRNA directed to a gene or the mRNA of a gene may be a siRNA that recognizes the mRNA of the gene and directs a RNA-induced silencing complex (RISC) to the mRNA, leading to degradation of the mRNA.
  • RISC RNA-induced silencing complex
  • a siRNA directed to a gene or the mRNA of a gene may also be a siRNA that recognizes the mRNA and inhibits translation of the mRNA.
  • a siRNA may be chemically modified to increase its stability and safety. See, e.g. Dykxhoorn and Lieberman (2006) Annu. Rev. Biomed. Eng. 8:377-402 and U.S. Patent Application Publication No.: 2008/0249055.
  • dsRNA Double stranded RNAs
  • dsRNA double stranded RNA molecules that may be of any length and may be cleaved intracellularly into smaller RNA molecules, such as siRNA.
  • longer dsRNA such as those longer than about 30 base pair in length, may trigger the interferon response.
  • dsRNA may be used to trigger specific RNAi.
  • miRNAs refer to single-stranded RNA molecules of 21 -23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down- regulate gene expression.
  • mRNA messenger RNA
  • siRNA, dsRNA, and miRNA to inhibit gene expression can be designed following procedures known in the art. See, e.g., Dykxhoorn and Lieberman (2006) Annu. Rev. Biomed. Eng. 8:377-402; Dykxhoorn et al. (2006) Gene Therapy 13:541 - 52; Aagaard and Rossi (2007) Adv. Drug Delivery Rev. 59:75-86; de Fougerolles et al. (2007) Nature Reviews Drug Discovery 6:443-53; Krueger et al. (2007)
  • siRNA, dsRNA or miRNA Delivery of siRNA, dsRNA or miRNA to a cell can be carried out with methods known in the art. See, e.g., Dykxhoorn and Lieberman (2006) Annu. Rev. Biomed. Eng. 8:377-402; Dykxhoorn et al. (2006) Gene Therapy 13:541 -52; Aagaard and Rossi (2007) Adv. Drug Delivery Rev. 59:75-86; de Fougerolles et al. (2007) Nature Reviews Drug Discovery 6:443-53; Krueger et al. (2007) Oligonucleotides 17:237-250; U.S. Patent Application Publication No.: 2008/0188430; and U.S. Patent Application Publication No.: 2008/0249055.
  • Antisense oligonucleotides have nucleotide sequences complementary to the protein coding or "sense” sequence. Antisense RNA sequences function as regulators of gene expression by hybridizing to complementary mRNA sequences and arresting translation (Mizuno et al. (1984) PNAS 81 :1966; Heywood et al. (1986) Nucleic Acids Res. 14:6771 ). An antisense polynucleotide comprising the entire sequence of the target transcript or any part thereof can be synthesized with methods known in the art. See e.g., Ferretti et al. (1986) PNAS 83:599.
  • the antisense polynucleotide can be placed into vector constructs, and effectively introduced into cells to inhibit gene expression (Izant et al. (1984) Cell 36:1007). Generally, to assure specific hybridization, the antisense sequence is substantially complementary to the target sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence.
  • polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid
  • sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to the gene is retained as a functional property of the polynucleotide.
  • antisense nucleic acids can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art.
  • antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning.
  • an antisense RNA can be made by inserting (ligating) a gene sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid).
  • the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.
  • Ribozymes can also be inhibitory to a gene (see, e.g., Cech (1995)
  • RNA enzyme an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own
  • a ribozyme can be a triplex ribozyme.
  • Tripleplex ribozymes allow for increased target cleavage relative to conventionally expressed ribozymes.
  • Examples of triplex ribozymes include hairpin ribozymes and hammerhead ribozymes. Methods of making and using triplex ribozymes are found in, e.g., Aguino-Jarguin et al. (2008)
  • the anti-HIV polynucleotide in one embodiment, encodes an antibody that specifically recognizes a gene involved in HIV infection, such as CCR5.
  • Methods of preparing an antibody are generally known in the art and antibodies are
  • an anti-HIV polynucleotide encodes a
  • transdominant protein of a protein involved in HIV infection such as CCR5.
  • transdominant proteins of CCR5 is provided, for example, in Benkirane et al. (1997) J. Biol. Chem., 272:30603-6.
  • a "mutant anti-HIV polynucleotide or polypeptide” is a mutant of a gene that is involved in HIV infection, which mutation results in reduction or elimination of the function or activity of the gene. Therefore, a cell having the mutant polynucleotide or polypeptide has a defective HIV infection and thus is resistance to HIV infection.
  • mutants of CCR5 can be readily designed by, for example, deleting or changing a domain that is involved in maintaining CCR5's stability or activity.
  • One such example is the CCR5 A32-bp deletion (Hutter et al. N Engl J Med; 360: 692-8 (2009)).
  • the iPSC can be prepared from a MSC or HSC isolated from a donor who has such a mutant.
  • a mutation can be generated with methods known in the art, such as using a knock-out procedure or a zinc-finger nuclease.
  • ZFNs Zinc-finger nucleases
  • Zinc finger domains can be engineered to target desired DNA sequences which enables zinc- finger nucleases to target unique sequence within a complex genome. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Cathomen and Joung (2008) Molecular Therapy 16(7):1200-7.
  • ZFNs for examples, have been designed to specifically mutate CCR5.
  • Perez et al. (2008) Nature Biotechnology, 26:808-16 describes a CD4+ T cell with a mutant CCR5 generated with ZFNs.
  • Another example of an anti-HIV polynucleotide encodes a Tripartite motif- containing protein 5 (Trim5a).
  • Trim5a is a retrovirus restriction factor and can mediate species-specific, early block to retrovirus infection.
  • an anti-HIV stem cell contains two or more anti-HIV polynucleotides or polynucleotides, each of which can be on a single vector or separate vectors.
  • vector refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a cell, for example by a process of transformation.
  • Vectors may be viral or non-viral.
  • Viral vectors include retroviruses, adenoviruses, herpesvirus, papovirus, or otherwise modified naturally occurring viruses.
  • non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA- protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
  • Non-viral vector may include plasmid that comprises a heterologous polynucleotide capable of being delivered to a target cell, either in vitro, in vivo or ex- vivo.
  • the heterologous polynucleotide can comprise a sequence of interest and can be operably linked to one or more regulatory elements and may control the transcription of the nucleic acid sequence of interest.
  • a vector need not be capable of replication in the ultimate target cell or subject.
  • the term vector may include expression vector and cloning vector.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
  • Gene delivery vehicles also include DNA liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., a cell surface marker found on stem cells or cardiomyocytes.
  • cell surface antigens e.g., a cell surface marker found on stem cells or cardiomyocytes.
  • direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this invention are other non-limiting techniques.
  • Proteins or antibodies have been described that have the ability to translocate desired nucleic acids across a cell membrane.
  • such proteins have amphiphilic or hydrophobic subsequences that have the ability to act as membrane-translocating carriers.
  • homeodomain proteins have the ability to translocate across cell membranes.
  • the shortest internalizable peptide of a homeodomain protein, Antennapedia was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz (1996) Current Opinion in Neurobiology 6:629-634.
  • Another subsequence, the h (hydrophobic) domain of signal peptides was found to have similar cell membrane translocation
  • Such subsequences can be used to translocate oligonucleotides across a cell membrane. Oligonucleotides can be conveniently derivatized with such sequences.
  • a linker can be used to link the oligonucleotides and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker or any other suitable chemical linker.
  • a method for preparing an HIV- resistant pluripotent cell comprising, or alternatively consisting essentially of, or yet alternatively consisting of, transducing a mesenchymal stem cell or a hematopoietic stem cell with a recombinant or mutant anti-HIV polynucleotide and one or more polynucleotides encoding OCT4, SOX2, KLF4 and c-MYC, thereby preparing the HIV-resistant pluripotent cell.
  • anti-HIV polynucleotides are provided above.
  • the hematopoietic stem cell is CD34+ or
  • the present disclosure further in another embodiment, provides a method for preparing an HIV-resistant pluripotent cell, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting a mesenchymal stem cell or a hematopoietic stem cell with a polynucleotide encoding OCT4, SOX2, KLF4, c- MYC and an anti-HIV polypeptide or nucleic acid.
  • a method for preparing an HIV-resistant pluripotent cell comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting a mesenchymal stem cell or a hematopoietic stem cell with a polynucleotide encoding OCT4, SOX2, KLF4, c- MYC and an anti-HIV polypeptide or nucleic acid.
  • anti-HIV anti-HIV
  • polynucleotides are provided above.
  • the present disclosure further in another embodiment, provides a method for preparing an HIV-resistant pluripotent cell, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, transducing a stem cell comprising an anti-HIV nucleic acid with a polynucleotide encoding OCT4, SOX2, KLF4 and c- MYC.
  • the stem cell is a mesenchymal stem cell or a hematopoietic stem cell.
  • the hematopoietic stem cell is CD34+ or
  • CD34+/CD133+ examples of anti-HIV polynucleotides (nucleic acids) are provided above.
  • the anti-HIV polynucleotide is integrated at a location that does not activate an oncogene or disrupt a housekeeping gene.
  • the one or more polynucleotide encoding OCT4, SOX2, KLF4 and c-MYC is omitted and/or substituted by a small molecule, so long as the cell can be induced into a iPSC.
  • the present disclosure provides methods for preventing or treating HIV infection in a patient, comprising administering to the patient an effective amount of an isolated cell or cell population of any of the above embodiments.
  • the isolated cell or cell population displays a human leukocyte antigen (HLA) type that is the same as the HLA type of the patient.
  • the method further comprises administering to the patient an effective amount of anti-HIV therapy.
  • compositions described herein for a therapeutic use may be any compositions described herein for a therapeutic use.
  • “pharmaceutical carriers” are well known to those of skill in the art and can include, but not be limited to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions, such as oil/water emulsions and various types of wetting agents.
  • administering for in vivo and ex vivo purposes means providing the subject with an effective amount of the nucleic acid molecule or polypeptide effective to prevent or inhibit a disease or condition in the subject.
  • compositions are well known to those of skill in the art and include, but are not limited to, microinjection, intravenous or parenteral administration.
  • the compositions are intended for topical, oral, or local administration as well as intravenously, subcutaneously, or intramuscularly.
  • Administration can be effected continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the vector used for therapy, the polypeptide or protein used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. For example, the compositions can be administered prior to a subject already suffering from a disease or condition that is linked to apoptosis.
  • kits for use in preventing or treating HIV infection comprising, or alternatively consisting essentially of, or yet alternatively consisting of, an effective amount of a composition comprising an anti- HIV stem cell or cell population and instructions to use.
  • anti-HIV stem cell or cell population for the manufacture of a medicament for preventing or treating HIV infection.
  • HSCs human hematopoietic stem cells
  • iPSCs express anti-HIV genes for HIV gene therapy.
  • HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5a gene.
  • a robust quantity of colony forming CD133+ HSCs were obtained.
  • These cells were further differentiated into functional end stage macrophages which displayed a normal phenotypic profile.
  • the anti-HIV iPSC derived macrophages Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection from HIV-1 infection. This example demonstrates the ability of iPSCs to develop into HIV- 1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.
  • a selfinactivating lentiviral vector CCLc-MNDU3-x-PGK-EGFP
  • CCLc-MNDU3-x-PGK-EGFP a selfinactivating lentiviral vector
  • human TRIM5a cDNA was cloned into pCR2.1 (Invitrogen, Carlsbad, CA). Site directed mutagenesis was performed to generate the chimeric human/rhesus macaque TRIM5a gene (HRH), as described previously [39], and subsequently inserted under the control of the MNDU3 promoter in the CCLc-MNDU4-x-PGK- EGFP lentiviral vector.
  • HRH human/rhesus macaque TRIM5a gene
  • the U6-CCR5shRNA expression cassette was PCR amplified using a human genomic U6 promoter sequence and a long DNA oligo corresponding to the CCR5shRNA sequence, as described previously [12] (FIG. 8). The U6- CCR5shRNA expression cassette was then cloned downstream of the MNDU3-HRH expression cassette.
  • HEK-293T cells were transfected by lipofection with 25 g of the lentiviral transfer vectors (OCT4, SOX2, KLF4, c-MYC, EGFP-alone (control vector), or the combination anti-HIV), 25 g of pCMV-A8.9 (packaging plasmid expressing gag and pol), and 5 g of pMDG-VSVG (Vesicular Stomatitis Virus glycoprotein-pseudotyping envelope). Two days
  • vector supernatants were collected, concentrated by ultrafiltration, and stored at -80 °C.
  • CD34+ cells were cultured for two days in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum, and 50 ng/ml of Flt3, SCF and TPO.
  • IMDM Iscove's Modified Dulbecco's Medium
  • CD34+ cells were transduced with all four of the individual doxycyclineinducible lentivectors expressing human OCT4, SOX2, KLF4, and c-MYC alone or in addition to either the EGFP-alone
  • control vector or the anti-HIV combination vector at a 1 :1 :1 :1 :1 ratio at an MOI of 16.
  • Karyotyping To determine the chromosomal and genetic stability of the HSC derived iPSCs, karyotyping was performed. The iPSCs were treated with Colcemid, a mitotic inhibitor, for 30 minutes at 37°C to arrest the cells in metaphase followed by treatment with cell stripper for 10 minutes at 37°C. The cell suspension was then treated with KCL hypotonic solution and subsequent 3:1 methanol :acetic acid fixative solutions. Karyotyping slides were made and Giemsa banded.
  • Karyotyping was performed on an Olympus Bx41 microscope with a DP20 camera. Analysis was performed with an Applied imaging System.
  • Immunofluorescence To determine whether the derived iPSCs displayed normal pluripotency and self renewal markers, immunofluorescence was performed. iPSCs and hESCs were plated in 12-well plates, cultured for 2 days, then rinsed with phosphate buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS for 10 minutes at room temperature. Cells were washed with PBS and
  • RNA-Stat-60 Tel-Test, Friendswood, TX
  • cDNA was synthesized using an Applied Biosystems Taqman Reverse Transcription Reagents kit (Applied Biosystems, Carlsbad, CA).
  • RT-PCR reactions were performed using 200 ng of cDNA for each reaction with primer sets specific for each gene of interest: OCT4(f) 5'-AAACCCTGGCACAAACTCC-3' OCT4(r) 5'-GACCAGTGTCCTTTCCTCTG-3' SOX2(f) 5'-
  • TRIM5a was detected using the SYBR Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA) with a primer pair specific to the chimeric TRIM5a gene and not the native human TRIM5a gene: (f) 5'- CTGGGTTGATGTGACAGTGG-3' and (r) 5'-CGTGAGTGACGGAAACGTAA-3'.
  • a QuantiMir RT kit System Biosciences, Mountain View, CA was used according to the manufacturer's protocol.
  • the forward primer used for detection of the CCR5 shRNA was 5'- GTAGATGTCAGTCATGCTC-3'. These experiments were performed in triplicate.
  • Directed differentiation of iPS cells For directed differentiation of iPSCs to the hematopoietic lineage, hESCs and iPSCs were co-cultured on OP9 mouse stromal cells. One day before co-culture, 1 .2 x 10e OP9 cells were plated onto gelatinized 10-cm dishes. Cells were added to OP9 cultures at a density of 1 .6 x 10e cells/dish in 20 ml of differentiation medium: a-MEM supplemented with 10% FBS and 50 ng/ml BMP-4. Half media changes were performed every three days. Cells from the cystic bodies which formed in the co-cultures were harvested at day 9 and single cell suspensions were prepared by treatment of the co-cultures with
  • Collagenase IV (BD Biosciences, San Jose, CA) (I mg/ml in a MEM) for 10 minutes at 37°C followed by treatment with 0.05% trypsin-EDTA for 3 minutes at 37 °C. Cells were washed twice with PBS, filtered through a cell strainer, and used for FACS analysis and HSC cell purification.
  • CD133 magnetic antibodies Single cell suspensions from the hESC and iPSC OP9 co-cultures were labeled with CD133 magnetic antibodies using a Direct CD133 microbead isolation kit (Miltenyi Biotech, Auburn, CA) and processed through two LS separation column.
  • myelomonocytic colonies were harvested from the Methocult cultures and plated in complete DMEM containing 10% FBS and 10 ng/ml each of GM-CSF and M-CSF. The macrophages were then used for phenotypic, functional, and HIV-1 challenge experiments.
  • Flow cytometry To determine the levels of HSC generation in the hESC and iPSC OP9 co-cultures, single-cell suspensions obtained from the OP9 co- cultures were labeled with a PE-conjugated antibody specific for human CD133 (Miltenyi Biotec, Auburn, CA). Control cord blood CD34+ HSCs were labeled with the CD133-PE antibody and also with an APC-conjugated antibody specific for human CD34 (BD Biosciences, San Jose, CA). The CD34+ HSC derived iPSCs were labeled with the CD133-PE and CD34-APC antibodies to determine if they retained expression of HSC markers upon induction of pluripotency.
  • iPSC and cord blood CD34+ derived macrophages were labeled with PE-conjugated antibodies specific for human CD14, CCR5, CD4, and CD68 (BD Biosciences, San Jose, CA). FACS analyses were performed on a Beckman Coulter FC500 and analyzed on Expo32 software. Experiments were performed in triplicate.
  • HIV-1 challenge of iPSC derived macrophages To determine whether the expression of the anti-HIV genes in the anti-HIV iPSC derived macrophages conferred resistance to HIV-1 infection, cells were challenged with an R5-tropic strain of HIV-1 , BaL-1 , at an MOI of 0.05. On various days post-infection, challenge supernatants were sampled and analyzed with an HIV-1 p24 antigen ELISA kit (Zeptometrix Corp., Buffalo, NY). Experiments were performed in triplicate. Results:
  • iPSC induced pluripotent stem cell
  • iPSCs are similar to human embryonic stem cells (hESCs) in their capacity to differentiate into cells of all three germ layers, the ectoderm, mesoderm, and endoderm [26].
  • hESCs human embryonic stem cells
  • Various cell types have been derived from iPSCs including cardiomyocytes, mesenchymal stem cells, hepatic endoderm, and hematopoietic cells [30-34].
  • iPSCs like hESCs, are also capable of indefinite growth and self-renewal with detected telomere elongation [35]. They have enormous potential as a source of autologous cells for experimental and therapeutic use.
  • iPSCs have the potential to generate a continuous supply of anti-HIV HSCs.
  • cells differentiated from iPSCs not only provide a source for cellular therapies but also hold the potential to correct genetic defects and inhibit infectious diseases such as HIV.
  • This example has generated anti-HIV gene expressing iPSCs from cord blood CD34+ "starter cells".
  • the example has utilized a combination of anti-HIV genes including a CCR5 shRNA and a chimeric human/rhesus TRIM5a molecule.
  • the iPSC lines were continually growing and did not contain any HSCs.
  • the anti-HIV iPSCs generated a robust quantity of colony forming hematopoietic progenitors which subsequently developed into phenotypically and functionally normal macrophages which were resistant to HIV-1 infection.
  • the results presented in this example are the first to generate HIV-1 resistant immune cells from iPSCs and highlight the potential use of iPSC technology for HIV gene and cellular therapies.
  • This vector contains a human/rhesus macaque TRIM5a isoform under the control of the modified retroviral MNDU3 promoter directly followed by a CCR5 shRNA under the control of a U6 pol-lll promoter.
  • Another self-inactivating lentiviral vector, CCLc-TRE-PGK-rtTA which is doxycycline inducible, was utilized to construct the iPSC reprogramming vectors OCT4, SOX2, KLF4, and c-MYC.
  • the transduction and expression of the anti-HIV genes did not adversely affect either iPSC generation or the morphology of the anti-HIV iPSC colonies as the colonies formed at the same time (9 days) as the control cells and also displayed similar morphology.
  • the newly generated iPSC lines have retained their pluripotent and "embryonic-like"
  • Anti-HIV iPSCs express pluripotency markers: To determine whether the anti-HIV iPSCs were fully reprogrammed and expressed pluripotency markers, immunofluorescence was performed. iPSCs were stained with antibodies specific for various pluripotency markers including OCT4, SOX2, NANOG, and SSEA4. As displayed in FIG. 2, all iPSC lines, WT, EGFP, and anti-HIV expressed all
  • pluripotency markers analyzed as compared to control H9 hESCs.
  • Reverse transcriptase PCR was also performed to detect the gene expression of various pluripotency genes including OCT4, SOX2, c-MYC, NANOG, TDGF1 , and REX1 .
  • RT-PCR Reverse transcriptase PCR
  • all iPSCs expressed the pluripotency genes as compared to control H9 hESCs.
  • Cord blood CD34+ cells (CB) used as a negative control, expressed lower levels of the pluripotency markers as compared to the iPSCs or the H9 hESCs.
  • GAPDH was used as an internal control.
  • Another characteristic of true pluripotent stem cells is their ability to differentiate into cells and tissues of all three germ layers, the ectoderm, mesoderm, and endoderm. To determine if the anti-HIV iPSCs were capable of such
  • RT-PCR was performed to detect expression of specific genes from all three germ layers including alphafetoprotein (AFP), CK8, brachyury (BRACHY), MSX1 , and PAX6.
  • AFP alphafetoprotein
  • CK8 brachyury
  • MSX1 brachyury
  • PAX6 PAX6.
  • Cells were allowed to differentiate on mouse OP9 stromal cells followed by analysis by RT-PCR.
  • FIG. 3b upon differentiation (D), all iPSC lines expressed similar expression levels of each gene analyzed as compared to the positive control H9 hESC differentiated cells. Expression of the differentiated genes was not detected in undifferentiated (U) iPSCs or H9 hESCs. GAPDH was used as an internal control.
  • FIG. 4a top right panel
  • the CD34+ cell derived iPSCs did not express CD34 or CD133 upon reprogramming. This was in comparison to cord blood (CB) CD34+ cells which expressed 94.1 % CD34 and 65.9% CD133 upon immunomagnetic bead purification (FIG. 4a, top middle panel).
  • CB cord blood
  • FIG. 4a top middle panel
  • the iPSC derived CD133+ cells formed morphologically normal myeloid CFUs as compared to H9 hESC derived CD133+ HSCs and cord blood CD34+ HSCs (Figs. 1a,b).
  • EGFP and anti-HIV iPSC derived CFUs were EGFP positive confirming continued expression of the EGFP and anti-HIV transgenes upon differentiation of the iPSCs into HSCs (FIG. 1a).
  • the iPSC derived CD133+ HSCs were also evaluated to determine whether their proliferation potential was comparable to cord blood CD34+ cells. After 9 days of culture in Methocult media, total cell numbers were counted and compared to the initial cell input. As displayed in FIG. 4b, iPSC derived CD133+ HSCs, whether WT, EGFP, or anti-HIV, increased approximately ⁇ 40-fold in total cell numbers.
  • Anti-HIV iPSC derived phenotypically and functionally normal macrophages To evaluate the capacity of anti-HIV iPSC derived HSCs to develop into phenotypically and functionally normal macrophages, CFUs were further cultured in a macrophage specific differentiation media containing GM-CSF and MCSF. After four days, morphologically distinct macrophages had attached to the culture plates in the WT, EGFP, and anti-HIV iPSC derived macrophage cultures (FIG. 1a). This was similar to cord blood CD34+ cell derived macrophage cultures which also displayed macrophage development in four days (FIG. 1 b).
  • CCR5 cell surface expression was dramatically decreased (6.7% positive) in the anti-HIV iPSC derived macrophages compared to CB (68.2% positive), WT iPSC (63.0% positive), and EGFP iPSC (62.3% positive) derived macrophages (FIG. 5a).
  • the decrease in CCR5 expression was due to the presence of the CCR5 shRNA anti-HIV gene and demonstrated the constitutive expression of this transgene upon differentiation from iPSC to end-stage macrophages.
  • EGFP (99.9%) and anti-HIV (99.8%) iPSC derived cells also remained EGFP positive throughout the
  • FIG. 1a The iPSC and cord blood CD34+ HSC derived macrophages were stimulated with
  • IL-6 lipopolysaccharide
  • IL-10 lipopolysaccharide
  • TNFa TNFa
  • FIG. 6 the iPSC derived macrophages were functional and secreted substantial amounts of IL-6 (FIG. 6a), IL-10 (FIG. 6b), and TNFa (FIG. 6c) upon stimulation compared to non-stimulated (NS) macrophages.
  • NS non-stimulated
  • the secretion of IL-2, IL-4, and IFNy were not detected and were used as negative controls (data not shown).
  • Cytokine secretion from the iPSC derived macrophages were directly compared to cord blood (CB) derived macrophages.
  • CB cord blood
  • the goal of the study was to derive end-stage macrophages from iPSCs which were resistant to HIV infection. Therefore, to determine whether the anti-HIV iPSC derived macrophages were capable of blocking HIV-1 infection, the cells were challenged with an R5-tropic BaL-1 strain of HIV-1 at a multiplicity of infection (MOI) of 0.05. On various days post-infection, culture supernatants were sampled and analyzed by p24 antigen ELISA. As displayed in FIG. 7A, potent inhibition of HIV-1 infection was observed. By day 15 post-infection, over a 3-log reduction in p24 antigen was detected in the anti-HIV macrophage cultures as compared to the WT and EGFP macrophage cultures. These results demonstrate that HIV-resistant macrophages can be developed from anti-HIV gene expressing iPSCs.
  • MOI multiplicity of infection
  • Anti-HIV gene expressing iPSCs were also capable of generating a robust quantity of anti-HIV HSCs which were further differentiated into macrophages which inhibited HIV-1 infection.
  • Current HIV stem cell gene therapy protocols rely on the apheresis of a patient's mobilized peripheral blood stem cells, ex vivo manipulation of these cells, and transplantation of the gene transduced cells back into the patient [7-1 1 ].
  • a major disadvantage with this strategy is that the quantity of cells obtained for ex vivo manipulation is limited.
  • starter cells have been utilized to generate iPSC lines including fibroblasts, lymphocytes, and hematopoietic stem cells [26-27,29].
  • starter cells include fibroblasts, lymphocytes, and hematopoietic stem cells [26-27,29].
  • the initial hypothesis for utilizing HSCs as "starter cells” to derive the anti-HIV iPSCs stemmed from the final goal of deriving anti-HIV HSCs from the reprogrammed cells.
  • the expression of the anti-HIV transgenes did not show any adverse effect on iPSC generation or on the morphology of the iPSCs as displayed in FIG. 1a.
  • the anti-HIV iPSCs had similar morphology as compared to H9 hESCs displaying defined round colonies and tight edges.
  • expression of the anti-HIV genes in the iPSCs also did not have an effect on the expression of pluripotency markers as measured by immunofluorescence and RT-PCR.
  • the anti-HIV iPSCs displayed normal expression of the various pluripotency markers analyzed.
  • the anti-HIV iPSCs displayed similar growth kinetics compared to control iPSCs as determined by visualization after every passage. These data confirmed that the anti- HIV genes introduced into CB CD34+ cells at the same time as the reprogramming factors was feasible, did not produce toxicity, and did not have a negative effect on development, growth kinetics, or directed differentiation into HSCs and macrophages.
  • anti-HIV HSCs differentiated from the anti-HIV iPSCs need to harness the capacity for cell proliferation and differentiation into phenotypically and functionally normal immune cells.
  • the anti-HIV HSCs derived from the anti-HIV iPSCs were capable of generating a robust quantity of CD133+ HSCs and hematopoietic progenitors as compared to H9 hESC derived CD133+ cells.
  • the anti-HIV iPSC derived HSCs were capable of developing into normal macrophages at a rate similar to cord blood CD34+ cells.
  • Anti-HIV iPSC derived macrophages were phenotypically normal and expressed cell surface markers similar to cord blood CD34+ cell derived macrophages. Additionally, they also secreted normal macrophage cytokines upon stimulation with LPS.
  • anti-HIV iPSC derived HSCs have the potential to bridge the gap between current HIV gene therapy protocols and the successful suppression of HIV replication in the HIV infected patient who received an allogeneic bone marrow transplant from a donor homozygous for the CCR5 A32-bp deletion [24].
  • Another advantage in utilizing clonal anti-HIV iPSCs for HIV gene therapy is that each iPSC line can be fully characterized for its safety and analysis of the vector integration site.
  • the integration site of the therapeutic vector can be defined and the iPS lines with "safe harbor" sites can be selected, expanded, and further characterized for their anti-HIV efficacy. As demonstrated in FIG.
  • CD34+ HSCs or bone marrow MSCs are mobilized from HIV infected patients or from individuals who are not infected with HIV from different HLA types. Donors are screened before their cells are used to make sure as many HLA types are covered as possible. The cells are isolated and re-programmed into iPSCs using the various reprogramming factors including a lentiviral vector encoding a combination of anti-HIV genes (CCR5 siRNA and TRIM5oc).
  • anti-HIV iPSCs are screened for safe harbor integration sites of the anti-HIV genes. iPSC factors are then excised using Cre recombinase. The screened/excised anti-HIV iPSC lines are then directly differentiated into anti-HIV CD133+ HSCs which can be frozen down as stocks for future use or directly used for autologous transplantation into the patient from which the "starter" CD34+ HSCs or MSCs were isolated from.
  • the frozen stocks of anti-HIV iPSC lines or the differentiated anti-HIV HSCs can be used in future allogeneic transplantations with individuals who are HLA matched.
  • the stocks of all of the different anti-HIV iPSC derived HSCs with the various HLA types are stored for worldwide shipment to patients who match each individual cell line.
  • reprogramming factors are inserted into these stem cells, and at the same time the anti-HIV genes with a 2nd lentiviral vector are also introduced. After deriving multiple iPSC colonies, the reprogramming factor vector from these multiple colonies are excised and the iPSCs are propagated would human embryonic stem cells (hESCs).
  • hESCs human embryonic stem cells
  • the HIV infected patient receives a stem cell transplant with his or her own, anti-HIV gene containing HSCs.
  • Al HSCs that the patient receives contain anti HIV genes.
  • All HIV target cells are made by HSCs.
  • All newly arising immune cells formerly victims of HIV, are now resistant to HIV.
  • a few weeks after the stem cell transplant HIV does not have any cells to grow in anymore, since HIV resistant immune cells have taken over the immune system, and HIV resistant, new immune system cells are able to eliminate and control the last portion of HIV surviving in the patient.
  • the patient has a new, HIV resistant immune system for the rest of his or her life, hence there is no need for any other HIV treatment.
  • a one time treatment cures the patient from HIV and saves the patient's life.
  • This example demonstrates preclinical evaluation of a combination anti-HIV lentiviral vector, in vivo, in humanized RAG1 -/-y-/-IL2R knockout mice.
  • This combination vector which previously displayed strong pre-integration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5alpha isoform, a CCR5 shRNA, and a TAR decoy.
  • Multi-lineage hematopoiesis from anti-HIV lentiviral vector transduced human CD34+ HSCs was observed in the peripheral blood and in various lymphoid organs including the thymus, spleen, and bone marrow of engrafted mice.
  • Anti-HIV vector transduced CD34+ cells displayed normal development of immune cells including T cells, B cells, and macrophages.
  • the anti-HIV vector transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA.
  • CCR5 shRNA After in vivo challenge with either an R5- tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1 , maintenance of human CD4+ cell levels and a selective survival advantage of anti-HIV gene modified cells was observed in engrafted mice.
  • the data provided in this example confirms the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and suggests its application in future clinical trials.
  • Lentiviral vector design and production The construction of the combination anti-HIV lentiviral vector has been described previously (Anderson et al. Mol. Ther. 17:2103-14 (2009)). Briefly, a third-generation self-inactivating lentiviral vector, pCCLc-MNDU3-x-PGK-EGFP, which contains an EGFP reporter gene was utilized to construct the combination anti-HIV vector (FIG. 9a).
  • the chimeric human/rhesus macaque TRIM5a gene under the control of the MNDU3 promoter, a CCR5 shRNA under the control of a human polymerase-lll U6 promoter, and a TAR decoy under the control of a human polymerase-lll U6 promoter were inserted upstream of the PGK driven EGFP reporter gene to derive pCCLc-Combination- PGK-EGFP (FIG. 9a) (45-46,64,67).
  • Lentiviral vectors were generated in the packaging cells, HEK-293T.
  • ⁇ 8.9 containing gag and pol genes
  • 25ug of pCCLc-MNDU3-x-PGK-EGFP control empty vector
  • pCCLc- Combination-PGK-EGFP transfer vector
  • 5ug of VSVG envelope
  • Vector supernatants were collected at 48 hours post-transfection and concentrated by ultrafiltration 100-fold.
  • Vectors were subsequently tittered on HEK-293T cells and titers obtained ranged from 2x10 9 to 6x10 9 transducing units/ml.
  • CD34+ hematopoietic stem cells were isolated from umbilical cord blood (NDRI, Philadelphia, PA) by Ficoll-Paque (GE Healthcare, Piscataway, NJ) and purified by magnetic bead column separation (Miltenyi Biotec, Auburn, CA). CD34+ cell isolation purity (>93%) was routinely obtained.
  • Total CD34+ cells were cultured in complete IMDM media containing 10% FBS and supplemented with 50 ng/ml stem cell factor (SCF), Flt-3 ligand, and thrombopoietin (TPO). Cells were transduced with the lentiviral vectors EGFP-alone or the anti-HIV combination vector (MOI 10) for three hours at 37°C with 8 g/ml protamine sulfate.
  • SCF stem cell factor
  • TPO thrombopoietin
  • RAG1 -/-Y-/-IL2R mice were obtained from The Jackson Laboratory (Sacramento, CA) and were used in compliance with institutional and IACUC guidelines and regulations. Two to five day old newborn RAG1 -/-Y-/-IL2R pups were sublethally irradiated with 200 rads of gamma irradiation. Nontransduced, EGFP-alone transduced, or anti-HIV vector transduced HSCs (3 * 10 5 total cells/mouse) were injected intrahepatically into irradiated pups.
  • mice were bled retro-orbitally and the peripheral blood was analyzed by FACS for EGFP and human leukocytes with a PE-CY7 conjugated anti-human CD45 antibody (BD Biosciences, San Jose, CA).
  • FACS analysis of engrafted human immune cells To evaluate multi- lineage hematopoiesis in transplanted RAG1 mice, cells from the peripheral blood and various lymphoid organs including the thymus, spleen, and bone marrow were stained with anti-human antibodies and analyzed by FACS. T cells were stained with an APC-conjugated CD3, a PE-conjugated CD4 antibody, or a PE-CY7 conjugated CD8 antibody (BD Biosciences, San Jose, CA). B cells were stained with a PE-conjugated CD19 antibody (BD Biosciences, San Jose, CA).
  • Macrophages were stained with a PE-conjugated CD14 antibody (BD Biosciences, San Jose, CA ). To detect cell surface expression of CCR5, cells were stained with a PE-conjugated anti-human antibody (BD Biosciences, San Jose, CA). Cells were also evaluated for EGFP expression to determine the levels of engraftment of vector transduced cells. FACS analysis was performed on a Beckman Coulter FC-500.
  • In vivo HIV-1 challenge of engrafted RAG1 mice To determine whether the anti-HIV gene modified cells were resistant to HIV-1 infection, engrafted mice were challenged in vivo with either an R5-tropic BaL-1 or an X4-tropic NL4-3 strain of HIV-1 .
  • mice were infected intravenously with 200,000 total infectious units.
  • peripheral blood draws were taken and analyzed for total human CD4+ cell percent by FACS and for HIV plasma viremia by quantitative- PCR (QPCR).
  • FACS analysis the cells were stained with a PE-CY7-conjugated CD45 antibody, an APC-conjugated CD3 antibody, and a PE-conjugated CD4 antibody (BD Biosciences, San Jose, CA). EGFP percent was also analyzed to determine the levels of vector transduced cells.
  • FACS analysis was performed on a Beckman Coulter FC-500.
  • nontransduced or EGFP+ anti-HIV gene modified cells (5x10 5 cells/well) were stimulated with 1 g/ml IL-2 and 1 g/ml phytohemagglutinin.
  • the cells were challenged at an MOI of 0.05 with either an R5-tropic BaL- 1 or an X4-tropic NL43 strain of HIV-1 .
  • Cytokine secretion from in vivo derived T cells Splenocytes from engrafted mice were isolated and sorted based on EGFP expression.
  • CD3+ T cells either control nontransduced or EGFP+ anti-HIV gene modified cells (1 x10 6 cells/well) were stimulated with 1 g/ml IL-2 and 1 g/ml phytohemagglutinin. On day 3 post-stimulation, culture
  • IL-4, IL-6, IL- 10, TNFa, and IFNy were collected and analyzed by FACS for expression of IL-4, IL-6, IL- 10, TNFa, and IFNy using a BD Cytokine Bead Array kit (BD Biosciences, San Jose, CA).
  • the cell suspension was then treated with KCL hypotonic solution and subsequent 3:1 methanol :acetic acid fixative solutions.
  • Karyotyping slides were made and Giemsa banded.
  • Karyotyping was performed on an Olympus Bx41 microscope with a DP20 camera. Analysis was performed with an Applied imaging System.
  • the average engraftment of EGFP-alone vector transduced cells was 25.3% with a standard deviation of 12.9 and the average engraftment of anti-HIV vector transduced cells was 27.22% with a standard deviation of 17.5.
  • Representative FACS plots from each mouse cohort display levels of EGFP+ human leukocytes of 46.8% for EGFP-alone and 36.8% for anti-HIV vector transduced cell engrafted mice. The percentages obtained were from total human leukocytes stained with an anti-human CD45 antibody.
  • Human CD4+ T cells (gated on the CD3+ T cell population) were also observed at normal levels in anti-HIV vector transduced cells compared to control nontransduced and EGFP-alone vector transduced cells.
  • the average level of anti-HIV vector transduced CD4+ cells was 51 .2% of CD3+ T cells (standard deviation of 17.8) compared to nontransduced cells (average of 54.7% CD4+ T cells with a standard deviation of 12.0) and EGFP-alone vector transduced cells (average of 44.9% CD4+ T cells with a standard deviation of 1 1 .0).
  • Representative FACS plots from each cohort of eight mice are displayed in FIG. 9c.
  • Multi-lineage human hematopoiesis from anti-HIV vector transduced cells in lymphoid organs To determine if normal engraftment and multi-lineage hematopoiesis of anti-HIV vector transduced cells had occurred in the lymphoid organs of transplanted RAG1 mice, FACS analysis was performed. Single cells were isolated from the thymus, spleen, and bone marrow of engrafted mice and stained with respective antibodies as described in the Methods section. Successful engraftment and development of anti-HIV gene modified T cells was observed in the thymus of anti-HIV vector transduced mice (41 .0% EGFP+ of total CD3+ T cells).
  • Splenocytes were harvested from transplanted mice and analyzed by FACS for the engraftment of anti-HIV T cells (CD3+/CD4+) and B cells (CD19+).
  • mice were sacrificed at week 14 post-infection and human CD3+ T cells were analyzed from the spleen for the levels of human CD4+ T cells. As displayed in FIG. 11c, normal human CD4+ T cell levels were maintained in the spleen (average of 71 .1 % of human CD3+ T cells with a standard deviation of 1 1 .7) of anti-HIV vector transduced cell engrafted mice.
  • mice engrafted with control EGFP-alone vector transduced cells which displayed a low level of CD4+ human CD3+ T cells in the spleen (average of 26.6% with a standard deviation of 14.5).
  • Representative FACS plots from eight mice from each cohort are displayed in FIG. 11c.
  • mice engrafted with control EGFP-alone vector transduced cells did not display a substantial increase in total human EGFP+/CD4+ cells after infection with either a BaL-1 (FIG. 12a) or an NL43 (FIG. 12b) strain of HIV-1 .
  • mice engrafted with anti-HIV vector transduced cell did not display a decrease in HIV-1 plasma viremia over the course of infection.
  • Levels of HIV-1 RNA copies/ml remained between 7 and 8 logs in the anti-HIV cell engrafted mice which were similar to control cell engrafted mice (FIG. 13).
  • EGFP+ and EGFP- human CD3+ T cells were sorted from the spleens of successfully engrafted mice. Upon stimulation with IL-2 and PHA for 3 days post-sorting, the cells were challenged with either R5-tropic BaL-1 (FIG. 13c) and X4-tropic NL43
  • FIG. 13d At an MOI of 0.05.
  • transduced cells when faced with an HIV-1 viral load, display potent resistance to infection, coupled with strongly diminished HIV-1 production.
  • Anti-HIV gene modified cells are functional and retain a normal karyotype: To determine whether the anti-HIV gene modified cells were functionally normal, the levels of secretion of specific cytokines were measured. T cells from the spleens of engrafted mice were purified and sorted based on EGFP and human CD3 expression. The T cells were stimulated with IL2 and PHA and cultured for three days. On day 3 post-stimulation, culture supernatants were collected and analyzed by FACS for expression of IL-4, IL-6, IL-10, TNFa, and IFNy using a BD Cytokine Bead Array. As displayed in FIG. 14a, the anti-HIV gene containing T cells were functional and secreted similar levels of the various cytokines as compared to control nontransduced cells. These data show that transduction had not perturbed normal cytokine secretion from the T cells.
  • FIG. 9 After transduction of CD34+ HSCs with the anti-HIV vector and injection into newborn RAG1 -/-y-/- pups, successful engraftment of combination anti-HIV vector transduced cells was achieved (FIG. 9).
  • FIG. 9b In vivo development of transduced CD45+ human leukocytes (FIG. 9b) and human T cells (FIG. 9c) was observed in the peripheral blood similar to control vector transduced cells.
  • the levels of anti-HIV vector transduced cell engraftment correlated with the initial transduction efficiencies of the CD34+ HSCs. Before injection, CD34+ cell transduction efficiencies with the anti-HIV vector ranged between -5-50%.
  • the major goal of HIV stem cell gene therapy is to transplant anti-HIV gene modified HSCs into infected patients which would further develop into HIV resistant immune cells capable of blocking HIV infection and fostering in the face of a viral load. Therefore, to evaluate the levels of protection from HIV-1 infection of the anti-HIV gene modified immune cells which developed in the RAG1 mice, both total human CD4+ cell percentages and plasma viremia were measured in HIV-1 infected mice. These two measurements were chosen since a decline in CD4+ cells and a rise in plasma viremia are hallmark characteristics of HIV disease progression. The results demonstrated that anti-HIV gene modified human CD4+ cells, indeed, were protected from HIV-1 infection in the face of a viral load.
  • mice which, upon infection, demonstrated a steady decline of human CD4+ T cells due to their inability to resist infection (FIG. 11 ).
  • the ability of the anti-HIV gene modified cells to survive during HIV-1 infection was also observed in the spleens of infected mice.
  • the maintenance of human CD4+ T cells in mice engrafted with the anti-HIV CD34+ HSCs was due to a selective survival advantage of the protected cells.
  • an increase in EGFP+ cell percentages was observed in mice engrafted with the anti-HIV CD34+ HSCs.
  • the selective pressure of HIV- 1 on the anti-HIV gene modified cells enabled them to be selected for expansion and proliferation, however, there were still unprotected cells for the virus to continually infect and produce plasma viremia (FIG. 13). Therefore, higher transduction efficiencies and in vivo engraftment percentages may need to be achieved to reach an optimal level of HIV-resistant immune cells to suppress HIV-1 replication and decrease plasma viremia. This was observed when this example challenged a sorted and pure population of anti-HIV gene modified human T cells from the spleen of engrafted mice.
  • a pure population of anti-HIV T cells was capable of resisting HIV-1 infection and displayed a significant decrease in HIV-1 p24 output compared to nontransduced human T cells (FIG. 13).
  • the human immune system of an infected individual may come to the aid to decrease viral load. If HIV-specific immune cells survive and are not killed off by HIV, over the long term (a time period that cannot be easily tested in a short term animal model) the viral load may be substantially reduced since HIV-producing cells from the viral reservoirs can be safely eliminated by immune cells. Even though this example did not observe a decrease in viral load, it was able to demonstrate that the cells expressing the anti-HIV genes were capable of resisting infection in vivo. These cells were able to maintain normal human CD4+ cell levels due to a selective survival advantage upon HIV challenge in vivo.
  • this example evaluated the ability of human T cells which engrafted in the spleen of RAG1 humanized mice to secrete normal levels of human cytokines upon stimulation. Even though the CD34+ HSCs were transduced with a triple combination anti-HIV vector and had undergone multi-lineage
  • the anti-HIV vector transduced CD34+ HSCs also did not appear to have any chromosomal abnormalities or gene rearrangements upon transduction with the anti-HIV vector and expansion (FIG. 14).
  • This example demonstrated the safety and efficacy of this combination anti- HIV lentiviral vector in a humanized mouse model which is capable of demonstrating multi-lineage hematopoiesis from engrafted human CD34+ HSCs.
  • the subsequent protection and expansion of the HIV resistant immune cells in the face of an HIV viral load establishes the utility of this vector for use in future clinical trials.
  • SCID-hu mice transplanted with human fetal tissue.
  • lymphocytes lymphocytes.
  • Nef and Vpr proteins contribute to disease progression by promoting depletion of bystander cells and prolonged survival of HIV-infected cells? Biochem Biophys Res Commun; 267; 677-685.
  • CD34+(+) cell-derived macrophages in vitro and in T cells in vivo in severe combined immunodeficient (SCID-hu) mice transplanted with human fetal tissue.
  • SCID-hu severe combined immunodeficient mice transplanted with human fetal tissue.
  • cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World Monkeys. Nature 427:848-853.

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Abstract

L'invention concerne des cellules souches hématopoïétiques (HSC) qui sont programmées pour générer des cellules souches pluripotentes induites (iPSC) qui expriment des polynucléotides anti-VIH, comprenant un ARNsh CCR5 et un gène TRIM5? chimère humain/rhésus et, facultativement, un leurre Tar. Des HSC CD133+ formant des colonies ont été obtenues suite à la différenciation dirigée des iPSC anti-VIH vers le lignage hématopoïétique. Ces cellules ont été ultérieurement différenciées en des macrophages fonctionnels de stade terminal qui présentaient un profil phénotypique normal. Suite à une stimulation virale in vitro et in vivo, les macrophages issus des iPSC anti-VIH ont présenté une forte protection contre une infection par VIH-1 et peuvent être utilisés pour la prévention ou le traitement d'une infection par le VIH chez des patients.
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016063192A1 (fr) * 2014-10-21 2016-04-28 Consiglio Nazionale Delle Ricerche Nouveaux procédés et agents pour la reprogrammation de cellules
WO2017139065A1 (fr) * 2016-02-08 2017-08-17 American Gene Technologies International Inc. Vaccination et immunothérapie contre le vih
WO2017143266A1 (fr) * 2016-02-19 2017-08-24 The Regents Of The University Of California Arn court en épingle à cheveux (shrna734) et son utilisation pour sélectionner positivement et éliminer des cellules génétiquement modifiées
US9834790B1 (en) 2016-01-15 2017-12-05 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10036038B2 (en) 2016-07-08 2018-07-31 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US10137144B2 (en) 2016-01-15 2018-11-27 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10548914B2 (en) 2008-10-17 2020-02-04 American Gene Technologies International Inc. Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
US10767183B2 (en) 2016-03-09 2020-09-08 American Gene Technologies International Inc. Combination vectors and methods for treating cancer
US11352646B2 (en) 2018-11-05 2022-06-07 American Gene Technologies International Inc. Vector system for expressing regulatory RNA
US11583562B2 (en) 2016-07-21 2023-02-21 American Gene Technologies International Inc. Viral vectors for treating Parkinson's disease
US11820999B2 (en) 2017-04-03 2023-11-21 American Gene Technologies International Inc. Compositions and methods for treating phenylketonuria
US11976292B2 (en) 2016-06-08 2024-05-07 American Gene Technologies International Inc. Non-integrating viral delivery system and methods related thereto
US11980663B2 (en) 2015-07-08 2024-05-14 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008150814A2 (fr) * 2007-05-29 2008-12-11 Reid Christopher B Procédés de production et d'utilisations de populations de cellules multipotentes, pluripotentes, différentiées et résistantes au vih
US20100041054A1 (en) * 2008-08-12 2010-02-18 Amanda Mack Methods for the production of ips cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008150814A2 (fr) * 2007-05-29 2008-12-11 Reid Christopher B Procédés de production et d'utilisations de populations de cellules multipotentes, pluripotentes, différentiées et résistantes au vih
US20100041054A1 (en) * 2008-08-12 2010-02-18 Amanda Mack Methods for the production of ips cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDERSON, J. S. ET AL.: 'Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy' MOL THER. vol. 17, no. 12, 18 August 2009, pages 2103 - 14 *
KAMATA, M. ET AL.: 'Generation of human induced pluripotent stem cells bearing an anti-HIV transgene by a lentiviral vector carrying an internal murine leukemia virus promoter' HUM GENE THER. vol. 21, no. 11, 05 October 2010, pages 1555 - 67 *

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