WO2010051521A1 - Produit de thérapie cellulaire pour le traitement de l'infection par le vih - Google Patents

Produit de thérapie cellulaire pour le traitement de l'infection par le vih Download PDF

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WO2010051521A1
WO2010051521A1 PCT/US2009/062909 US2009062909W WO2010051521A1 WO 2010051521 A1 WO2010051521 A1 WO 2010051521A1 US 2009062909 W US2009062909 W US 2009062909W WO 2010051521 A1 WO2010051521 A1 WO 2010051521A1
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sequence
cell
hiv
encodes
binds
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PCT/US2009/062909
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Boro Dropulic
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Lentigen Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • HIV infection in humans is considered pandemic by the WHO. From 1981 to 2006, AIDS killed more than 25 million people. Approximately 40 million people worldwide are infected with HIV. At the end of 2006, an estimated 1.1 million persons in the United States were living with HIV infection, with 21% undiagnosed. It has been estimated that approximately 56,300 people were newly infected with HIV in 2006. There is still significant progression from infection to progression to AIDS, with an estimated 37,041 new AIDS cases in 2007. The total estimated number of persons living with AIDS in the U.S. and dependent areas was 468,578. In 2007, the estimated number of deaths of persons with AIDS was 14, 561.
  • HIV/ AIDS is an ideal candidate for a novel gene therapy approach, since it is an incurable and ultimately fatal disease.
  • FIGURES Figure 1 shows a lentiviral vector expressing anti-CCR5, env anti-sense, and MGMT. It has a standard LTR (comprised of U3, R, and U5 regions), and contains splice donor (SD), splice acceptor sites (SA), central poly-purine tract (cPPT), and the psi packaging signal (not shown).
  • the MGMT transgene will be driven from the native LTR, as will the shRNA construct (solid arrow), encoding the antiCCR5 shRNA sequence.
  • TAR- dependent LTR transcription will express the anti-HIV anti-sense (AS) RNA.
  • FIG. 2 shows examples of seven different lentiviral vectors, two designed for hematopoietic stem cells (HCSs), two designed for T-cells, and three designed for mesenchymal stem cells (MSCs).
  • the first two constructs transduce HSCs. They contain an anti-HIV antisense sequence (targeted to any part of HIV genome that is not in the vector), a RNAi sequence targeted to the CCR5 gene (the CCR5delta32 mutant protein could also be expressed from this construct and the region that the RNAi would bind would be made resistant to the effects of the RNAi by codon degeneration), and the MGMT fate-controlling gene, that in the presence of Temozolomide, facilitates LV-transduced HSC expansion in vivo.
  • HCSs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • the second two constructs transduce T-cells. They contain an anti-HIV antisense as described above, a RNAi sequence targeted to the CCR5 gene (the CCR5delta32 mutant protein could also be expressed from this construct and the region that the RNAi would bind would be made resistant to the effects of the RNAi by codon degeneration), and two fate- controlling genes, one that functions to delete the cells (TMPK) if required (useful, for example, in allogeneic transplantation) and an shRNA targeted to the Program Cell Death gene 1 (PD-I), which facilitates expansion of antigen-stimulated T cells.
  • TMPK RNAi sequence targeted to the CCR5 gene
  • PD-I Program Cell Death gene 1
  • the three final constructs transduce MSCs or any substrate cell type that can be used to support the engraftment and/or expansion of HSCs.
  • These vectors contain, in addition to any of the above combination of anti-HIV genes and fate-controlling genes, genes that promote HSCs or T cells - examples being genes or factors that facilitate engraftment, homing, survival, function and/or expansion of HSCs or T cells.
  • Non-limiting examples of such genes are SCF, TPO, EPO, G-CSF, FLT-3L, H0X-B4.
  • the invention relates to cell therapy for the treatment of HIV infection in humans.
  • HIV includes all clades and/or strains of human immunodeficiency virus 1 (HIV-I) and human immunodeficiency virus 2 (HIV -2).
  • the invention provides a composition of genetically modified human cells for introduction into the body of a person infected with HIV, whether or not the person is actually suffering from AIDS, to reduce the person's viral load and to provide and reconstitute T-cells that are resistant to HIV infection.
  • the composition comprises allogeneic or autologous human CD4 + T-cells, allogeneic or autologous human hematopoietic stem cells (HSCs), and allogeneic or autologous human mesenchymal stem cells (MSCs).
  • Each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence.
  • the each cell type is either all allogeneic or all autologous, but all three cell types need not be either all allogeneic or all autologous. In one embodiment, all of the cell types are allogeneic.
  • the heterologous anti-HIV sequence is any DNA sequence, which has been inserted into the genome of the cells, that results in the cells being resistant to HIV infection and replication.
  • Messenger RNA (mRNA) transcribed from the sequence, or a protein or polypeptide encoded by the sequence acts, within the cells to inhibit HIV from infecting the cells or from replicating in the cells.
  • the DNA sequence encodes an antisense mRNA transcript that is complementary to part of the HIV genome and binds (hybridizes) to it, which inhibits the virus from replicating in the cell.
  • the words “encode” and “encodes” refer to the production of RNA transcripts from a DNA sequence as well as the production of polypeptides and proteins from the sequence.
  • the antisense mRNA sequence hybridizes to all or part of the HIV gag, pol, nef, and/or env gene. In another aspect, it hybridizes to all or part of the HIV env gene.
  • the appropriate length of the mRNA sequence can vary. In one aspect, the antisense sequence is about 500 bases to about 3000 bases long. Generally, it is about 1 kilobase to about 2 kilobases long.
  • the DNA sequence encodes an interfering RNA sequence (RNAi).
  • the RNAi comprises a short hairpin RNA (shRNA) embedded within a microRNA (miRNA) that down-regulates at least one of the cells' HIV receptors.
  • shRNA down-regulates the cells' CCR5 receptors.
  • the shRNA is embedded within another RNA sequence, preferably a miRNA.
  • Cells process this shRNA-miRNA by enzymes, such as DROSHA and DICER, to ultimately give rise to a short interfering RNA (siRNA).
  • the siRNA interferes with mRNA that encodes CCR5 protein, thereby down-regulating it.
  • the heterologous fate-controlling sequence allows the cells to be selected, expanded in number, or killed. This can be achieved when contacted with certain chemicals directly in vitro or indirectly in vivo when those chemicals are administered to the human to whom the cellular composition of the invention has been administered.
  • drugs are not always necessary and other methods for controlling cell fate can be accomplished by techniques known in the art.
  • the control gene can be self limiting.
  • An example of this is the PD-I gene that is active in T-cells where the T-cell receptor (TCR) is HIV specific and during a period where there is ample HIV antigen stimulating the cells. Once HIV antigen is abated, then the PD-I gene would not be active (since it depends upon TCR signalling to facilitate cell expansion) and therefore would be self limiting.
  • TMPK refers to a nucleic acid sequence that encodes this modified human TMPK protein
  • TK refers to a nucleic acid sequence that encodes the TK protein
  • dCK refers to a nucleic acid sequence that encodes the dCK protein.
  • the fate-controlling sequence comprises a cell selection sequence.
  • the cell selection sequence encodes a protein that permits expansion of the genetically modified cells in vitro or in vivo by either expressing or inhibiting a cellular gene. This can be done in the presence of a pro-drug or without a pro-drug. Examples are well- known to those skilled in the art and include multidrug resistance protein 1 (MDR-I), dihydro folate reductase (DHFR), and the mutated version of methylguanine-DNA methyltransferase (also refered to as O 6 -alkylguanine-DNA alkyltransferase). This last protein and nucleic acid sequences encoding it are disclosed in U.S. Pat. No.
  • the fate-controlling sequence comprises an anti-apoptosis sequence.
  • This is a sequence that encodes a protein or a mRNA transcript, such as a RNAi, that inhibits or prevents the cell from undergoing apoptosis in circumstances where it would otherwise undergo apoptosis.
  • the sequence encodes a shRNA that targets the programmed cell death 1 gene (PDl gene).
  • the shRNA is embedded within a miRNA.
  • the MSCs of the composition include at least one heterologous sequence that encodes a stem cell promotion factor.
  • stem cell promotion factor is any protein, polypeptide, or other molecule produced within the cells that promotes engraftment and/or proliferation of the MSCs in vitro or within the human to whom the cellular composition has been administered.
  • the stem cell promotion factor comprises a cytokine. Cytokines are well-known to those skilled in the art, and the selection of the appropriate ones are within the skill on those in the art.
  • Cytokines that are particularly appropriate for the MSCs of the invention include thrombopoietin (TPO), stem cell factor (SCF), erythropoietin (EPO), FLT3-ligand (FLT-3L), interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • TPO thrombopoietin
  • SCF stem cell factor
  • EPO erythropoietin
  • FLT3-ligand FLT3-ligand
  • IL-3 interleukin-3
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the cytokines are SCF, TPO, FLT-3L, and/or G-CSF.
  • the stem cell promotion factor comprises H0X-B4.
  • either or both of the T-cells and the HSCs within the cellular composition comprise two different anti-HIV sequences that inhibit HIV from infecting those cells or from replicating in the infected cells.
  • one anti-HIV sequence encodes an mRNA that is an antisense sequence that binds to at least part of the HIV genome and the other encodes an miRNA that down-regulates at least one of the cell's HIV co-receptors.
  • the antisense sequence can hybridize to at least part of the HIV gag, pol, nef, and/or env gene, and the miRNA can be an shRNA that down-regulates the cells' CCR5 HIV co-receptors.
  • the cellular composition is administered to people who are infected with HIV.
  • the invention provides a method of lowering the number of HIV particles in a human comprising administering to the human an effective amount of the composition sufficient to permit engraftment and proliferation of the genetically modified cells to sufficient levels in the body so as to dramatically impact HIV viral load and disease progression to AIDS. Since it is widely known that there is a direct correlation between viral load and disease progression, lowering the viral load will postpone the development of full-blown AIDS, possibly indefinitely. The hope of this therapy is to decrease the HIV viral load to levels that are not conducive to the development of AIDS.
  • composition comprises three types of modified human cells: CD4 + T-cells, HSCs, and MSCs.
  • CD4 + T-cells CD4 + T-cells
  • HSCs HSCs
  • MSCs MSCs.
  • Each type of cell has been genetically engineered to include and express the DNA sequences discussed above.
  • the therapy can use one, two, all three, or any combination of the types of genetically modified cells described herein.
  • the allogeneic or autologous human CD4+ T-cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell, and it comprises at least one heterologous fate-controlling sequence.
  • the cell comprises two different heterologous anti-HIV sequences. In a further embodiment, it also comprises two different heterologous fate-controlling sequences.
  • the T-cells are obtained from human blood. Autologous cells are obtained from the blood of the same person to whom the modified T-cells will be administered. Allogeneic cells are obtained from a genetically different person. Generally, standard techniques in the art are used to obtain allogeneic cells that best match the tissue type of the intended recipient.
  • the anti-HIV sequence can be a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an shRNA that down-regulates at least one of the cell's HIV co-receptors.
  • the antisense sequence binds to at least part of the HIV gag, pol, nef , and/or env genes. In a further aspect, it binds to at least part of the HIV env gene.
  • the antisense sequence is about 500 bases to about 3000 bases long and typically about one kb to about two kb long.
  • a shRNA that is embedded within a miRNA down-regulates the cell's HIV CCR5 co-receptors.
  • the heterologous fate-controlling sequence is selected from the group consisting of a cell-killing sequence and an anti-apoptosis sequence.
  • the cell-killing sequence comprises TK or dCK.
  • it comprises TMPK.
  • the anti-apoptosis sequence comprises a sequence that encodes a shRNA that targets the PDl gene.
  • the shRNA is embedded within another RNA sequence to enhance the processing of the shRNA.
  • this sequence is an miRNA.
  • the allogeneic or autologous human HSC comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell, and it comprises at least one heterologous fate-controlling sequence.
  • the cell comprises two different heterologous anti-HIV sequences.
  • it also comprises two different heterologous fate-controlling sequences.
  • the anti-HIV sequences are the ones described above.
  • the HSCs are obtained from human bone marrow, human blood, human umbilical cord, or human umbilical cord blood. Generally, allogeneic cells are matched to the tissue type of the intended recipient.
  • the heterologous fate-controlling sequence is a cell-killing sequence or a cell selection sequence.
  • the cell-killing sequence is as described for the T-cell.
  • the cell selection sequence is a sequence encoding neomycin resistance protein, MDRl, or mutant DHFR.
  • the cell selection sequence comprises MGMT.
  • the allogeneic or autologous human MSC comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell and at least one heterologous fate-controlling sequence.
  • the anti-HIV sequence comprises the various anti-sense sequences described above.
  • the fate-controlling sequence comprises a cell-killing sequence as described above.
  • the MSCs are obtained from various human tissues, including bone marrow, blood, umbilical cord, and umbilical cord blood. Generally, allogeneic cells are matched to the tissue type of the intended recipient.
  • the MSC further comprises a sequence that encodes a stem cell promotion factor.
  • the stem cell promotion factor comprises a cytokine.
  • the cell can include one or more heterologous sequences encoding one or more of the following cytokines: TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.
  • the cytokine comprises one or more of SCF, TPO, FLT-3L, and G-CSF.
  • the stem cell promotion factor comprises H0X-B4.
  • modified cell types may be made by various techniques known to those skilled in the art. In one embodiment, they are made by transducing the unmodified cells with lentiviral vectors to introduce the desired sequences into the genomes of the cells.
  • the lentiviral vector of the invention comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell, and it comprises at least one heterologous fate-controlling sequence.
  • the anti-HIV sequence comprises an antisense sequence to at least part of the HIV genome.
  • the antisense sequence binds to at least part of the HIV env, gag, pol, and/or nef gene.
  • the antisense sequence is about 500 bases to about 3000 bases long. Generally, it is about one kb to two kb long. In a still further aspect, the antisense sequence binds to at least part of the
  • the anti-HIV sequence encodes an shRNA that down- regulates at least one of the cell's HIV receptors.
  • the receptor is CCR5.
  • the shRNA is embedded within another sequence, which is optionally a miRNA.
  • the heterologous fate-controlling sequence is one or more of the following: 1) a cell-killing sequence, 2) a cell selection sequence, 3) an anti-apoptosis sequence, and 4) a sequence encoding a stem cell promotion factor.
  • the cell- killing sequence comprises TK, dCK, or TMPK. In a preferred aspect, it comprises TMPK.
  • the cell selection sequence comprises a sequence encoding MDR-I, neomycin resistance protein, and DHFR. In a preferred aspect, it comprises MGMT.
  • the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene. In a preferred aspect, the shRNA is embedded within a miRNA.
  • the sequence encoding a stem cell promotion factor encodes one or more cytokines. In one aspect, the cytokine is one or more of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.
  • the cytokine is one or more of SCF, TPO, FLT-3L, and G-CSF.
  • the stem cell promotion factor comprises HOX-B4.
  • the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) an anti-HIV sequence that encodes an miRNA embedded shRNA that down-regulates the CCR5 HIV co-receptors on the surface of a cell that can be infected by HIV, and 3) a cell selection sequence comprising MGMT.
  • the second vector comprises the sequences of the first vector plus a cell-killing sequence comprising TMPK.
  • the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) a cell-killing sequence that comprises TMPK, and 3) an anti-apoptosis sequence comprising a sequence that encodes an miPvNA embedded shRNA that targets the PD-I gene.
  • the second vector comprises the sequences of the first vector plus an anti-HIV sequence that encodes a shRNA that down- regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV.
  • the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) a cell-killing sequence that comprises TMPK, and 3) sequences encoding the cytokines TPO, SCF, and FLT-3L.
  • the second vector comprises the antisense sequence and the cell-killing sequence of the first vector plus a sequence encoding G-CSF.
  • the third vector comprises the antisense sequence and the cell-killing sequence of the first vector plus a sequence encoding H0X-B4.
  • the lentiviral vectors are constructed by techniques known to those skilled in the art. Such techniques are disclosed in U.S. Patent Application No. 11/884,639, published as US 2008/0254008 Al, and in U.S. Patent Nos. 5,994,136, 6,013,516, 6,165,782, 6,294,165 Bl, 6,428,953 Bl, 6,797,512 Bl, 6,863,884 B2, 6,924,144 B2, 7,083,981 B2, and 7,250,299 Bl, the disclosures of which are incorporated herein by reference in their entireties. Generally, plasmids are prepared that contain one or more of the heterologous sequences disclosed herein.
  • the plasmids are transfected into packaging cells that contain an HIV gag-pol sequence and a sequence that encodes a pseudo-typed envelope protein, such as the vesicular stomatitis virus protein G (VSV-G).
  • VSV-G vesicular stomatitis virus protein G
  • These cells are mammalian cells, generally human cells. In one aspect of the invention, they are human embryonic kidney cells, such as HEK 293 cells.
  • the producer cells are cultured under standard conditions for such cells, and the vectors are recovered from the supernatant.
  • the plasmid of the invention comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell, and it comprises at least one heterologous fate-controlling sequence.
  • the sequences are those described above with respect to the lentiviral vectors.
  • the plasmids that produce the vectors shown in Figure 2 contain the same sequences. The sequences can all be in one plasmid or they can be in multiple plasmids.
  • Lentiviral vector LG896 is designed to render the immune system free of the pathologic effects of HIV infection by the expression of three distinct transcripts in genetically modified CD4 progenitors by transducing CD34+ hematopoietic stem cells
  • HSC HSC-Coactivated HSC
  • the first anti-HIV transcript encoded by the LV will serve to decrease the infectability of target cells by down-modulating the CCR5 co- receptor for HIV.
  • CCR5 blockade on its own has shown great potential for protecting cells from infection [I].
  • the second anti-HIV transcript is a 1 kb anti-sense sequence to the HIV env sequence. This transcript will limit productive HIV infection in instances where CCR5 blockade (encoded by the first transcript) was overcome.
  • the therapeutic env anti-sense transcript will rapidly associate with the single-stranded RNA genome of the virus and any mRNA env transcripts. Once bound, the double-stranded RNA will be rapidly degraded by the host cell.
  • the use of a long transcript 1 kb sequence helps to avoid problems with sequence variability [2].
  • Lentiviral vectors (LV) are ideally suited for the transduction of HSC given the permanency of expression of the integrated transgene and the superior safety profile of LV over onco- retroviral vectors. While HSC can be harvested from mobilized peripheral blood, successful long-term engraftment of gene modified autologous cells requires "space" to be created in the bone marrow. This normally requires full lymphodepletion (as occurs during bone marrow transplantation, HSCT). That is why MGMT is incorporated into the LV.
  • HIV/ AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease.
  • highly active anti-retroviral therapy has significantly improved the survival of HIV-infected individuals, the duration of response is limited by development of drug-resistant viruses, long-term toxicities and a substantial reservoir of latently infected T-cells [9] [1O][I I].
  • Tremendous advances have been made over the past several years in understanding HIV pathogenesis. Following primary infection, the virus replicates in local lymph nodes, then disseminates in a massive viremia.
  • HIV- 1 elicits a strong immune response in most infected individuals, the virus almost invariably escapes immune containment [12].
  • Persistent infection is characterized by the gradual decrease of CD4+ T cells, ultimately leading to AIDS.
  • Tat will drive the expression of the anti-en v transcript.
  • the splice donor and acceptor sites present in the vector backbone will allow expression of downstream MGMT and shRNA sequences (for anti-CCR5).
  • the shRNA sequence specific for CCR5 will be expressed in the context of a micro-RNA sequence.
  • This miRNA expression system for shRNA is the basis for the SMART vectorTM program marketed by Thermo Scientific. Diminished cell surface CCR5 expression, caused by anti-CCR5 shRNA expression, will result in diminished HIV infectability of the target cell.
  • Tat-mediated expression of the antisense transcript will render the infection in that cell non-productive.
  • the third element encoded by the vector, MGMT is a well-described tool for expanding the percentage of transduced HSC in the marrow of treated subjects. Subsequently, the LG896 vector will be tested in clinical trials involving HIV/ AIDS patients and will significantly challenge existing therapies, which are currently inadequate, for providing stable and prolonged therapeutic activity.
  • HIV-I infection is characterized by a host- virus relationship in which the virus utilizes the host cell's macromolecular machinery and energy supplies to produce progeny virus[14].
  • HIV-I alters the cell's physiological state, leading to disruption of immune responses and cell death.
  • Specific viral and cellular proteins are known to play crucial roles in the alteration of cellular functions. Examples include the HIV-I accessory proteins and host cell chemokine coreceptors, CCR5 and CXCR4, which are essential for HIV- 1 infection [ 16] [ 17] .
  • HIV-I accessory proteins (Tat, Rev, Nef, Vif, Vpu and Vpr) are regulatory proteins encoded by HIV-I, which are involved in HIV infectivity, transcription, replication and/or pathogenesis [22] [23]. Functional inhibitors of these viral regulatory proteins are not currently available.
  • HAART anti-retroviral drugs
  • This therapy has led to maintenance of very low viral load in the majority of treated patients [24]. This therapy is not, however, able to induce sustained suppression or cure. Even if the plasma viral level falls below the current level of detection, HIV-I continues to replicate at very low levels or persist in a reservoir of latently infected T cells [25].
  • Therapies in the experimental stages of testing include vaccines based on engineered gpl20-CD4-CCR5 fusion proteins, which have been shown to elicit antibodies capable of neutralizing infectivity of primary HIV-I isolates [26].
  • Latently infected resting CD4 T cells provide the major reservoir for HIV in individuals on highly active antiretro viral therapy (HAART) [27].
  • HAART highly active antiretro viral therapy
  • Two distinct forms of latency are thought to occur.
  • Pre-integration latency involves recently infected resting CD4 T cells that harbor partially or completely reverse transcribed HIV DNA in a labile pre- integration complex [28]. Although pre -integration latency is the most prevalent form of latency in untreated HIV infection, it is highly labile, decaying with a half- life of 1-6 days. If these cells are activated before the pre -integration complex becomes non-functional, then nuclear import, integration, and productive infection will occur [29][30][28].
  • Eliminating latently infected CD4 T cells Reactivation of pre- and post- integration latency has been ttempted in vitro by stimuli that trigger T cell activation (i.e. antibodies to CD3 orthe PMA homologue prostatin, or specif ⁇ ccytokines) [34][35][36][37][38]. Control and, ultimately, cure of HIV infection requires a therapy either kills the latently infected cell or, disrupts the ability to infect and replicate in the na ⁇ ve CD4 T cell population. Killing ofat least some latently infected cells has been achieved in vitro by an anti-CD45RO immunotoxin, although levels of CD45RO are too low for detection on many infected cells [39] [40].
  • stimuli that trigger T cell activation i.e. antibodies to CD3 orthe PMA homologue prostatin, or specif ⁇ ccytokines
  • Lentiviral vectors provide highly efficient and stable gene expression.
  • Lentivirus gene therapy vectors promise to be safer than the murine oncoretroviral-based (MLV) vectors recently used in the successful treatment of disease in three gene therapy trials to date, including an X-linked SCID trial [65][66][67].
  • MLV murine oncoretroviral-based
  • LV are not associated with oncogenesis and therefore may represent a safety advantage over oncoretroviral gene therapy vectors [68][69][70][71][72][73].
  • LV are thought not to have the same enhancer activity as oncoretrovirus-based vectors.
  • the long terminal repeat has a low basal rate of activity and expression is driven by Tat binding to the TAR element in the LTR to facilitate elongation of HIV RNA, rather than the upregulation of transcriptional activity via enhancer-DNA binding protein complexes.
  • HIV- based LV have less poly- A read through into neighboring genes [74].
  • leukemia is not a recognized side effect of HIV patients and is extremely rare, even though memory T- cells are known to harbor integrated virus for years and many of these pro viruses are defective and therefore do not kill the host cell [75][32][76].
  • none of the patients in the recently finished Phase I clinical trial of the HIV-based LV have experienced any adverse events due to treatment, thus supporting the safety of LV vectors for applications in humans [13].
  • a LV expressing: a) a miRNA that down regulates CCR5 by expression of a CCR5-specific shRNA, b) a 1 kb anti-sense transcript to the HIV viral genome, and c) the MGMT gene in Lentigen's LentiMax system.
  • the lentiviral vector genome as developed into a gene delivery system by Lentigen, can be manipulated in a number of ways in order to express new RNAs or proteins in vector-treated host cells.
  • the LentiMax system should efficiently express antisense RNA for env, the MGMT gene and shRNA for the knock-down of CCR5 from the vector shown schematically in Figure 1.
  • the advantage of this vector is that different amounts of the three RNA species will be expressed depending upon whether or not the target cell becomes infected with HIV. In non-infected cells there will be a significant level of CCR5- specific shRNA and MGMT transcripts formed from the activity of the native LTR promoter.
  • Transcripts from the native LTR will initiate in the LTR, then utilize the splice donor and acceptor sites, (SA, SD) resulting in the transcription of MGMT and miRNA from backbone sequence that follows the SA sequence. If a transduced cell becomes infected with HIV, the Tat transcriptional regulatory protein and Rev post-transcriptional regulatory proteins will also be expressed. Tat will bind to the TAR element, which upregulates transcription and, in concert with nuclear-exporter Rev, expresses the anti-sense transcript (that is located between the splice donor (SD) and splice acceptor (SA) sites) for export and targeting of wt-HIV genomic RNA.
  • SA splice donor and acceptor sites
  • CCR5-specif ⁇ c shRNA and MGMT are expressed prior to prevent HIV infection and generate HIV-resistant cells.
  • the long lkb anti-HIV antisense is expressed in Tat and Rev dependent manner to prevent productive HIV replication.
  • the long antisense is not expressed constitutively prior to HIV infection, but only after HIV breakthrough infection, needing the in-coming wt-HIV s Tat and Rev to express the anti-HIV antisense sequence, target wt-HIV RNA and interfere with productive HIV replication.
  • the vector backbone produced in Aim Cl .A. will be used to transduce 293 cells (by calcium phosphate transduction, along with packaging and VSV-G envelope constructs) for the purpose of producing infectious lentiviral vector particles (LV).
  • LV infectious lentiviral vector particles
  • supernatant will be collected 24 and 48 hours post transfection, pooled and concentrated by high-speed centrifugation at 9,500 rpm for 14 hours.
  • Vector will be resuspended in approximately 1/500 of its original volume in a vector storage buffer (65 mM NaCl and 20 mM HEPES) at pH 7.22, and frozen at -80 C.
  • Dilutions of the harvested supernatant will then be used to infect new cultures of HEK 293 cells in order to determine lentiviral vector titer.
  • Titer will be determined by real-time PCR.
  • a 100 bp specific DNA sequence is part of the Lentigen vector system that was introduced into the vector specifically for quantitation.
  • the 5' forward and 3' reverse primers that will be used are: 5'-CCACTCCTGACAACTACTCT, and 5'- GGAGTTGAGACCAGTGTAGT.
  • the real-time PCR probe is 5'- CAGTAGGTGAAGGAGTCGTAGTTG.
  • the genomic DNA of transduced cells will be isolated using the DNAeasy kit (Qiagen) and real-time PCR performed on normalized (for DNA concentration) samples using primers and probe.
  • Typical titers range between 1-7 xl O 9 transducing units (TU) per ml. The titer will then be used to determine the amount of vector needed to transduce a high proportion of target cells in subsequent studies.
  • retrovirus-based gene delivery vectors One primary concern for retrovirus-based gene delivery vectors is the development of RCL, replication competent retrovirus.
  • the concentrated lentiviral vector preparation will be tested on C8166-45 indicator cells.
  • An attenuated HIV unable to express the HIV-I accessory proteins vif, vpr, vpu, and nef will be used as the positive control. This will be an appropriate control because none of these HIV-I accessory genes are expressed in the vector or packaging constructs, and therefore would not be present in RCL generated during vector production. This attenuated control will be measured for TCID50 on the C8166 indicator cell line.
  • Real-time PCR using a specific TaqMan probe will be used as one method to measure the positive control and putative RCL in the supernatant of the indicator cell lines. Specifically, HIV gag and VSV-G RNA will be analyzed after passage of the vector on the indicator cell line. The assay will also be used to detect residual VSV-G DNA in the vector prep. We will also measure p24. P24 production in the supernatant of C8166 cells will be measured by p24 ELISA (ABL, Inc.) according to manufacturer's specifications.
  • Human CD34+ hematopoietic stem cells will be obtained from left over cord blood samples Primary human T cells will be obtained from healthy volunteers. Human CD34 will be cultured for 2 days in growth factors in vitro and transduced on each of two consecutive days with a range of MOI (1 to 75) in triplicate wells. On day 3, HSC will then be cultured in methylcellulose (MethoCult, StemCEll Technologiers, Vancouver, BC, with hemin, SCF, IL-3, GMCSF and epo [82]) and assayed for colony progenitor (HPC) cell activity.
  • HPC activity is standard, as is the quantification of percent transduction of HSC by RT-PCR for MGMT by plucking individual colonies from culture 13 days after plating.
  • Human PBMC will stimulated with rIL-2, and anti- CD3/anti-CD28 beads (Dynabeads ClinExVivo CD3/CD28 in Optimizer media, as per Invitrogen), in retronectin-coated cell processing bags in the presence of LV-containing supernatant [13].
  • the three parameters that will be tested are: a) the ability of transduced lymphocytes to expand in vitro upon subsequent re-stimulation, and b) the expression of each vector-encoded RNA in the transduced cell population as determined by RT-PCR, and c) the expression of CCR5 on the surface of transduced lymphocytes as determined by flow cytometry.
  • RNA encoding anti-sense env and anti-CCR5 shRNA will be tested in two immortalized cell lines that express CCR5, monocyte-derived macrophages, and primary T cells. Documented expression of LV-encoded transgenes will justify testing the construct for the ability to prevent HIV infection in future clinical studies.
  • Suptl/CCR5 T cells and PMl cells are immortalized cell lines that express CCR5 and are cultured in standard media.
  • Monocytes will be purified from normal human volunteers by CDl 1 b+ cell sorting and then differentiated into macrophages with GM-CSF and M-CSF as described by Oberling et al., [83][84].
  • CD4 cells will be expanded from density gradient purified lymphocytes using anti- CD3/anti-CD28 beads (as above).
  • CCR5 on the cell surface will be determined by flow cytometry (BD Pharmingen), as described in Cordelier, etal [42].
  • Each cell type will be transduced with LG896 at an MOI of 50 for two consecutive days, cultured for 3-4 days, and then stained for surface expression of CCR5.
  • GFP expressing vector will serve as a positive control for transduction of each cell type, as well as a test for non-specific CCR5 down- regulation.
  • HIV-I infection assays will be carried out initially with HIV-I Ba-L (0.05-1.5 ng p24 equivalents of cell free virus) but then with other strains (NL4-3; X4 strain etc). After overnight incubation, cells will be washed and every 2-3 days thereafter and assayed for expression of phenotypic markers. At each sub-culture step, 100,000 infected and non- infected cells will be harvested for quantification of LV-transduction levels (% transduction), HIV-I infection (of integrated genomes) by DNA PCR, and for LG896 encoded RNA for MGMT, anti-sense env, and shRNA for CCR5 by RT-PCR. The transduction level over time will be determined by RT-PCR wherein copies of integrated DNA are correlated to the overall genome number. To quantify the amount of virus produced from naive, mock-infected, and HIV-I infected cells.
  • T lymphocytes will be activated with anti-CD3/anti-CD28 beads and transduced with vector on days 0 and 1. Viability will be assessed by flow cytometric analysis by Annexin-V and 7- AAD staining every 2-3 days along with phenotypic staining for CD3, CD4, and CCR5.
  • HPCs vs. LG896 transduced CD34+ HSCs
  • the presence of the MGMT gene in the LG896 vector should allow for the in vivo selection of transduced cells by treatment with BCNU. Having demonstrated that LG896 does not negatively impact NOD/SCID repopulating cells in vitro, the ability to specifically select for and expand MGMT-expressing cells in vivo will be demonstrated.
  • Sorrentino BP Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug- resistant hematopoietic stem cells. Blood 2003, 102:506-513.
  • Fauci AS Host factors and the pathogenesis of HIV -induced disease. Nature 1996, 384:529-534.
  • Fusion-competent vaccines broad neutralization of primary isolates of HIV. Science 1999,
  • GP Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type- 1 -infected patients.
  • Cavazzana-Calvo M Insertional oncogenesis in 4 patients after retro virus-mediated gene therapy of SCID-Xl. J Clin Invest 2008, 118:3132-3142.

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Abstract

Cette invention concerne la thérapie cellulaire pour le traitement d'une l'infection par le VIH chez l'homme. Elle concerne une composition de cellules humaines génétiquement modifiées destinées à être introduites dans le corps d'un sujet infecté par le VIH pour réduire la charge virale dudit sujet et générer et reconstituer des cellules T résistantes à l'infection par le VIH. La composition comprend de cellules T CD4+ humaines allogéniques ou autologues, des cellules souches hématopoïétiques (CSH) humaines allogéniques ou autologues, et des cellules souches mésenchymateuses (CSM) humaines allogéniques ou autologues. Chaque cellule comprend au moins une séquence anti-VIH hétérologue qui empêche le VIH d'infecter les cellules ou de se répliquer dans les cellules et au moins une séquence hétérologue pour contrôler le sort. Cette invention concerne également des vecteurs lentiviraux pour préparer les cellules, des plasmides et des cellules productrices pour préparer les vecteurs, et des procédés pour préparer les cellules génétiquement modifiées.
PCT/US2009/062909 2008-10-31 2009-11-01 Produit de thérapie cellulaire pour le traitement de l'infection par le vih WO2010051521A1 (fr)

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WO2012159120A2 (fr) * 2011-05-19 2012-11-22 University Of Florida Research Foundation, Inc. Stratégie basée sur la thérapie génique pour traiter l'infection par le vih
CN104498444A (zh) * 2014-12-10 2015-04-08 浙江大学 一种阻断hiv-1感染的ccr5δ32慢病毒及其制备方法
WO2015105999A1 (fr) 2014-01-08 2015-07-16 Immunovative Therapies, Ltd. Traitement du virus de l'immunodéficience humaine/du syndrome de l'immunodéficience acquise
WO2015191874A1 (fr) * 2014-06-12 2015-12-17 Children's National Medical Center Génération de cellules immunitaires contre le virus et généralement spécifiques ciblant plusieurs antigènes du vih pour une utilisation prophylactique et thérapeutique
WO2017007994A1 (fr) * 2015-07-08 2017-01-12 American Gene Technologies International Inc. Pré-immunisation et immunothérapie du vih
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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
WO2023125822A1 (fr) * 2021-12-31 2023-07-06 北京三诺佳邑生物技术有限责任公司 Lymphocytes t récepteurs d'antigènes chimériques ciblant des cellules infectées par le vih
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
KR102663972B1 (ko) 2017-01-09 2024-05-23 아메리칸 진 테크놀로지스 인터내셔널 인코포레이티드 예비-면역화 단계가 없는 hiv 면역요법

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