Methods and compositions for non-myeloablative bone marrow reconstruction

Abstract

The present invention provides a method and composition for performing bone marrow transplantation. [Solution] This disclosure relates, as a whole, to methods and compositions for performing bone marrow transplantation using non-myeloablative chemotherapeutic agents and chemotherapeutic-resistant cells. Using the methods and compositions described herein, a patient's bone marrow can be reconstituted, and the patient can avoid adverse side effects, including myeloablative and / or immune system disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 586,813, filed November 15, 2017, which is incorporated herein by reference in its entirety.

[0002] Technical field This disclosure provides methods and compositions for non-myeloablative bone marrow transplantation, including in the treatment of various diseases such as HIV, cancer (e.g., hematological cancers), and similar conditions. In some embodiments, modified stem cells are provided herein, which are grafted into the patient's bone marrow and enable bone marrow reconstitution without the negative side effects experienced in conventional bone marrow transplantation. These cells can also be used to express proteins of interest that may be inherently therapeutic. [Background technology]

[0003] Bone marrow transplantation (BMT) is a procedure that replaces damaged or destroyed bone marrow with healthy bone marrow stem cells isolated from either the patient (autologous) or another person (allogeneic). BMT is used to treat not only leukemia but also many other diseases, including severe aplastic anemia, lymphoma, multiple myeloma, immunodeficiency disorders, and some solid tumor cancers. [Overview of the project] [Problems that the invention aims to solve]

[0004] Myeloablative bone marrow cell therapy (BMT) involves first treating the patient to kill cells in the bone marrow (both normal and abnormal cells), and then injecting healthy bone marrow cells. The first step requires high doses of chemotherapy and / or radiation to kill cells, followed by the introduction of allogeneic or autologous cells. This process of eradicating the patient's bone marrow is called myeloablative disruption. Because this process kills not only unhealthy cells but also healthy immune cells and stem cells, patients are highly susceptible to infection and often require multiple antibiotics and continued presence in a sterile environment. Patients remain at high risk of infection until the bone marrow is reconstituted, and the recovery period can last up to six months. During this time, patients are advised to stay near the hospital or clinic where they are receiving treatment, especially if complications arise. In addition to acute toxicity, myeloablative chemotherapy is associated with many other side effects, including cataracts, growth retardation, cardiotoxicity, and endocrine and reproductive problems. Younger patients are particularly susceptible to these effects. Therefore, novel compositions and methods for performing BMT are needed. This disclosure satisfies these requirements. [Means for solving the problem]

[0005] [Once the scope of the patent claims is finalized, it will be included in its entirety.] [Brief explanation of the drawing]

[0006] [Figure 1]This figure shows the percentage of live GFP (green fluorescent protein)-positive (GFP+) granulocytes in the peripheral blood of mice that were administered bone marrow cells transduced with a lentiviral vector expressing EGFP ("control vector") or a lentiviral vector expressing EGFP+ALDH1A1 ("test vector", SEQ ID NO: 2, Figure 4), and treated with intraperitoneal (ip) cyclophosphamide (CTX) at the indicated concentrations once daily. Blood was collected by inducing hemorrhage posterior to the orbit on days 23, 35, and 42 of the study (corresponding to days 16, 28, and 35 of CTX administration, respectively), and the percentage of live GFP+ granulocytes in the peripheral blood was assessed by flow cytometry. There were n=3 in each of the groups without post-transplant CTX treatment and n=6 in all other groups. [Figure 2A] This figure shows the percentage of GFP+ cells in the bone marrow, as assessed by flow cytometry, in mice that were administered bone marrow cells transduced with a control vector or a test vector and subjected to a once-daily regimen of CTX at the indicated concentrations. There were n=3 in each of the groups that did not receive post-transplant CTX treatment, and n=6 in all other groups. Figure 2A shows the percentage of total (dead and live) GFP+ cells. [Figure 2B] This figure shows the percentage of GFP+ cells in the bone marrow, as assessed by flow cytometry, in mice that were administered bone marrow cells transduced with a control vector or a test vector and subjected to a once-daily regimen of CTX at the indicated concentrations. There were n=3 in each of the groups that did not receive post-transplant CTX treatment, and n=6 in all other groups. Figure 2B shows the percentage of live GFP+ granulocytes. [Figure 3]This figure shows the white blood cell (WBC) count in mice that were administered bone marrow cells transduced with a control vector or bone marrow cells transduced with a test vector and subjected to a once-daily regimen of CTX at the indicated concentrations. For each group, n=3 was present in the group that did not receive post-transplant CTX treatment, and n=5 in all other groups. [Figure 4] This figure shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 5] This figure shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 6] This figure shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 7] This figure shows a non-limiting schematic diagram of a study design for investigating dose ranges and the efficacy of lentiviral vectors. [Figure 8] This figure shows a non-limiting schematic diagram of a study design for the efficacy of lentiviral vector expression and shRNA knockdown studies. [Figure 9] This figure shows a non-limiting schematic diagram of a study design for transplanting HIV-resistant and CTX-resistant cells into HIV+ patients according to one embodiment of the present disclosure. [Figure 10] This figure shows a non-limiting schematic diagram of a study design in which HIV+ patients are treated with CTX once daily after transplanting cells that are multi-resistant to HIV and resistant to CTX chemotherapy into the patient. The possibility of applying the same study design to HIV-need subjects for HIV prevention will be explored. [Figure 11] This figure shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 12] This figure shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Modes for carrying out the invention]

[0007] While not always explicitly stated, it is understood that all numerical specifications are preceded by the term “about.” Where used herein, the term “about” means that the numerical value is an approximation and that slight variations will not significantly affect the implementation of the disclosed embodiments.

[0008] When used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" should be understood to include plural referents. Thus, for example, a reference to "cells" includes multiple cells.

[0009] definition As used herein, the following terms have the following meanings:

[0010] The term "approximately" when used before numerical specifications, such as temperature, time, quantity, concentration, and others, including ranges, indicates an approximate value that may vary by (+) or (-) 20%, 10%, 5%, or 1%.

[0011] Similarly, as used herein, “and / or” means any and all possible combinations of one or more of the related enumerated items, as well as the absence of any combination, as interpreted by the choice ("or").

[0012] The terms "administering" and "dosing" refer to introducing an agonist (e.g., cells) to a target. Typically, an effective dose is administered, which is determined by the physician administering the procedure. Any route of administration can be used, including local, subcutaneous, peritoneal, intravenous, intra-arterial, inhalation, vaginal, rectal, nasal, oral, intraoral, introduction into cerebrospinal fluid, or intravenous infusion into a body compartment. The terms and expressions "administering" and "administering ~" when used in relation to a composition (and its grammatical equivalents) refer to both direct administration, which may be administration to a patient by a medical professional or by self-administration by the patient, and / or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer an agonist (e.g., cells) and / or provides the patient with instructions about the drug is administering the agonist to the patient. "Intermittent administration" or "regular administration" refers to multiple procedures performed on a principle of once a day, once a week, or once a month. Intermittent administration may also refer to the administration of the agonist once, twice, three times, or more times per day.

[0013] As used herein, the terms “comprising” (and any form of “comprising,” e.g., “comprise,” “comprises,” and “comprised”), “having” (and any form of “having,” e.g., “have,” and “has”), “including” (and any form of “including,” e.g., “includes,” and “include”), or “containing” (and any form of “containing,” e.g., “contains,” and “contain”) are comprehensive or unrestrictive and do not exclude any further unlisted elements or processes. Any process or composition using the transitional phrase “comprise” or “comprising” may also be said to be described using the transitional phrase “consisting of” or “consists.”

[0014] An "effective dose" is the amount of an agent or compound (e.g., cells or a population of cells) sufficient to produce a favorable or desired result. An effective dose may be in one or more administrations, applications, or doses. Determining these parameters is well within the scope of the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard texts.

[0015] As used herein, the term “to bring into contact” means to bring two elements together in an in vitro or in vivo setting. For example, bringing a virus into contact with a cell. "Contact" with an individual, patient, or cells includes administering a virus to an individual or patient such as a human, and introducing a compound into a sample containing, for example, a cell preparation or purified preparation containing the cells of interest.

[0016] The term “heterogeneous” refers, when referring to nucleic acid molecules, proteins, vectors, or expression cassettes, to nucleic acid molecules, proteins, vectors, or expression cassettes that are expressed in cells through user manipulation and are not native. For example, heterogeneous genes refer to genes expressed by vectors or other media introduced into cells, or genes in the genome that have been modified via gene editing methods such as CRISPR or other recombination techniques for replacing genes within cells. Those skilled in the art will understand that the term “heterogeneous” does not refer to native genes in the genome of cells that have not been modified. “Heterogeneous” can also be called “exogenous.”

[0017] When used herein in relation to nucleic acids such as DNA or RNA, the term “isolated” refers to a molecule separated from other DNA or RNA present in a naturally occurring source of macromolecular weight. The term “isolated” also, when used herein, refers to nucleic acids or peptides that substantially lack cellular material, viral material, or culture media when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Furthermore, “isolated nucleic acids” include nucleic acid fragments that do not exist naturally as fragments and are not found in their natural state. “Isolated cells,” for example, isolated bone marrow cells, are cells that substantially lack other cellular material, tissue, or culture medium in the environment in which they naturally occur.

[0018] The term "myeloablative" refers to a treatment that causes prolonged (usually irreversible) pancytopenia, kills cells in the bone marrow within 1 to 3 weeks of administration, and prevents autologous hematological recovery. (Bacigalupo et al., Biol Blood Marrow Transplant. 2009, 15(12): pp. 1628-1633). Examples of myeloablative doses of cyclophosphamide include, but are not limited to, 2.5 mg / kg / day of CTX or higher over a period of time that results in cumulative toxicity (McKinley et al., Clin J Am Soc Nephrol. 2009, 4: pp. 1754-1760).

[0019] The term "non-myeloablative" refers to a treatment that does not cause cytopenia, causes minimal cytopenia, or causes reversible cytopenia, and is minimally toxic. Non-myeloablative regimens are immunodesorptions. Examples of non-myeloablative doses include, but are not limited to, approximately 1.3 mg / kg / day over a period of time, or 1.0 to 1.5 mg / kg / day over 2 to 4 months, which do not cause cumulative toxicity (McKinley et al., Clin J Am Soc Nephrol. 2009, 4: pp. 1754-1760). Other non-myeloablative doses are described throughout and are included in the definition of non-myeloablative doses. An agonist or dose of an agonist that produces "cumulative toxicity" refers to a dose that produces toxicity in a patient over time. For example, administering cyclophosphamide to a human at a dose of 2.5 mg / kg / day over several weeks produces cumulative toxicity.

[0020] "Subject," "individual," or "patient" are used without distinction herein and refer to vertebrates, e.g., primates, mammals, or preferably humans. Mammals include, but are not limited to, horses, dogs, cattle, sheep, mice, rats, monkeys, and humans.

[0021] The term "sequence identity" in relation to protein sequences or amino acid sequences (or DNA sequences or RNA sequences) refers to the specific protein or amino acid sequence in a candidate sequence after aligning the sequence, introducing gaps as necessary to obtain the maximum possible sequence identity percentage, and without considering any conservative substitutions as part of the sequence identity. Alignment refers to the percentage of identical amino acid residues (or nucleotide residues) to a specific amino acid residue (or nucleotide residue in a particular DNA or RNA sequence) within a sequence. Alignment can be performed by any method known to those skilled in the art, for example, by using publicly available programs such as BLAST and EMBOSS. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared, although default parameters are used in some embodiments. Programs can be accessed, for example, from the National Center for Biotechnology Information.

[0022] The term “mutant,” as used herein, refers to a nucleic acid or protein that differs from a reference nucleic acid or reference protein (i.e., calmodulin or a fragment thereof) but retains its fundamental properties (i.e., biological activity). A typical variant of a polynucleotide differs in its nucleotide sequence from another reference polynucleotide. A change in nucleotide sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. A change in nucleotides can result in amino acid substitutions, additions, deletions, fusions, and / or truncations in the polypeptide encoded by the reference sequence.

[0023] The term “vector” is used herein to refer to a nucleic acid molecule capable of moving or carrying another nucleic acid molecule. The moved nucleic acid is generally ligated to, for example, the nucleic acid molecule of the vector. A vector may contain a sequence that directs autonomous replication in a cell, or a sequence sufficient to enable integration into cellular DNA. Examples of vectors include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial or yeast artificial chromosomes, and viral vectors. Useful viral vectors include, for example, adenoviruses, retroviruses, particularly replication-deficient retroviruses, and lentiviruses. In some embodiments, a vector has the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, a vector contains the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or any combination thereof.

[0024] The expression “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or drug form that falls within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit-risk ratio.

[0025] Methods for performing bone marrow transplantation This specification provides methods for performing bone marrow transplantation in patients in need. It also provides methods for replacing target bone marrow cells with a population of cells expressing one heterologous nucleic acid molecule expression cassette or multiple heterologous expression cassettes, or with genome-edited cells that differ from the target genome. In some embodiments, these methods include administering one or more chemotherapy-resistant modified cells to a patient and administering at least one dose of a chemotherapeutic agent. In certain embodiments, the dose is a non-myeloablative dose of the chemotherapeutic agent. In some embodiments, the amount of cells is a therapeutically effective dose.

[0026] In some embodiments of the methods provided herein, the patient has HIV.

[0027] The chemotherapy-resistant modified cells for use in the disclosed method may be any suitable cells known to those skilled in the art. For example, the cells may be stem cells or immune cells. Non-limiting examples of stem cells include umbilical cord blood cells, embryonic stem cells, and embryonic stem cells (ESCs). These include hematopoietic stem cells (HSCs), hematopoietic progenitor cells, pluripotent stem cells (PSCs), artificial PSCs (iPSCs), or cells derived therefrom. In some embodiments, the immune cells are T cells. In some embodiments, the cells are CD34+ and / or CD4+. In some embodiments, the cells are mesenchymal stem cells, stromal stem cells, umbilical cord blood-derived hematopoietic stem cells / hematopoietic progenitor cells, umbilical cord tissue-derived stem cells / progenitor cells, iPSCs, HESCs, fetal tissue-derived stem cells, CD4+ cells, and similar. In some embodiments, the stem cells are CD34+.

[0028] Chemotherapy resistance The chemotherapy-resistant cells for use in this method can be generated using any method known in the art for conferring chemotherapy resistance. In certain embodiments, the bone marrow transplantation method provided herein comprises modifying one or more cells to be chemotherapy-resistant. For example, in certain embodiments, a method is provided for performing a bone marrow transplant in a patient in need, comprising generating chemotherapy-resistant modified cells, administering an effective amount of the chemotherapy-resistant modified cells to the patient, and administering at least one dose of a chemotherapeutic agent. In certain embodiments, the dose is a non-myeloablative dose of the chemotherapeutic agent. In some embodiments, the chemotherapy-resistant cells are resistant to cyclophosphamide. In some embodiments, the chemotherapy-resistant cells are resistant to a non-myeloablative amount of cyclophosphamide.

[0029] In some embodiments, cells can be modified to express an exogenous chemotherapy resistance gene (i.e., a transduced gene), for example, the exogenous chemotherapy resistance gene may be a cyclophosphamide resistance gene, a variant thereof, or a nucleic acid sequence encoding a portion thereof. In some embodiments, the cyclophosphamide resistance gene is aldehyde dehydrogenase 1 (ALDH1). In some embodiments, ALDH1 is a nucleic acid molecule containing the sequence shown in SEQ ID NO: 1 or a variant thereof. In some embodiments, ALDH1 is expressed in a lentiviral vector containing the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a variant thereof. Cells modified with ALDH1 but resistant to cyclophosphamide may remain susceptible to other non-cyclophosphamide chemotherapy agents (i.e., the cells do not become multidrug resistant).

[0030] Exogenous chemotherapy resistance genes can be expressed using any modification method known to those skilled in the art, including viral vectors (e.g., retroviruses such as adenoviruses, replication-deficient retroviruses, and lentiviruses), non-viral vectors (e.g., episomal plasmids), or transposon systems (e.g., Sleeping Beauty or PiggyBac). In some embodiments, the vector has the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the chemotherapy resistance gene is synthetic messenger RNA (mRNA). Synthetic mRNA provides the genetic information to produce the protein of interest and can be chemically modified to avoid eliciting an immune response. Zangi et al. (2013) Nature Biotech 31: pp. 898-907. Because mRNA is not integrated into the host cell genome, synthetic RNA acts for a period of time and then disappears when the cell divides. In some embodiments, synthetic mRNA is modified with, for example, pseudouridine and / or 5-methylcytidine to reduce the intrinsic antiviral response to single-stranded RNA. In some embodiments, synthetic RNA encodes ALDH (e.g., ALDH1) and / or their respective equivalents.

[0031] In some embodiments, chemotherapy resistance, such as cyclophosphamide resistance, is transiently expressed by modified cells. Sphamide is expressed by modified cells over periods of approximately 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, or 3 years. Transient expression refers to the persistence of expression of the gene or protein that confers resistance. Transient means that the resistance is not permanent.

[0032] In some embodiments, exogenous chemotherapy resistance genes, such as cyclophosphamide resistance genes, are introduced into cells using one of a variety of well-known techniques, such as nonviral-based transfection of cells. Intracellular introduction can be carried out by any nonviral-based transfection method known in the art, for example, but not limited to, electroporation, calcium phosphate-mediated transfection, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated transfection, microinjection, microparticle gun-mediated transfection (nanoparticles), cationic polymer-mediated transfection (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), and similar), or cell fusion. Other transfection methods include transfection reagents such as Lipofectamine®, Dojin Chemical Laboratories' Highlymax®, Fugene®, jetPEI®, Effectene®, and DreamFect®.

[0033] Cell isolation and / or purification The chemotherapy-resistant modified cells for use in this method may be patient cells (i.e., autologous cells), donor cells (i.e., allogeneic cells), or any combination thereof, that have been modified to confer chemotherapy resistance. In certain embodiments, the method provided herein further comprises isolating and / or purifying cells from a patient or donor. In certain embodiments of these, the method further comprises modifying cells to be chemotherapy-resistant. For example, in certain embodiments, a method is provided for performing a bone marrow transplant in a patient in need, comprising isolating and / or purifying one or more cells from a patient or subject, modifying one or more cells to be chemotherapy-resistant as described herein, administering an effective amount of one or more chemotherapy-resistant modified cells to the patient, and administering at least one dose of a chemotherapeutic agent.

[0034] Cells can be isolated by any method known to those skilled in the art, for example, based on the expression / absence of certain markers, proliferation rate, and potential for differentiation. In some embodiments, cells may be isolated by any method known to those skilled in the art, such as CD34, CD4, Sca-1, CD38, CD123, CD90, CD45, CD133, antigen-presenting cell markers (CD8, CD8 alpha, CD11b, CD11c, CD103, CD205, CD24, CD115, CD117, CD135, CD11c low CD45RA, CD123, ILT-7, MHC Class II, MHC Class II low Cells are isolated based on the presence of specific markers or combinations of markers, including TLR7 and / or TLR9. In some embodiments, cells are isolated based on the absence of specific markers, e.g., CD3, CD14, CD19, CD56, and / or CD66b. In other embodiments, negative selection is performed for markers of, for example, T cells, B cells, granulocytes, and / or myelomonocytes. In some embodiments, cells are isolated based on the presence of Thy-1, either alone or in combination with any other marker. In some embodiments, HSCs are isolated based on the presence of Lin - Thy1 + Sca-1 + They are isolated based on their expression profile. In some embodiments, mouse HSCs have an expression profile of CD34 - , Sca1 + c-kit + They can be isolated by [method]. In some embodiments, human HSCs can be isolated based on CD34 expression.

[0035] Chemotherapy agents In some embodiments, the methods provided herein involve administering one or more doses of a chemotherapeutic agent to a patient. In some embodiments, the dose is a non-myeloablative dose of the chemotherapeutic agent. The chemotherapeutic agent may be any suitable chemotherapeutic agent known to those skilled in the art. Non-limiting examples of chemotherapeutic agents include actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine (BCNU), cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothirone, etoposide, fluorouracil, gemcitabine, hydroxy This includes siurea, idarubicin, imatinib, irinotecan, lomustine (CCNU), mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nimustine (ACNU), oxaliplatin, paclitaxel, pemetrexed, temezolamide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, barrubicin, vemurafenib, vinblastine, vincristine, vindesine, and vinorelbine.

[0036] Disease-specific modifications In certain embodiments of the methods provided herein, chemotherapy-resistant modified cells may include one or more further modifications unrelated to chemotherapy resistance. For example, in certain embodiments, cells are further modified to express further HIV / disease-specific modifications. Thus, in certain embodiments, the bone marrow transplantation method provided herein further includes incorporating one or more further modifications, including one or more HIV / disease-specific modifications. For example, in certain embodiments, a method is provided for performing a bone marrow transplant in a patient in need, comprising isolating and / or purifying one or more cells from a patient or subject, modifying one or more cells to be chemotherapy-resistant as described herein, incorporating one or more further modifications into one or more cells, administering an effective amount of one or more chemotherapy-resistant modified cells to the patient, and administering at least one dose of a chemotherapeutic agent. In some embodiments, the dose is a non-myeloablative dose of the chemotherapeutic agent.

[0037] In some embodiments, modified cells are further modified to be HIV-resistant. For example, modified cells can be further modified to express at least one mutant HIV coreceptor that confers resistance to HIV infection, one or more mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor, or any combination thereof. In some embodiments, cells are modified to express a molecule that inhibits or reduces the expression of HIV coreceptors. In some embodiments, the molecule is an antisense molecule. In some embodiments, cells are modified to express shCCR5, shCXCR4, a GP-41 fusion inhibitor, a C46 fusion inhibitor, a C34 fusion inhibitor, any other C-peptide fusion inhibitor, or any combination thereof. In some embodiments, the CCR5 mutation is a CCR5-delta32 mutation. In some embodiments, both copies of the CCR5 gene in the cell are replaced with a CCR5-delta32 mutation. In some embodiments, one copy of the CCR5 gene is replaced with a CCR5-delta32 mutation.

[0038] This disclosure provides cells modified to have chemotherapy resistance, e.g., cyclophosphamide resistance, and HIV resistance. In some embodiments, cells can be modified to have cyclophosphamide resistance and HIV resistance. HIV resistance is achieved by reducing the expression of at least one HIV coreceptor, or by eliminating one protrusion of at least one HIV coreceptor. HIV resistance is conferred by a natural mutation, multiple mutations, the expression of at least one HIV fusion inhibitor, or any combination thereof. HIV resistance is conferred by reduced expression of the CCR5 HIV coreceptor, reduced expression of the CXCR4 coreceptor, the expression of a C-peptide fusion inhibitor (e.g., a C46 fusion inhibitor or a C34 fusion inhibitor), or any combination thereof.

[0039] Cells can also be modified to express any desired molecule. The desired molecule can be modified according to the determination by the user or according to the specific patient's requirements.

[0040] Administration to a patient In some embodiments, the methods provided herein include administering to a subject an effective amount of chemotherapy-resistant modified cells and a non-myeloablative dose of a chemotherapeutic agent. The modified cells and the chemotherapeutic agent can be administered by any suitable route that will be apparent to those skilled in the art, depending on the disease or condition being treated. Exemplary routes of administration include intravenous, intraarterial, intramuscular, subcutaneous, intracranial, intranasal, intradermal, oral, or intraperitoneal routes.

[0041] In some embodiments, per 1 m of the subject's body surface area 2 about 1×10 8 to about 1×10 11 cells are administered to the subject. The cells can be administered to an individual on an absolute cell number basis, e.g., the individual can be administered from about 1000 cells / injection to up to about 10 billion cells / injection, e.g., per injection, about, at least about, or up to, about 1×10 8 、1×10 7 、5×10 7 、1×10 6 、5×10 6 、1×10 5 、5×10 <00> 5 、1×10 4 、5×10 4 、1×10 3 、5×10 3 cells (etc.), or any range between any two of these values, including the endpoints, can be administered. In some embodiments, about 5×10 6 cells / kg to about 10×10 6 cells / kg are used for HSC transplantation. <>

[0042] In other embodiments, the subject is administered about 1000 cells / injection / m 2From up to approximately 10 billion cells / injection / m 2 It can be administered, for example, per injection, about, at least about, or at most about 1 × 10 8 pieces / m 2 , 1 x 10 7 pieces / m 2 , 5×10 7 pieces / m 2 , 1 x 10 6 pieces / m 2 , 5×10 6 pieces / m 2 , 1 x 10 5 pieces / m 2 , 5×10 5 pieces / m 2 , 1 x 10 4 pieces / m 2 , 5×10 4 pieces / m 2 , 1 x 10 3 pieces / m 2 , 5×10 3 pieces / m 2 Cells of (etc.), or any range between any two of these values, including the endpoint, can be administered.

[0043] In other embodiments, cells can be administered to such an individual on a relative number basis, for example, the individual can be administered with about 1,000 to a maximum of about 10 billion cells per kilogram of the individual, for example, about, at least about, or at most about 1 × 10¹⁶ cells per kilogram of the individual. 8 pieces, 5×10 7 pieces, 1×10 7 pieces, 5×10 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 1×10 5 pieces, 5×10 4 pieces, 1×10 4 pieces, 5×10 3 pieces, 1×10 3 Individual cells, or any range between any two of these values, including the endpoint, can be administered.

[0044] In some embodiments, at least one dose of a non-myeloablative chemotherapeutic agent is administered to the patient. The administration of the chemotherapeutic agent can be done simultaneously with or consecutively with the administration of the modified cells. In some embodiments, at least one dose of a non-myeloablative chemotherapeutic agent is administered after the administration of the modified cells. In some embodiments, a pre-treatment step (also referred to herein as the “pre-conditioning step”) is performed before the administration of the cells, in which at least one dose of a chemotherapeutic agent, such as fludarabine or cyclophosphamide, is administered to the patient before the administration of the modified cells. In some embodiments, pre-conditioning The preparation step is a pre-preparation step with a non-myeloablative chemotherapeutic agent. In some embodiments, the pre-preparation step is not performed before administering the cells. It is believed that the cells of this disclosure can still be efficiently grafted into the patient's bone marrow even without a pre-preparation step (e.g., fludarabine) before administering the cells.

[0045] In some embodiments, at least one non-myeloablative dose of a chemotherapeutic agent for human subjects or patients is a non-myeloablative dose of cyclophosphamide. In some embodiments, the non-myeloablative dose of cyclophosphamide is approximately 0.15 mg / kg / day to less than 2.5 mg / kg / day, approximately 0.4 mg / kg / day to approximately 1.7 mg / kg / day, or approximately 0.8 mg / kg / day to approximately 1.5 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide is approximately 0.15 mg / kg / day, approximately 0.2 mg / kg / day, approximately 0.25 mg / kg / day, approximately 0.3 mg / kg / day, approximately 0.35 mg / kg / day, approximately 0.4 mg / kg / day, approximately 0.45 mg / kg / day, approximately 0.5 mg / kg / day, approximately 0.55 mg / kg / day, approximately 0.6 mg / kg / day, approximately 0.65 mg / kg / day, approximately 0.7 mg / kg / day, approximately 0.75 mg / kg / day, approximately 0.8 mg / kg / day, approximately 0.85 mg / The dosages are approximately kg / day, about 0.9 mg / kg / day, about 0.95 mg / kg / day, about 1.0 mg / kg / day, about 1.1 mg / kg / day, about 1.2 mg / kg / day, about 1.3 mg / kg / day, about 1.4 mg / kg / day, about 1.5 mg / kg / day, about 1.6 mg / kg / day, about 1.7 mg / kg / day, about 1.8 mg / kg / day, about 1.9 mg / kg / day, about 2.0 mg / kg / day, about 2.1 mg / kg / day, about 2.2 mg / kg / day, about 2.3 mg / kg / day, or about 2.4 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide is about 1.3 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide ranges from approximately 0.8 mg / kg / day to approximately 1.6 mg / kg / day, approximately 0.8 mg / kg / day, approximately 0.98 mg / kg / day, approximately 1.3 mg / kg / day, approximately 1.5 mg / kg / day, or approximately 1.6 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide ranges from approximately 0.5 to approximately 2 mg / kg / day.

[0046] In some embodiments, a non-myeloablative dose of the chemotherapeutic agent is administered daily for a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, or longer.

[0047] In some embodiments, a non-myeloablative dose is provided over a period of time that does not cause cumulative toxicity. For example, the period of time that does not cause cumulative toxicity is a period of time such as less than about 1 year, less than about 6 months, less than about 3 months, less than about 2 months, less than about 1 month, less than about 3 weeks, less than about 2 weeks, less than 1 week, less than about 6 days, less than about 5 days, less than about 4 days, less than 3 days, or less than 2 days.

[0048] In some embodiments, there is at least one interruption of a certain period between the administration of cyclophosphamide-resistant modified cells and the administration of at least one dose of a non-myeloablative chemotherapeutic agent. For example, the interruption may be about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, or longer. In some embodiments, the interruption may be about 3 days, about 7 days, about 10 days, and about 14 days.

[0049] In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about one year. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about six months. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about five months. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about four months. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about three months. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about two months. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about one month. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about two weeks. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells within about one week.

[0050] In some embodiments, the patient does not experience bone marrow destruction and / or immunodeficiency during the procedure. In some embodiments, the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, low platelet count, low leukocyte count, low erythrocyte count, or any combination thereof, or related symptoms.

[0051] In another embodiment, treatment with the cells and chemotherapeutic agents of this disclosure may result in one or more of the following outcomes: (i) an increase in white blood cells of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual percentage change, or median percentage change); (ii) an increase in granulocytes of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual percentage change, or median percentage change); (iii) an increase in neutrophils of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual percentage change, or median percentage change); (iv) Compared to the control, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least 60%, at least 65%, at least 70%, at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, and This is an increase of at least 99% (actual change, or median change) in lymphocytes; (v) An increase in eosinophils of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) or median change %) compared to the control; (vi) an increase in monocytes of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) or median change %) compared to the control group; (vii) An increase in basophils of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) or median change %) compared to the control; (viii) Red blood cell increase of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) or median change %) compared to the control; (ix) An increase in all three blood cellular components (red blood cells, white blood cells, and platelets) by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) or median change %) compared to the control; (x) No recurrence for at least approximately 6 months, approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 15 years, approximately 20 years, approximately 25 years, approximately 30 years, approximately 35 years, approximately 40 years, approximately 45 years, approximately 50 years, approximately 55 years, approximately 60 years, or longer; (xi) Increased recurrence-free survival rate in patients for at least approximately 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, or longer, compared to the control group; (xii) Increased patient survival rate compared to the control group for at least approximately 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, or longer; (xiii) Compared to controls, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least 60%, at least 65%, at least 70%, at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) of HIV intracellular lifespan Shortened form; (xiv) A reduction of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %, or median change %) in HIV-carrying hosts compared to controls; and (xv) Exhaustion of viral DNA of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least 60%, at least 65%, at least 70%, at least approximately 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (actual change %) of the control.

[0052] In some embodiments, the control may be a subject treated with placebo, a baseline control, or a subject treated with unmodified cells.

[0053] In some embodiments, modified cells are administered to a subject for a period effective in reducing at least one symptom of HIV by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to a control. The control group may consist of subjects treated with placebo, baseline controls, or subjects treated with unmodified cells.

[0054] Non-specific symptoms include fever, headache, lethargy, skin rash, skin pain, glandular hypertrophy, infection (e.g., pneumonia, tuberculosis, hepatitis C), night sweats, diarrhea, nausea and vomiting, weight loss, severe headache, joint pain, muscle pain, and chronic cough.

[0055] In some embodiments, modified cells are administered in conjunction with at least one other HIV therapy. Suitable other HIV therapies include any HIV therapy known to those skilled in the art. Non-limiting examples of other HIV therapies include combination drugs (e.g., efavirenz / emtricitabine / tenofovir disoproxil fumarate (Atripla®), emtricitabine / rilpivirine / tenofovir disoproxil fumarate (Complera®), elvitegravir / cobicistat / emtricitabine / tenofovir disoproxil fumarate (Stribild®), and abacavir / dolutegravir / lamivudine (Triumeq®))), nucleoside / nucleotide reverse transcriptase inhibitors (NRT) I) (For example, abacavir (Ziagen®), efavirenz / emtricitabine / tenofovir disoproxil fumarate (Atripla®), lamivudine / zidovudine (Combivir®), emtricitabine / rilpivirine / tenofovir disoproxil fumarate (Complera®), emtricitabine (Emtriva®), lamivudine (Epivir®), abacavir / lamivudine (Epzicom®), zidovudine (Retrovir®), abacavir / lamib Trizivir (zidovudine), emtricitabine / tenofovir disoproxil fumarate (Truvada®), didanosine (Videx®), didanosine sustained-release (Videx EC®), tenofovir disoproxil fumarate (Viread®), and stabudine (Zerit®)), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (e.g., tipranavir (Aptivus®), indinavir (Crixivan®), atazanavir / cobicistat (Evotaz®), saquinavir (Invirase®), lopinavir / ritonavir (Kaletra®) (Registered Trademark), fosamprenavir (Lexiva®), ritonavir (Norvir®), darunavir / cobicistat (Prezcobix®), darunavir (Prezista®), atazanavir (Reyataz®), nelfinavir (Viracept®), entry inhibitors (e.g., enfuvirtide (Fuzeon®)), integrase inhibitors (e.g., raltegravir (Isentress®)), dollar These include tegravir (Tivicay®) and elvitegravir (Vitekta®), chemokine coreceptor antagonists (CCR5 antagonists) (e.g., maraviroc (Selzentry®) or bicriviroc), cytochrome P4503A inhibitors, and immunotherapy (e.g., hydroxychloroquine sulfate (Plaquenil)). In some embodiments, modified cells and at least one other HIV therapy are administered simultaneously. In other embodiments, modified cells and at least one other HIV therapy are administered sequentially. In some embodiments, administration of at least one of the above other HIV therapies is explicitly excluded, for example, in some embodiments, NRTIs are explicitly excluded. In some embodiments, no other HIV therapy other than the modified cells and at least one dose of a non-myeloablative chemotherapeutic agent (e.g., cyclophosphamide) disclosed herein is administered.

[0056] Cells, chemotherapeutic agents, and, as may, other HIV therapies may be administered to a patient with HIV either once or multiple times during the course of treatment, for example, once every hour, every two hours, every three hours, every four hours, every five hours, every six hours, every seven hours, every eight hours, every nine hours, every ten hours, every eleven hours, every twelve hours, every thirteen hours, every fourteen hours, every fifteen hours, every sixteen hours, every seventeen hours, every eighteen hours, every nineteen hours, every twenty hours, every twenty-one hours, every twenty-two hours, or every twenty-three hours; or once every day, every two days, every three days, every four days, every five days, every six days, or every seven days; or once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, or every more than that; or once every range between any two of these values, including the endpoint.

[0057] In some embodiments, a method is provided for treating a patient having HIV. In some embodiments, the method involves treating the patient's CD34 + This involves recruiting stem cells from the bone marrow to the periphery. In some embodiments, cells are recruited by administering G-CSF (granulocyte colony-stimulating factor). G-CSF can be administered, for example, as a regimen of 1, 2, 3, 4, or 5 days. In some embodiments, G-CSF is administered for 3 to 5 days. The recruited cells can be captured using methodologies such as apheresis. In some embodiments, for example, cell isolation by apheresis is performed using CD34 + The number of cells is 10.0 to 20.0 × 10⁶ per kg of body weight. 6 The procedure is performed when the number of cells reaches or exceeds this value. In some embodiments, the cell count is 5.0 to 25.0 × 10⁶ per kg of body weight. 6 or exceeding. Cd34+ cells are used as markers to capture cells for transduction, but other cell markers, such as those described herein, may also be used. For example, certain markers or ma Cells used for isolation based on the presence of marker combinations include, for example, CD34, CD4, Sca-1, CD38, CD123, CD90, CD45, CD133, and antigen-presenting cell markers (CD8, CD8 alpha, CD11b, CD11c, CD103, CD205, CD24, CD115, CD117, CD135, CD11c). low CD45RA, CD123, ILT-7, MHC Class II, MHC Class II low This includes TLR7 and / or TLR9). In some embodiments, cells are isolated based on the absence of specific markers, e.g., CD3, CD14, CD19, CD56, and / or CD66b. In other embodiments, negative selection is performed for markers of, for example, T cells, B cells, granulocytes, and / or myelomonocytes. In some embodiments, cells are isolated based on the presence of Thy-1, either alone or in combination with any other marker. In some embodiments, HSCs are Lin - Thy1 + Sca-1 + They are isolated based on their expression profile. In some embodiments, mouse HSCs have an expression profile of CD34 - , Sca1 + c-kit + They can be isolated by [method]. In some embodiments, human HSCs can be isolated based on CD34 expression. In some embodiments, the isolated cells are CD34+ or CD4+, or any combination thereof.

[0058] In some embodiments, the method includes centrifugation of the recovered cells. This is done, for example, to develop a cell-rich pellet. The cells are then resuspended in a cryopreservation solution and frozen. In some embodiments, the cryopreservation solution includes a heparinized plasmalyte solution and a 10% DMSO (dimethyl sulfoxide) solution. In some embodiments, the cells are first stored at -4°C, and then the sample is frozen by lowering it to a target temperature of -156°C (when stored in the vapor phase) to -196°C (when stored in the liquid phase).

[0059] In some embodiments, the method involves transduction of isolated cells to induce resistance to chemotherapeutic agents such as cyclophosphamide. As described herein, chemotherapeutic resistance is achieved by the expression of ALDH1. ALDH1 can be introduced into selected cells through the use of a vector (described throughout this specification), such as the use of a lentiviral vector. ALDH1 can be functionally ligated to a cell-specific promoter. In some embodiments, the promoter is a CD34 promoter. In some embodiments, the promoter is an hCD34 promoter. In some embodiments, the promoter has the sequence of SEQ ID NO: 12. In some embodiments, the promoter is an hCD4 promoter, such as the one provided in SEQ ID NO: 8. In some embodiments, the sequence of ALDH1 is expressed as a protein provided in SEQ ID NO: 10. In some embodiments, ALDH1 is encoded by a nucleic acid molecule containing the sequence of SEQ ID NO: 1. Due to the degenerate nature of genetic coding, the sequence of SEQ ID NO: 1 is provided as a non-limiting example, and other nucleic acid molecules may also be used to encode the expression of a protein containing SEQ ID NO: 10. In some embodiments, ALDH1 contains 1 to 10 conservative substitutions that do not alter the function of ALDH1. In some embodiments, the expressed ALDH1 is at least 95% homologous or identical to SEQ ID NO: 10.

[0060] ALDH1 expression in the vector is also driven by an enhancer element. For example, the enhancer element may be a CD3E enhancer. In some embodiments, the CD3E enhancer includes the sequence of SEQ ID NO: 9.

[0061] In some embodiments, CD34 + Cells can be isolated by magnetic bead separation. Human CD34 via lentiviral vector + Cell transduction can be performed, for example, by cytokine stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L This may include a 24-hour preliminary stimulation of cells in a medium supplemented with ), thrombopoietin (TPO), IL-6, IL-2, IL-3, fibronectin, or any combination thereof. In some embodiments, cells are then contacted (infected) with a lentivirus expressing ALDH1. In some embodiments, the vector contains the sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5. Contact is performed in serum-free X-Vivo 10 medium with cytokines SCF, FLT3L, and TPO (100 ng·ml each). -1 This can be done in the presence of ( ). The cells may then be frozen or not, depending on the circumstances. In some embodiments, the cells are not brought into contact with the AAV or AV vector.

[0062] In some embodiments, the method involves injecting transduced cells into a target. In some embodiments, the target has HIV. In some embodiments, the target does not have HIV but is at high risk of contracting HIV and therefore desires to become HIV resistant.

[0063] In some embodiments, after injecting modified cells, a non-myeloablative dose of a chemotherapy drug such as cyclophosphamide is administered. In some embodiments, the dose is 50 to 200 mg and is administered once daily. In some embodiments, the non-myeloablative dose of cyclophosphamide ranges from about 0.15 mg / kg / day to less than 2.5 mg / kg / day, from about 0.4 mg / kg / day to about 1.7 mg / kg / day, or from about 0.8 mg / kg / day to about 1.5 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide is approximately 0.15 mg / kg / day, approximately 0.2 mg / kg / day, approximately 0.25 mg / kg / day, approximately 0.3 mg / kg / day, approximately 0.35 mg / kg / day, approximately 0.4 mg / kg / day, approximately 0.45 mg / kg / day, approximately 0.5 mg / kg / day, approximately 0.55 mg / kg / day, approximately 0.6 mg / kg / day, approximately 0.65 mg / kg / day, approximately 0.7 mg / kg / day, approximately 0.75 mg / kg / day, approximately 0.8 mg / kg / day, approximately 0.85 mg / The dosages are approximately kg / day, about 0.9 mg / kg / day, about 0.95 mg / kg / day, about 1.0 mg / kg / day, about 1.1 mg / kg / day, about 1.2 mg / kg / day, about 1.3 mg / kg / day, about 1.4 mg / kg / day, about 1.5 mg / kg / day, about 1.6 mg / kg / day, about 1.7 mg / kg / day, about 1.8 mg / kg / day, about 1.9 mg / kg / day, about 2.0 mg / kg / day, about 2.1 mg / kg / day, about 2.2 mg / kg / day, about 2.3 mg / kg / day, or about 2.4 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide is about 1.3 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide ranges from about 0.8 mg / kg / day to about 1.6 mg / kg / day, about 0.8 mg / kg / day, about 0.98 mg / kg / day, about 1.3 mg / kg / day, about 1.5 mg / kg / day, or about 1.6 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide ranges from about 0.5 to about 2 mg / kg / day. Doses may be administered as indicated herein. While not bound by any particular theory, once-daily oral cyclophosphamide to facilitate grafting increases genetically modified myeloid cells. Modified CD34 +The patient has HIV at the time the cells are injected. + It is conceivable that in this case the cells are functioning to treat and / or cure HIV, or modified CD34 + The patient has HIV at the time the cells are injected. - It is conceivable that this could be the case, and in this instance, the cells are functioning to prevent future HIV infection. + Figures 9 and 10 show non-definitive schematic diagrams of the patient's treatment, but the patient has HIV. - But it is understood that this is possible.

[0064] In some embodiments, the subjects are also treated with fludarabine before the modified cells are injected. In some embodiments, on day 2 after harvesting (or day 5 before transplantation), the patient is given fludarabine (15 mg / m²) for 5 days (up to day 1 before transplantation). 2 ) is administered. In some embodiments, instead of fludarabine, the patient is 4 days before transplantation. Treatment can be performed with busulfan at a dose of mg / kg. In some embodiments, patients are treated with a single dose of 1000 mg / m2 of cyclophosphamide on day 2 before transplantation. However, after cell infusion, subjects are treated with the non-myeloablative doses of cyclophosphamide as indicated herein.

[0065] As described herein, the vector may also include other expression cassettes that express shCCR5 or fusion inhibitors, such as C44, C46, ​​or others as described herein. The fusion inhibitor may be a fusion of a GPI anchor and an HIV fusion inhibitor. In some embodiments, the fusion inhibitor is encoded by the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the fusion inhibitor is a protein containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion inhibitor is encoded by the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the fusion inhibitor is a protein containing the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion inhibitor is immobilized to the membrane by a GPI anchor. In some embodiments, the anchor is encoded by a nucleic acid molecule containing the sequence of SEQ ID NO: 16. In some embodiments, the anchor contains the sequence of SEQ ID NO: 17. In some embodiments, the fusion-anchor protein contains an IgG hinge region. In some embodiments, the IgG hinge region is IgG3. In some embodiments, the fusion inhibitor protein contains the sequence of SEQ ID NO: 19. In some embodiments, the fusion inhibitor protein is encoded by a nucleic acid molecule containing the sequence of SEQ ID NO: 18. In some embodiments, the anchor is GP41.

[0066] In some embodiments, the fusion inhibitor is controlled by a promoter different from that of the ALDH1 promoter. In some embodiments, the promoter is the EFS promoter. In some embodiments, the promoter is the CD4 promoter, for example, the CD4 promoter described herein.

[0067] In some embodiments, the vector transduced into cells as provided herein expresses an antisense molecule that reduces or inhibits CCR5 expression. In some embodiments, the vector encodes an shCCR5 inhibitor molecule. In some embodiments, the lentiviral vector encodes the sense sequence of hCCR5 shRNA of SEQ ID NO: 6, or its complement, and / or the antisense sequence of hCCR5 shRNA of SEQ ID NO: 7, or its complement. In some embodiments, the sequences may also be in reverse order. In some embodiments, the vector includes a mir30 expression cassette. In some embodiments, the mir30 expression cassette encodes hCCR5 shRNA. In some embodiments, the mir30 construct includes the sequence of SEQ ID NO: 13. The antisense molecules that can be used to inhibit CCR5 expression are non-limiting examples, and other antisense molecules that target CCR5 may also be used.

[0068] Therefore, in some embodiments, nucleic acid molecules comprising the sequences of SEQ ID NO: 6, SEQ ID NO: 7, and / or SEQ ID NO: 13 are provided.

[0069] In some embodiments, the Disclosure provides proteins comprising SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, and / or SEQ ID NO: 19.

[0070] In some embodiments, nucleic acid molecules are provided that include SEQ ID NO: 1 or its variants, SEQ ID NO: 3 or its variants, SEQ ID NO: 6 or its variants, SEQ ID NO: 7 or its variants, SEQ ID NO: 13 or its variants, SEQ ID NO: 14 or its variants, SEQ ID NO: 16 or its variants, SEQ ID NO: 18 or its variants, or any combination thereof. In some embodiments, nucleic acid molecules are provided that include SEQ ID NO: 1 or its variants, SEQ ID NO: 6 or its variants, SEQ ID NO: 7 or its variants, and SEQ ID NO: 3, sequence number This includes one or more of sequence number 14 and sequence number 18. In some embodiments, nucleic acid molecules encoding proteins are provided, including sequence number 2 and one or more of sequence number 11, sequence number 15, and sequence number 19.

[0071] In some embodiments, the disclosure provides nucleic acid molecules encoding fusion inhibitors, including ALDH1 or its variants, the shCCR5 molecule or its variants, and / or immobilized fusion inhibitors.

[0072] In some embodiments, a single nucleic acid molecule, such as a single vector, is used to encode or express each of the nucleic acid molecules or proteins provided herein. In some embodiments, a single lentivirus comprises a nucleic acid sequence provided herein. In some embodiments, a lentivirus is provided comprising a single expression construct encoding each of ALDH1 or its variants, the shCCR5 molecule or its variants, and / or a fusion inhibitor. Non-limiting examples of vectors comprising the various elements described herein are shown in Figures 4, 5, 6, 11, and 12. The promoters and response elements functionally linking the nucleic acid molecules encoding ALDH1, shCCR5, and the fusion inhibitor (including immobilized fusion inhibitors) are non-limiting, and other promoters and response elements may also be used. Those skilled in the art will understand that the different promoters described are interchangeable.

[0073] Non-limiting examples of nucleic acid sequences that can be used as viral vectors or as a basis for forming lentiviruses include, for example, the nucleic acid sequences containing SEQ ID NOs. 2, 4, and 5.

[0074] In some embodiments, the nucleic acid molecule includes 5'LTR and 3'LTR adjacent to the nucleic acid molecule encoding ALDH1, shCCR5, and / or a fusion inhibitor protein. To leave no doubt, the shCCR5 sequence and the fusion inhibitor sequence can be replaced with other sequences of interest used for co-expression with ALDH1. Thus, in some embodiments, the nucleic acid molecule includes the sequence encoding ALDH1 and the sequence of interest, which may be, for example, any other protein, antisense, miRNA, or other nucleic acid molecule desired for expression in bone marrow or the cell types provided herein.

[0075] Therefore, in some embodiments, a method is provided which includes administering cells expressing ALDH1 and the molecule of interest to an individual, and administering a non-myeloablative dose of a chemotherapeutic agent (e.g., cyclophosphamide) to the target.

[0076] In some embodiments, a method is provided for treating HIV in a subject, comprising administering to a population of cells heterologously expressing ALDH1 and one of the following: i) at least one HIV coreceptor mutant, one or more mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of the HIV coreceptor, or heterologous nucleotide molecules encoding any combination thereof. In some embodiments, the method comprises administering at least one dose of a non-myeloablative chemotherapeutic agent. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are homogeneous to the subject. In some embodiments, the cells express shCCR5, shCXCR4, and / or C-peptide fusion inhibitors. In some embodiments, the cells include nucleic acid molecules containing sequences 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof. In some embodiments, the cells include nucleic acid molecules encoding sequences 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof. In some embodiments, the cells are CD34+ and / or CD4+, or as specified herein. It is something else that is offered.

[0077] In some embodiments, a method is provided for expressing the molecule of interest in a subject, comprising administering ALDH1 and cells heterologously expressing the molecule of interest to the subject, and administering a non-myeloablative dose of cyclophosphamide. In some embodiments, the cells are CD34+ and / or CD4+, or other as provided herein. In some embodiments, the molecule of interest is a molecule that reduces the expression of CCR5; a molecule that reduces the expression of CXCR4; a molecule that encodes the expression of a C-peptide fusion inhibitor; or any combination thereof. In some embodiments, the molecule of interest that reduces the expression of CCR5 is shCCR5. In some embodiments, the molecule includes a nucleic acid molecule that includes or encodes SEQ ID NO: 6 and / or SEQ ID NO: 7. In some embodiments, the C-peptide fusion inhibitor includes the sequence of SEQ ID NO: 11, the sequence of SEQ ID NO: 15, the sequence of SEQ ID NO: 19, or any combination thereof.

[0078] It will be understood that a variety of sequences are provided herein. In addition to the exact sequences, sequences that are variants of the disclosed sequences are also provided. In some embodiments, sequences are provided that have at least, about, or exactly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology or identity with respect to the sequences mentioned. Those skilled in the art will readily understand how to determine the homology of two proteins or nucleic acids. For example, homology can be calculated after aligning the two sequences so that their homology is at its highest level. In some embodiments, the calculation of homology can be performed by the published algorithm. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Adv.Appl.Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J.MoL Biol.48:443 (1970), the search for similarity method of Pearson and Lipman, Proc.Natl.Acad.Sci.USA85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection. The same type of homology can be obtained for nucleic acids by algorithms disclosed, at least for materials relating to nucleic acid alignment, as incorporated herein by reference, for example, Zuker, M. Science 244: pp. 48-52, 1989; Jaeger et al., Proc. Natl. Acad. Sci. USA 86: pp. 7706-7710, 1989; Jaeger et al., Methods Enzymol. 183: pp. 281-306, 1989.For example, using Blastn or BlastP with default settings, you can align two sequences using the website maintained by the National Center for Biotechnology Information.

[0079] For example, as used herein, a sequence referred to as having a particular percentage of homology to another sequence refers to a sequence that has the homology referred to when calculated by one or more of the calculation methods described above. For example, if the first sequence is calculated to have 80 percent homology to the second sequence using Zuker's calculation method, then, even if the first sequence does not have 80 percent homology to the second sequence when calculated by any of the other calculation methods, the first sequence is defined herein as having 80 percent homology to the second sequence. As another example, using both Zuker's calculation method and the Pearson and Lipman calculation methods... If a first sequence is calculated to have 80 percent homology to a second sequence, then, as defined herein, the first sequence has 80 percent homology to the second sequence, even if calculations using the Smith and Waterman method, the Needleman and Wunsch method, the Jaeger method, or any other method do not show that the first sequence has 80 percent homology to the second sequence. As yet another example, if using each of the calculation methods, the first sequence is calculated to have 80 percent homology to the second sequence, then, as defined herein, the first sequence has 80 percent homology to the second sequence (however, in practice, different calculation methods often yield different results for the homology percentage).

[0080] Cells and compositions In certain embodiments, the above-mentioned chemotherapy-resistant modified cells related to the disclosed method, and the use of these cells in the disclosed method, are provided herein. Also provided are methods for generating these cells by incorporating one or more modifications conferring chemotherapy resistance into suitable cells, and optionally by incorporating one or more further modifications not related to chemotherapy resistance, such as further HIV / disease-specific modifications.

[0081] Furthermore, in certain embodiments, compositions are provided herein that include a composition for use in a method provided herein, comprising at least one chemotherapy-resistant modified cell provided herein. In some embodiments, the composition further includes pharmaceutically acceptable excipients, diluents, carriers, or any combination thereof.

[0082] The composition may include pharmaceutically acceptable excipients, pharmaceutically acceptable salts, diluents, carriers, media, and other inert agents known to those skilled in the art. Commonly used media and excipients in pharmaceutical preparations include, for example, talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, and the like. Solutions can be prepared using water or physiologically compatible organic solvents, such as ethanol, 1,2-propylene glycol, polyglycol, dimethyl sulfoxide, aliphatic alcohol, triglycerides, partial esters of glycerin, and the like. Compositions can be prepared using conventional techniques, including sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycol mixed with water, Ringer's solution, and the like. In one embodiment, colorants are added to facilitate the identification of the composition's location and precise placement of the composition at the intended treatment site.

[0083] The composition may contain preservatives and / or stabilizers. Non-limiting examples of preservatives include methylparaben, ethylparaben, propylparaben, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride, benzyl alcohol, thimerosal, phenylmercury salts, chlorhexidine, phenol, 3-cresol, quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, and imidourea.

[0084] To adjust the tonicity, the composition may contain physiological salts such as sodium salts. Sodium chloride (NaCl) is preferred, and its concentration can range from 1 to 20 mg / ml. Other possible salts include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, and calcium chloride.

[0085] The composition may contain one or more buffers. Typical buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer; or citrate buffer. The buffers are typically included at concentrations ranging from 5 to 20 mM. The pH of the composition is generally between 5 and 8, more typically between 6 and 8, for example, between 6.5 and 7.5, or between 7.0 and 7.8.

[0086] In some embodiments, the composition may include a cryoprotectant. Non-limiting examples of cryoprotectants include glycols (e.g., ethylene glycol, propylene glycol, and glycerin), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combination thereof.

[0087] In one embodiment, the cells are part of a population of cultured cells (i.e., in vitro). In another embodiment, the cells are part of a population of cells of interest (i.e., in vivo). For example, modified cells and / or non-myeloablative doses of chemotherapeutic agents are delivered to in vivo cells or to a population of in vivo cells forming a tissue or organ within interest for the purpose of treating or preventing HIV or the disease of interest. Alternatively, modified cells and / or non-myeloablative doses of chemotherapeutic agents are delivered to cultured cells or a population of cultured cells for the purpose of conducting experiments to study their effects on a particular type of cell.

[0088] The composition can be included in implantable devices. Suitable implantable devices considered in this invention include intravascular stents (e.g., self-expanding stents, balloon-expanding stents, and stent grafts), scaffolds, grafts, and similar devices. Such implantable devices may be coated on at least one side with a composition capable of treating or preventing HIV or other diseases, or may be impregnated with a composition capable of treating or preventing HIV or other diseases.

[0089] Sequences referred to herein are those in the following table or equivalent:

[0090] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0091] The composition can be administered to the subject by any suitable form and route. Non-limiting examples include internal, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intra-arterial, intramuscular, intraperitoneal, intradermal, and subcutaneous routes. The composition is also suitable for transdermal delivery as part of a cream, gel, or patch. Other forms of administration include tablets; capsules; pills; powders; aerosols; suppositories; parenteral drugs; and oral liquids including suspensions, solutions, and emulsions. Sustained-release forms of administration can also be used. Non-myeloablative doses of chemotherapeutic agents can be administered orally or by other methods provided herein.

[0092] In some embodiments, the embodiments provided herein also include, but are not limited to, the following:

[0093] 1. A method for performing bone marrow transplantation in a patient, comprising modification of cyclophosphamide resistance. The method comprises administering to a patient a population of cells and at least one non-myeloablative dose of cyclophosphamide.

[0094] 2. The method of Embodiment 1, wherein the population of cyclophosphamide-resistant modified cells contains a heterologous gene encoding aldehyde dehydrogenase 1 (ALDH1).

[0095] 3. The method of Embodiment 1, wherein a population of cyclophosphamide-resistant modified cells expresses ALDH1.

[0096] 4. The method of Embodiment 1, wherein cyclophosphamide resistance in modified cells is conferred by the expression of aldehyde dehydrogenase 1 (ALDH1).

[0097] 5. Any one of Embodiments 1 to 4, wherein more than 50% of the patient's bone marrow is replaced with cyclophosphamide-resistant modified cells or cells derived therefrom within 6 months.

[0098] 6. The patient has HIV, according to any one of embodiments 1 to 5.

[0099] 7. Cyclophosphamide resistance in modified cells is transient, according to any one of Embodiments 1 to 6.

[0100] 8. The method according to any one of Embodiments 1 to 7, wherein the cells are stem cells or immune cells.

[0101] 9. The method of Embodiment 8, wherein the stem cells are embryonic stem cells, umbilical cord blood-derived stem cells, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), artificial PSCs (iPSCs), embryonic stem cells (ESCs), or cells derived from these, such as CD34+ cells, CD90+ cells, CD45+ cells, CD17+ cells, CD45RA- cells, CD38- cells, or any combination thereof.

[0102] 10. The method of Embodiment 8, wherein the immune cells are T cells.

[0103] 11. The modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof, according to any one of Embodiments 1 to 10.

[0104] 12. Any one of Embodiments 1 to 11, further comprising contacting unmodified cells with an expression vector encoding ALDH1 expression to produce cyclophosphamide-resistant modified cells.

[0105] 13. The method of Embodiment 12, wherein the expression vector is a viral vector or a non-viral vector.

[0106] 14. The method of Embodiment 13, wherein the viral vector is a lentiviral vector or an adenovirus vector.

[0107] 15. The method of Embodiment 12, wherein the expression vector is a retrovirus, a transposon, an episomal expression vector, modified RNA, a plasmid, or any combination thereof.

[0108] 16. A non-myeloablative chemotherapeutic agent of at least one dose is administered after the administration of modified cells. One of the methods of embodiment 1 to 15, administered to [the subject].

[0109] 17. Any one of Embodiments 1 to 16, wherein at least one non-myeloablative dose of the chemotherapeutic agent is a non-myeloablative dose of cyclophosphamide.

[0110] 18. The method of Embodiment 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 0.16 mg / kg / day to less than 2.5 mg / kg / day.

[0111] 19. The method of Embodiment 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 0.41 mg / kg / day to approximately 1.63 mg / kg / day.

[0112] 20. The method of Embodiment 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 0.81 mg / kg / day to approximately 1.46 mg / kg / day.

[0113] 21. The method of Embodiment 17, in which the non-myeloablative dose of cyclophosphamide is approximately 1.3 mg / kg / day.

[0114] 22. The patient received 100 mg / m². 2 One of embodiments 17 to 21, wherein cyclophosphamide is not administered at a daily dose over a continuous period of 1 to 14 days.

[0115] 23. The patient should take 5-7 g / m². 2 One of embodiments 17 to 22, wherein a dose of cyclophosphamide is not administered over a period of 12 to 24 hours.

[0116] 24. A method according to any one of Embodiments 1 to 23, in which a non-myeloablative chemotherapeutic agent is administered daily for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months.

[0117] 25. A method according to any one of Embodiments 1 to 24, further comprising refraining from administering a non-myeloablative dose of a chemotherapeutic agent for a period of time between the administration of cyclophosphamide-resistant modified cells and the administration of at least one dose of a non-myeloablative chemotherapeutic agent.

[0118] 26. The method of Embodiment 25, wherein the period is selected from the group consisting of approximately 3 days, approximately 7 days, approximately 10 days, and approximately 14 days.

[0119] 27. Any one of Embodiments 1 to 26, wherein more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells.

[0120] 28. The method according to any one of embodiments 1 to 27, wherein the patient does not become myeloablative and / or immunodeficient as a result of being administered at least one dose of a non-myeloablative chemotherapeutic agent.

[0121] 29. The method according to any one of Embodiments 1 to 28, wherein the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, hypothrombocytopenia, hypoleukocytes, or any combination thereof or related symptoms.

[0122] 30. The method according to any one of Embodiments 1 to 29, wherein the pre-preparation step is performed before administering the cells.

[0123] 31. The method of Embodiment 30, wherein the pre-adjustment step is a non-myeloablative chemotherapy pre-adjustment step.

[0124] 32. The method according to any one of Embodiments 1 to 31, wherein the modified cells are resistant to HIV infection.

[0125] 33. The method of Embodiment 32, wherein the modified cells heterologously express at least one mutation of an HIV coreceptor that is resistant to HIV infection, one or more mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of an HIV coreceptor, or any combination thereof.

[0126] 34. The method of Embodiment 32, wherein modified cells heterologously express shCCR5, shCXCR4, a C-peptide fusion inhibitor, or any combination thereof.

[0127] 35. The method of Embodiment 32, wherein the modified cells do not express the HIV coreceptor.

[0128] 36. The method of Embodiment 32, wherein the modified cells either do not express CCR5, CXCR4, or express CCR5-Δ32, or a combination thereof.

[0129] 37. ALDH1, and i) a heteronucleotide molecule encoding at least one HIV coreceptor mutant, one or more mutations in at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of HIV coreceptors, or any combination thereof; and / or ii) one of the mutations or deletions of endogenous HIV coreceptors. Cells containing heterologous nucleotide molecules that encode the expression of [a specific gene / substance].

[0130] 38. Cells of Embodiment 37, comprising a heterogeneous nucleotide sequence that is i. encoding a molecule that reduces the expression of CCR5; ii. encoding a molecule that reduces the expression of CXCR4; iii. encoding the expression of a C-peptide fusion inhibitor; iv. comprising the sequence 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof; v. encoding the sequence 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof; or any combination thereof.

[0131] 39. Cells of Embodiment 37 or 38 expressing a C-peptide fusion inhibitor such as shCCR5, shCXCR4, and / or C44.

[0132] 40. Cells of Embodiment 37, modified to express heterologous nucleotide sequences using a nonviral gene transfer system.

[0133] 41. The nonviral gene transfer system is a transposon gene transfer system, in the cell of embodiment 40.

[0134] 42. The cell of Embodiment 37, wherein the transposon gene transfer system is the Sleeping Beauty gene transfer system or the PiggyBac transposon gene transfer system.

[0135] 43. A cell from any one of embodiments 37 to 42, containing the heterogeneous nucleic acid sequence of Sequence ID No. 4.

[0136] 44. A composition comprising one or more cells from any one of embodiments 37 to 43.

[0137] 45. ALDH1, and i) One heteronucleotide molecule encoding at least one HIV coreceptor mutant, at least one single mutation or multiple mutations in the HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of the HIV coreceptor, or any combination thereof. A nucleic acid molecule that codes for something.

[0138] 46. ​​A nucleic acid molecule of Embodiment 45, which encodes a molecule whose heterogeneous nucleotide sequence is i. a molecule that reduces the expression of CCR5, ii. a molecule that reduces the expression of CXCR4; a molecule that encodes the expression of a C-peptide fusion inhibitor; or any combination thereof.

[0139] 47. The nucleic acid molecule of Embodiment 45 or 46, which encodes the expression of shCCR5, shCXCR4, and / or a C-peptide fusion inhibitor.

[0140] 48. A nucleic acid molecule of Embodiment 45, wherein the molecule comprises the sequence 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

[0141] 49. A nucleic acid molecule of Embodiment 45, wherein the molecule comprises a nucleic acid molecule encoding sequence 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

[0142] 50. A vector containing one nucleic acid molecule from any one of embodiments 45 to 49.

[0143] 51. A vector of embodiment 50, which can be used to produce lentiviruses.

[0144] 52. A lentiviral vector, the vector of embodiment 50.

[0145] 53. A vector of Embodiment 50 comprising sequences of sequence numbers 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

[0146] 54. A vector of Embodiment 50 comprising a nucleic acid molecule encoding sequence numbers 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

[0147] 55. ALDH1, and i) One heteronucleotide molecule encoding at least one HIV coreceptor mutant, at least one single mutation or multiple mutations in the HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of the HIV coreceptor, or any combination thereof. A population of cells that heterologously express; and At least one non-myeloablative dose of chemotherapeutic agent A method of treating HIV in a subject, including administering it to the subject.

[0148] 56. The method of Embodiment 55, wherein cells express shCCR5, shCXCR4, and / or C-peptide fusion inhibitors.

[0149] 57. The method of Embodiment 55, wherein the cells comprise nucleic acid molecules comprising sequences of 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

[0150] 58. The method of Embodiment 55, wherein the cells include nucleic acid molecules encoding sequences of SEQ ID NOs: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

[0151] 59. A method for expressing a target molecule in a subject, comprising administering cells that heterologously express ALDH1 and the target molecule, and administering a non-myeloablative dose of cyclophosphamide.

[0152] 60. The method of Embodiment 59, wherein the cells are CD34+ and / or CD4+, or other as provided herein.

[0153] 61. The method of Embodiment 59, wherein the target molecule is one that reduces the expression of CCR5; reduces the expression of CXCR4; encodes the expression of a C-peptide fusion inhibitor; or any combination thereof.

[0154] 62. The method of Embodiment 61, in which the molecule intended to reduce the expression of CCR5 is shCCR5.

[0155] 63. The method of Embodiment 62, wherein the molecule comprises a nucleic acid molecule containing or encoding SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0156] 64. The method of Embodiment 61, wherein the C-peptide fusion inhibitor comprises the sequence of SEQ ID NOs: 11, 15, 19, or any combination thereof.

[0157] The following examples are illustrative and do not limit the compounds, compositions, and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments. [Examples]

[0158] Graft efficiency in autologous bone marrow transplantation in mice method Bone marrow isolation. Bone marrow was extracted from the femurs and tibias of syngeneic donor mice aged 8 to 16 weeks using Iskov-modified Dulbecco's medium (IMDM) containing 0.5 mM EDTA, 2% fetal bovine serum (FBS), and antibiotics. Cells were washed with a buffer containing 5 mM EDTA and bovine serum albumin (phosphate-buffered saline [PBS]).

[0159] Transduction of bone marrow cells. Mouse bone marrow cells were transduced at an MOI of 10 using either a lentiviral vector expressing only EGPF (i.e., pLV-Puro-EF1A-EGFP ("control vector")) or a lentiviral vector expressing EGFP and human ALDH1A (i.e., pLV-Puro-EF1A-EGFP-hALDH1A, Figure 4, Sequence ID No. 2 ("test vector")). Transduction efficiency was assessed 48 hours after transduction. The cells were then transplanted into irradiated mice as described below.

[0160] Transplantation. Syngeneic recipient mice (Balb / c) were pre-treated with 0.5 mg / kg fludarabine on day -5 and day -4, cyclophosphamide on day -2, and busulfan on day -1. On the day of transplantation (day 0), the mice were anesthetized and given 4 × 10⁴ 6 Individual bone marrow cells were transplanted via tail vein injection. The recovery of hematopoiesis in the transplanted tissue was monitored by GFP FACS analysis.

[0161] Treatment with cyclophosphamide. Seven days after transplantation, mice were given different doses of CTX: 0 mg / kg ("0 mg / kg post-transplant CTX") and 16 mg / kg ("16 mg / kg post-transplant CTX"). Post-transplant CTX (30 mg / kg) or 30 mg / kg ("30 mg / kg post-transplant CTX") was administered once daily via intraperitoneal (ip) injection. During this study, treatment consisted of 5 weeks of CTX, 1 week without CTX, and the following 1 week of CTX treatment, i.e., a total of 6 weeks of CTX treatment.

[0162] GFP analysis of peripheral blood. 50 microliters of peripheral blood from each mouse were collected from the retroorbital or tail vein and mixed with 1 ml of PBS containing 0.5 mM EDTA. Cells were erythrocyte-lysed, washed in PBS, and then further diluted in 400 μl of PBS. Live GFP + The percentage of granulocytes was analyzed using a flow cytometer.

[0163] Bone marrow terminal analysis. Six weeks after CTX treatment, animals were slaughtered and GFP was detected in the bone marrow. + The percentage of cells was assessed by flow cytometry. Granulocytes / neutrophils were identified using conventional forward scatter versus side scatter dispersion, where cells were plotted according to light reflection, size, and internal complexity (granularity). There were n=3 in each of the groups without post-transplant CTX treatment and n=6 in all other groups. After 6 weeks, the white blood cell count in the blood was determined for each mouse. The results showed the presence of outliers in each of the CTX-treated groups, and these data were excluded from the statistical analysis. Therefore, there were n=3 in each of the groups without post-transplant CTX treatment and n=5 in all other groups.

[0164] Results and Discussion When observing bone marrow regeneration after transplantation, peripheral blood can provide an indicator of transplant success, but it is necessary to understand the cellular complexity of immune cells before transplantation, as bone marrow produces various immune cells with different lifespans. For example, lymphocytes can live for about 180 days, while neutrophils live for only 5-7 days. Furthermore, monocytes can only survive for a few days in the periphery, but they can also become tissue-resident and extend their lifespan to several months. Therefore, when attempting to analyze lymphocyte populations for donor bone marrow reconstruction, an accurate reflection cannot be obtained until all of the donor's pre-transplant lymphocytes have died, i.e., six months after this procedure. To provide a more "real-time" reading of the recipient cell bone marrow graft, granulocyte populations provide the best indicator. Since all of the donor's neutrophils die within 7 days of transplantation, at 2 weeks post-transplant, these cells are the most direct reflection of the bone marrow environment and the success of the recipient transplant, which can also be measured by identifying the percentage of transduced neutrophils, which reflects the graft rate.

[0165] According to the above method, on the 23rd, 35th, and 42nd days of the study (the 16th, 28th, and 35th days after CTX administration, respectively), blood was collected by bleeding from behind the eye socket, and GFP in the peripheral blood of each mouse + The percentage of granulocytes was evaluated by flow cytometry.

[0166] The results showed that all mouse groups contained live GFP + granulocytes (Figure 1). Furthermore, on the 42nd day of the study, mice administered bone marrow cells transduced with the test vector (i.e., cells expressing EGFP + ALDH1A1) and treated with 16 mg / kg / day of CTX showed a similar percentage of live GFP + granulocytes compared to mice administered bone marrow cells transduced with the test vector and treated with 30 mg / kg / day of CTX (compare "test vector, CTX after transplantation at 16 mg / kg" and "test vector, CTX after transplantation at 30 mg / kg" in Figure 1).

[0167] After 6 weeks, the results demonstrated the presence of total GFP + cells (live and dead cells) (Figure 2A). The graft was shown by the percentage of the live GFP + granulocyte / neutrophil population (Figure 2B). Surprisingly, bone marrow cells transduced with the test vector (i.e., Both mice administered with EGFP+ALDH1A1-expressing CTX and treated with non-myeloablative 16 mg / kg / day CTX, and mice administered with transduced myelocytes using the test vector and treated with myeloablative 30 mg / kg / day CTX, demonstrated similar grafts of over 40% (Figure 2B, compare "Test vector, 16 mg / kg pt transplantation with CTX" and "Test vector, 30 mg / kg pt transplantation with CTX"). Furthermore, mice administered with transduced myelocytes using the test vector and treated with 16 mg / kg / day CTX had a significantly higher graft percentage compared to mice administered with transduced myelocytes using the control vector and treated with the same dose of CTX (i.e., 16 mg / kg / day CTX) (Figure 2B, compare "Test vector, 16 mg / kg pt transplantation with CTX" and "Control vector, 16 mg / kg pt transplantation with CTX").

[0168] White blood cells (WBCs) help fight infections caused by attacks from foreign factors entering the body. WBC counts can be useful in detecting hidden infections and / or susceptibility to infection. Peripheral blood WBC counts were determined for each study group after 6 weeks of CTX treatment. As shown in Figure 3, mice administered transduced bone marrow cells with either a control vector or a test vector and not treated with CTX had WBC counts within the normal range. There was one outlier in each CTX treatment group, which was removed in the statistical analysis. The high, normal, and low WBC ranges follow known ranges in the art, provided for male mice in Charles River Research Models (BALB / C Mouse Hematology, North American Colonies, January 2008–December 2012), http: / / www.criver.com / files / pdfs / rms / balbc / rm_rm_r_balb-c_mouse_clinical_pathology_data.aspx. In particular, mice administered bone marrow cells transduced with the test vector and treated with 16 mg / kg / day of CTX had a significantly larger number of WBCs compared to mice administered bone marrow cells transduced with the test vector and treated with 30 mg / kg / day of CTX (see Figure 3, compare "Test vector, 16 mg / kg pt transplantation with CTX" and "Test vector, 30 mg / kg pt transplantation with CTX"). [Examples]

[0169] In vivo humanized mouse research #1 Stem cell purification. Human PBMCs were collected from healthy donors treated with 5 mcg / kg / day of granulocyte colony-stimulating factor (G-CSF) (Amgen, Thousand Oaks, CA) for 5 days, followed by leukocytapheresis on days 5 and 6. Patients were then administered a regimen of 10 μg / kg of filgrastim or G-CSF to determine CD34 levels. + Stem cells were mobilized from the bone marrow to the periphery for isolation. CD34 in peripheral blood. +If the cell count exceeded 10.0 to 20.0×10 per kg of body weight, 6 apheresis was performed. CD34 + was purified using positive selection enrichment and then magnetic bead isolation was performed. The samples were washed in PBS containing 2% FBS, centrifuged, and resuspended in PBS. CD34+ cells were then tested for purity by FACS analysis with CD34 staining and viability was evaluated by trypan blue staining. The cells were then transplanted into mice immediately. Appropriate written informed consent in accordance with the Helsinki Declaration protocol was obtained from the donor prior to collection.

[0170] Transplantation of human HSCs into NOG mice. Adult mice were 10 - 12 week-old NOG mice. For neonatal mice, mice 1 - 2 days after birth were used. Mice were irradiated 1 day prior to cell transfer under SPF conditions at 2 - 2.5 Gy for adult mice and 1 Gy for neonates. Mice weighing less than 18 g may die with this dose of irradiation. For adult mice, 0.25 mL (1 - 0.5×10 4 cells) of the cell suspension was injected into the mice via the tail vein using a 1 mL syringe with a 27G needle or a microinjector syringe with a 29G needle while slightly anesthetizing with isoflurane.

[0171] Transduction of stem cells. CD34 + cells from the same donor were cultured in X-Vivo 10 medium (Lonza) coated with fibronectin in a test tube supplemented with stem cell factor (SCF) 100 ng / ml, thrombopoietin (TPO), and Fms-like tyrosine kinase 3 ligand (Flt-3L) (CellGenix, Freiburg, Germany), and optionally IL-2, IL-3, IL-6, or any combination thereof, at 37 °C and 5% CO2 for 16 hours at 1×10 6 cells / ml. The pre-stimulatory medium was then removed and the multiplicity of infection (MOI) corresponding to between MOI 2 and MOI 50, 1.35×10 8Fresh culture medium containing a tu / ml vector ("control vector" = pLV-Puro-EF1A-EGFP, or "test vector" = SEQ ID NO: 4, Figure 5) and 4 mg / ml of protamine sulfate (Sigma, Saint Louis, MO) was added. This transduction mixture was returned to the incubator for 12 to 48 hours. Transduced cells were detached with trypsin (Lonza, Walkersville, MD), washed, resuspended in PBS, and immediately transplanted into mice (without irradiation, as described above).

[0172] Peripheral blood GFP analysis, as well as terminal analyses of the spleen, blood, and bone marrow, were performed as described above. A schematic diagram of the study is shown in Figure 8, but this depiction is for illustrative purposes only, and other study designs may be used. [Examples]

[0173] In vivo humanized mouse research #2 Stem cell purification. Human PBMCs were collected from healthy donors treated with granulocyte colony-stimulating factor (G-CSF) (Amgen, Thousand Oaks, CA) 5 mcg / kg / day for 5 days, followed by leukocytapheresis on days 5 and 6. CD34 + The samples were purified by positive selection using immunomagnetic beads. The samples were washed in PBS containing 0.5% HSA, centrifuged, and resuspended in PBS. CD34 + The cells were then tested for purity by flow cytometry analysis with CD34 staining, and staining for viability was performed. The cells were then immediately transplanted into mice. Appropriate written informed consent was obtained from the donors prior to retrieval, in accordance with the Declaration of Helsinki protocol.

[0174] Transplantation of human HSCs into NOG mice. For adult mice, 10-12 week old NOG mice were used. For neonatal mice, mice 1-2 days after birth were used. Mice were irradiated with 2-2.5 Gy for adult mice and 1 Gy for neonatal mice under SPF conditions, one day before cell transfer. Mice weighing less than 18 g may die at this dose of radiation. For adult mice, 0.25 mL of cell suspension (1-0.5 × 10⁶) was used. 4 The individual doses were injected into mice via the tail vein using a 1 mL syringe with a 27G gauge.

[0175] Transfusion of stem cells. CD34 from the same donor. + Cells were incubated in fibronectin-coated test tubes in X-Vivo 10 medium (Lonza) supplemented with stem cell factor (SCF) 100 ng / ml, thrombopoietin (TPO), and Fms-like tyrosine kinase 3 ligand (Flt-3L) (CellGenix, Freiberg, Germany), and optionally IL-3 and IL-6, for 16 hours at 37°C and 5% CO2, at a rate of 1 × 10⁶ cells. 6 Cells were cultured at individual cells / ml. The preliminary stimulating medium was then removed, and 1.35 × 10⁶ cells were used, corresponding to the multiplicity of infection (MOI) between MOI 2 and MOI 50. 8 ul / ml vector ("control vector" = pLV-Puro-EF1A-EGFP) Fresh culture medium containing, or "test vector") was added. A suitable "test vector" contained one or a combination of the following:

[0176] [Table 14]

[0177] The transduction mixture was returned to the incubator for 12 to 48 hours. Transduced cells were harvested, washed, resuspended in PBS, and immediately transplanted into mice (without irradiation, as described above).

[0178] Peripheral blood GFP analysis, as well as terminal analyses of the spleen, blood, and bone marrow, were performed as described above. A schematic diagram of the study is shown in Figure 8, but this depiction is for illustrative purposes only, and other study designs may be used. [Examples]

[0179] Proof of Conceptual Study in Lentiviruses Lentiviral vector expression. Human CD34 + Cells were transduced using a lentiviral (LV) vector containing four expression cassettes. ALDH and C-peptide expression levels, shRNA-mediated CCR5 knockdown, and optionally shRNA-mediated CXCR4 knockdown were evaluated in the transduced cells. Cellular C-peptide expression was then correlated with CCR5 knockdown.

[0180] In short, recruited CSF, cryopreserved human CD34+ cells, were thawed at 37°C until the contents of the vial were completely liquid. The cells were transferred to sterile 15 mL conical tubes (Corning, Tewksbury, MA). Pre-warmed X-Vivo 10 serum-free medium (Lonza, Basel, Switzerland) was added dropwise and with gentle agitation up to 15 mL. The cells were then centrifuged at 200-300 × g for 5 minutes. After aspirating the supernatant, the pellet was resuspended in 10 mL of medium, and small aliquots were retained for counting and viability assessment. The cells were then centrifuged, the supernatant was aspirated, and 1.0 × 2.0 × 10¹⁶ cells were measured. 6 Resuspend to a density of individual cells / m, and then T75cm 3 The tissue was transferred to tissue culture flasks (Corning, Tewksbury, MA).

[0181] Cells were pre-stimulated by adding 100 ng / mL each of stem cell factor (SCF), thrombopoietin (TPO), and flt3 / flk2 ligand (Flt3L) (R&D Systems, Minneapolis, MN) to the culture medium, and then cultured in a 5% CO2 incubator at 37°C for 24 hours. The cells were then removed from the incubator and centrifuged. The cells were then subjected to 1.0 × 10⁶ doses in X-Vivo 10 medium supplemented with 100 ng / mL each of SCF and Flt3L, 10 ng / mL of TPO, and 60 ng / mL of IL-3. 6 The cells were resuspended at a density of individual cells / mL. Untreated retronectin (5 μg / cm³) was used to culture 12 wells of tissue. 2 ) Covering plate (Takara-bai O Corporation, Shiga Prefecture, Japan) 1.0 x 10 per well 6 Individual cells were then seeded.

[0182] Lentivirus particles were thawed at 37°C (CCR5.C peptide.LV or CCR5.C peptide.ALDH.LV), gently mixed during thawing. Each particle was added to the culture at approximately 2 to 50 MOI, e.g., 9, and mixed by gently swirling the plate. The cells were then returned to a 37°C 5% CO2 incubator and cultured for 24 to 48 hours. The samples were then retained for evaluation of cell number, viability, and transduction efficiency by flow cytometry and qRTPCR.

[0183] The remaining cells were centrifuged, the supernatant was gently aspirated, and 1 mL / well of fresh X Vivo 10 medium was added to each well. The plate was returned to the incubator. The cells were grown for up to 3 days after transduction.

[0184] Flow cytometry staining and analysis were performed immediately. If the LV construct contained a fluorescent marker, it was taken up using a MacsQuant FACS Analyzer. If the vector did not contain a fluorescent marker, the target protein or HIV-1 was identified. A staining procedure was performed to activate LTR transcription.

[0185] Further characterization can be performed using the cells. If further characterization is not required, immediately after transduction (or post-transduction growth), the cells should be placed in a control-rate freezer in a solution consisting of 50% IMEM (Thermo Fisher Scientific, Carlsbad, CA), 45% human serum albumin (HAS) (Sigma-Aldrich, St. Louis, MO), and 5% DMSO filtered through 0.2 μm, for a total of 5 × 10⁶ cells. 6 The cells were cryopreserved at a density of individual cells / mL. The cells can then be immediately transferred to liquid nitrogen or transported on dry ice.

[0186] Chemical protection. Human CD34 transduced by an LV vector expressing ALDH (e.g., SEQ ID NO: 2 or SEQ ID NO: 5). + Cells were treated with various doses of cyclophosphamide to demonstrate resistance to chemotherapeutic agents. The experiment can also be performed by treating mixed cultures of transduced and untransduced cells with various doses of cyclophosphamide. The viability of both transduced and untransduced cells was measured after treatment with each dose of cyclophosphamide.

[0187] In short, lyophilized maphosphamide was resuspended in sterile water in a 1 mg / mL working stock solution. Transduced cells were then washed once with standard medium (RPMI + 10% FBS), and transduced cells (both test vector (e.g., SEQ ID NO: 4) and control vector (e.g., SEQ ID NO: 5)) were placed in MethoCult 3330 medium (StemCell Technologies, Vancouver, BC, Canada) in a 0.5 × 10⁶ solution. 6The cells were resuspended to a density of individual cells / mL. The final solution consisted of 0.8% methylcellulose in α-agar supplemented with FBS, erythropoietin (1 U / ml), IL-3 (200 U / ml), IL-6 (200 U / ml), SCF (100 ng / ml), IL-1β (200 ng / ml), and 100 ng / ml granulocyte colony-stimulating factor (StemCell Technologies).

[0188] Maphosphamide was added to each set of dishes (control dish and test dish) to the following final concentrations: 0.15, 2.5, 5, 10, 15, and 20 μM. The plates were then incubated at 37°C and 5% CO2 for 14 days. After 14 days, colonies were scored in each plate using an inverted light microscope. Mann-Whitney U tests were then performed to differentiate cells expressing the test vector from those expressing the control vector. We determined whether there was any significant statistical difference in the number of colonies formed. [Examples]

[0189] Fusion Inhibition Research T cells isolated from the spleen and thymus of humanized mice were co-cultured with HIV strains as described above. C44 expression levels were assessed using an anti-Gp41 antibody that recognizes the C peptide, detected by flow cytometry. The C peptide expression levels (positive percentage and median fluorescence intensity) were then correlated with the degree of HIV fusion blockade.

[0190] Materials and reagents. 293T cells (ATCC); pAdVAntage(Promega Corporation); pCMV4-BlaM-Vpr (Addgene); pNL4-3 proviral DNA (NIH AIDS Reagent Program), or TN6-GFP encoding primary Env; DMEM (Mediatech, Cellgro®); RPMI 1640 (Mediatech, Cellgro®); 1× phosphate-buffered saline (PBS); fetal bovine serum (FBS); 100 U / ml penicillin and 100 U / ml streptomycin (Thermo Fisher Scientific, Gibco®); 2M CaCl2; Alliance HIV-I p24 ELISA kit (PerkinElmer) or FlaQ assay reagent (Hayden et al., 2003); peripheral blood lymphocytes (PBL); CCF2-AM substrate and loading solution (Thermo Fisher Scientific); CO2-independent medium (Thermo Fisher Scientific, Gibco®); probenecid (Sigma-Aldrich); mouse anti-human CD3 conjugated to APC-Cy7 and mouse anti-human CD4 conjugated to PE-Cy7 (BD Biosciences); BD CompBeads (BD Biosciences); 16% paraformaldehyde (Electron Microscopy Sciences); HBSS (see preparation method); Dulbecco's modified Eagle medium (DMEM) culture medium (see preparation method); Roswell Park Memorial Laboratory (RPMI) culture medium (see preparation method); CCF2 loading solution (see preparation method); stock solution of probenecid (250mM) prepared in 250mM NaOH (see preparation method); growth medium (see preparation method).

[0191] Vector: pLV[Exp]-H1>hCCR5[shRNA] EF1A>hALDH1A1[NM_000689.4](ns):T2A:{C peptide} (e.g., SEQ ID NO: 4)

[0192] Production method. HBSS (280 mM NaCl; 10 mM KCl; 1.5 mM Na2HPO4; 12 mM dextrose; 50 mM N-(2-hydroxyethylpiperazine)-N'-(2-ethanesulfonic acid) (HEPES) (pH 7.05); stored at -20°C).

[0193] Flow cytometry staining buffer (1× phosphate-buffered saline (PBS) without Ca++ and Mg++; 0.5% HSA; stored at 4°C).

[0194] Dulbecco's modified Eagle's medium (DMEM) culture medium (DMEM; heat-inactivated 10% fetal bovine serum; penicillin 100 U / ml; streptomycin 100 μg / ml).

[0195] RPMI culture medium (RPMI 1640; heat-inactivated 10% fetal bovine serum; penicillin 100 U / ml; streptomycin 100 μg / ml).

[0196] CCF2 loading solution: CCF2-AM was resuspended in dimethyl sulfoxide (solution A of the CCF2 kit) to obtain a stock solution (1 mM CCF2-AM). Aliquots were made and stored at -80°C in the dark. 1 μl of 1 mM CCF2-AM was mixed with 9 μl of a solution containing 100 mg / ml of pluronic-F127 and 0.1% acetic acid (solution B of the CCF2 kit) by vortex mixing. 1 ml of CO2-independent medium was added and vortexed again. Stock solution of probenecid (250 mM) prepared in 250 mM NaOH. Aliquots were made and stored at -20°C. Growth medium (2.5 mM probenecid; 10% fetal bovine serum in CO2-independent medium).

[0197]

[0198] ​Equipment: Culture flask with a T of 175 cm²; 96-well V-bottom plate (Corning Incorporated); 5 ml, 10 ml, and 25 ml pipettes; 50 ml Falcon tubing; 0.22 μm pore size Steriflip (EMD Millipore); 2 ml Nalgene tubing (Thermo Fisher Scientific); clear ultracentrifuge tubing (BD); incubator at 37°C, 5% CO₂; ultracentrifugation equipment with SW28 rotor; flow cytometer. For the fusion assay alone, a flow cytometer with violet laser excitation (405 nm) and two measurement parameters was required. A photomultiplier tube (PMT) with a 450 / 50 nm bandpass filter, commonly used for Pacific Blue detection, was used for the detection of the cleaved CCF2 substrate. Another PMT with a 515 / 20 nm bandpass filter, commonly used for Amcyan detection, was used for the detection of the uncleaved CCF2 substrate. Further PMT (Protein Mechanism Testing) was required to measure the fluorescent dyes associated with the CD3 and CD4 antibodies. APC-Cy7 was excited with a 633 nm red laser and detected with a PMT equipped with a 755 long-pass filter. PE-Cy7 was excited with a 531 nm yellow-green laser and detected with a PMT equipped with a 755 long-pass filter.

[0199] Software. FlowJoX software (Tree Star) or other FACS analysis software.

[0200] Fusion inhibition assay. 1.5 × 10⁻⁶ 7 293T cells were cultured in 20 ml of DMEM culture medium in a T175cm² chamber. 2The cells were seeded in a tissue culture flask and cultured overnight in a humidified incubator at 37°C with 5% CO2. 1.75 ml of H2O was prepared containing 60 μg of TN6-GFP provirus DNA, 20 μg of pCMV-BlaM-Vpr, and 10 μg of pAdVAntage vector. 2 ml of 2×HBSS was then slowly added and gently mixed by pipetting up and down. 250 μl of 2M CaCl2 was added dropwise. The DNA was precipitated by incubation at room temperature for 10 minutes. The 293T cell culture medium was replaced with 40 ml of fresh DMEM culture medium preheated to 37°C. Next, 4 ml of DNA precipitate was added and incubated at 37°C for 16 hours. The medium was then replaced with 40 ml of fresh DMEM culture medium preheated to 37°C and incubated at 37°C for 24 hours.

[0201] The supernatant from transfected 293T cells was collected in a 50 ml Falcon tube and centrifuged at room temperature for 10 minutes to remove cell debris. The clarified supernatant was then filtered through a steriflip. 36 ml of virion-containing supernatant was transferred to an ultraclear centrifuge tube. The tube was placed in a bucket of an SW28 rotor balanced with DMEM culture medium as needed and ultracentrifuged at 4°C without using a brake (72000 × g, 90 minutes). The supernatant was removed, the virus pellet was resuspended in 1 ml of DMEM, divided into 100 μl aliquots, and stored at -80°C.

[0202] p24 of virus preparations Gag The content was quantified by enzyme-linked immunosorbent assay or FlaQ assay (Maiti et al., 2014).

[0203] Wash the PBLs in RPMI culture medium, count the PBLs, and then add 2 × 10⁶ PBLs to the RPMI culture medium. 7 The cells were suspended at individual cells / ml. 100 μl (2 × 10⁶) of the cell suspension was placed in a V-bottom 96-well plate for each test condition. 6The cells were divided into individual aliquots. Two further cell aliquots were distributed into two wells to serve as compensation controls. One well was loaded with the CCF2 substrate, while the other was left unloaded. These two control wells were not stained with anti-CD3-APC-Cy7 antibody and anti-CD4-PE-Cy7 antibody.

[0204] 400ng p24 Gag An amount of HIV-1 virion containing BlaM-Vpr equal to 1 was added to all wells except for the "non-infected control" and two compensation controls, and incubated at 37°C for 2 hours.

[0205] The cells were then harvested by centrifugation at room temperature for 5 minutes, washed once with 200 μl of CO2-independent medium, and centrifuged at room temperature for 5 minutes. The pellet was resuspended in 100 μl of CCF2-AM loading solution and incubated at room temperature in the dark for 1 hour, with one well being resuspended in CO2-independent medium alone to ensure it remained unstained. The cells were harvested by centrifugation at 365 × g for 5 minutes at room temperature. The harvested cells were then washed with 200 μl of growth medium and centrifuged at room temperature for 5 minutes, and the pellet was resuspended in 200 μl of growth medium and incubated at room temperature in the dark for 16 hours.

[0206] Each of the BD compensation bead vials (negative control and anti-mouse IgK) was added dropwise to two empty wells. The cells and beads were then collected by centrifugation at 4°C for 5 minutes. The cells and beads were then washed once by adding 200 μl of FACS staining buffer and collected by centrifugation at 4°C for 5 minutes. The pellet was resuspended in 100 μl of immunostaining solution (in flow cytometry staining buffer) containing 1 / 100 dilution of anti-CD3-APC-Cy7 and 1 / 50 dilution of anti-CD4-PE-Cy7. Neither the two compensation controls nor the compensation beads were stained. The CompBeads were stained with flow cytometry staining buffer containing either 1 / 100 dilution of anti-CD3-APC-Cy7 or 1 / 50 dilution of anti-CD4-PE-Cy7. The samples were incubated at 4°C for 30 minutes.

[0207] After incubation at 4°C for 30 minutes, the cells and beads were collected by centrifugation at 4°C for 5 minutes. The cells and beads were then washed with 200 μl of flow cytometry staining buffer. The cells were fixed at 4°C for 24 hours using flow cytometry staining buffer supplemented with 1.2% paraformaldehyde.

[0208] Samples were acquired using a flow cytometer-MACSQuant instrument. The sample set included: unstained cells (unloaded), unstained cells loaded with CCF2, beads stained with CD3-APC-Cy7, beads stained with CD4-PE-Cy7, a non-infectious control loaded with CCF2 and immunostained, and an infected sample loaded with CCF2 and immunostained. Data were analyzed using FlowJo. [Examples]

[0209] HIV Challenge: Human CD4 transduced by lentiviral vectors + T cells GFP reporter CD4 transduced by LV containing CCR5shRNA / c-peptide / ALDH + T cells, or T cells transduced with LV containing an empty control vector, were cultured with R5 strain, X4 strain, or both experimental HIV strains. HIV infectivity was analyzed using a reporter marker (when using a T cell reporter system) or by p24 ELISA. [Examples]

[0210] Studies on dose range and efficacy of lentiviral vectors Sca1 transduced by lentivirus containing a CCR5shRNA / C-peptide / ALDH expression vector or an empty control vector + / c-kit - Stem cells and progenitor cells were transplanted into pre-adapted C57BL / 6 syngeneic mice.

[0211] In short, after 7 days, all mice were initiated with a once-daily regimen of cyclophosphamide (CTX). Both mouse groups were treated with varying doses of CTX (6 mice per group per dose): 0, 10, 13, 16, 19, and 40 mg / kg. One week after initiating CTX treatment, blood was collected from the mice on a weekly basis. A complete blood count (CBC) panel was performed to monitor for any cytotoxic effects of chemotherapy (graft efficacy: ALDH conferring CTX resistance (conference)), while flow cytometry analysis measured the expression of lineage-specific markers, as well as C-peptide and CCR5 expression in these cells (expression efficiency: the efficacy of shRNA knocking down target genes). The study continued for at least 10 weeks of CTX treatment or until a complete bone marrow graft was performed. A schematic diagram of the study is shown in Figure 7, and a more detailed protocol is provided below.

[0212] Materials and Reagents. EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (StemCell Technologies); Falcon 15 mL conical tubes (Corning); 5 mL (12×75 mm) polystyrene round-bottom tubes (Corning); StemSpan Serum-Free Medium (StemCell Technologies); Mouse Hematopoietic Stem Cell Expansion Kit Cytokine Panel (R&D); RetroNectin Recombinant Human Fibronectin Fragment (Clontec); RPMI-1640 (Thermo Fisher Scientific); Petri dishes (Thermo Fisher Scientific); HBSS (Thermo Fisher Scientific); 27G×1 / 2 needles (BD); Centrifuge (Thermo Fisher Scientific); Sorvall ST 40R Countess II Automated Cell Counter (Thermo Fisher Scientific); Trypan blue (Thermo Fisher Scientific); Lineage Cocktail (mCD3, mGr-1, mCD11b, mB220, mTer119) (including isotype control) (Biolegend); Ly-6A / E (Sca-1) (Thermo Fisher Scientific); CD117 (c-kit) (Thermo Fisher Scientific); viability dye eFluor 506 (Thermo Fisher Scientific); anti-mouse CD16 / 32 FC Block (Biolegend); Cell Staining Buffer (Biolegend); Balb / c female mice (Charles River).

[0213] Vectors. pLV[Exp]-H1>hCCR5[shRNA]- EF1A>hALDH1A1[NM_000689.4](ns):T2A:{C peptide} (e.g., SEQ ID NO: 4)

[0214] Isolation of Progenitor Cells from Bone Marrow (Number of Progenitor Cells Required). From the following table, the number of (donor) mice required to obtain the number of cells necessary for transduction was calculated.

[0215] [Table 15]

[0216] Mouse bone marrow isolation. BALB / c mice (female, 20-25g, 8-10 weeks old) were euthanized by CO2 asphyxiation. Subsequent experimental procedures were performed in a laminar flow air biosafety cabinet (BSC). Bone marrow cells were collected from the femurs. Briefly, the bone marrow contents were flushed with 2 ml of HBSS using a 1 ml insulin syringe with a 27 G × 1 / 2 needle. The contents were collected in a sterile 50 ml centrifuge tube. The BM cell suspension collected above was then diluted to a final volume of 7.5 ml in RPMI-1640. All clusters in the bone marrow suspension were dissociated by vigorous pipetting. The cells were then centrifuged, washed, and centrifuged again. Cell pellets from each femur were gently resuspended in 7.5 ml of RPMI-1640 to prepare a homogeneous suspension. Aliquots of the cell suspension were taken using an NC-200 automated cell counter to determine the total number and viability of cells.

[0217] Isolation of progenitor cells. Transfer the isolated cells to a new tube, rotate to settle, and resuspend in EasySep buffer (PBS + 2% FBS + 1 mM EDTA) in a volume range of 0.5–2 mL, then divide into 1 × 10⁻¹⁶ cells. 8 The concentration was adjusted to individual cells / mL. Rat serum was then added to the sample at 50 μL / mL, and the sample was transferred to a 5 mL (12 × 75 mm) polystyrene round-bottom tube. The EasySep mouse hematopoietic progenitor cell isolation cocktail was then added to the sample at 50 μL per 1 mL of sample. The samples were mixed and incubated at 4°C for 15 minutes. Rapid spheres were vortexed for 30 seconds and then added to the sample at 75 μL / mL. The samples were mixed and incubated at 4°C for 10 minutes, and the total volume was increased to 2.5 mL using EasySep buffer. Progenitor cells were isolated using magnetic separation.

[0218] Evaluation of isolated progenitor cells by flow cytometry. Cells were transferred to a new 96-well plate, rotated to precipitate, and resuspended in 50 μL of FACS buffer (PBS + 2% FBS + 1 mM EDTA). 3 μL of FC Block (anti-CD16 / 32) was added to each sample and incubated at 4°C for 15 minutes. The following panels and volumes were used:

[0219] [Table 16]

[0220] The controls included: a Fluorescence Minus One (FMO) control for CD117, Ly6A / E, and a system cocktail isotype control.

[0221] Each well was increased to a total volume of 100 μL and incubated in the dark at 4°C for 30 minutes. The cells were then washed with 200 μL of flow cytometer buffer and resuspended in a given volume. A MACSQuantCytometer was used for the flow cytometer. Purity was calculated by summing the percentages of Sca1- / c-kit+, Sca1+ / c-kit-, and Sca1+ / c-kit+ and multiplying by the percentage of live strain cocktail-negative events.

[0222] Lentivirus-induced transduction and proliferation of progenitor cells. On day 0, a 12-well or 24-well plate was coated with 0.3 ml of retronectin (100 ng / ml) and incubated at room temperature for at least 2 hours. The coating medium was then aspirated from the plate, and the plate was blocked with 2% BSA in 1× PBS at room temperature for at least 30 minutes, followed by three washes with 1× PBS. The plate was now ready for immediate use. It should be noted that the plate should not be allowed to dry out.

[0223] Fresh, isolated bone marrow progenitor cells are cultured in approximately 0.2-0.4 × 10⁶ cells per 1 ml of bone marrow progenitor cell culture medium. 6The cells were seeded between individual cells. Lentivirus was added to the cells for 24 hours (MOI=3).

[0224] On day 1, the cells were rotated to precipitate, the medium containing the lentivirus was removed, and fresh bone marrow progenitor cell culture medium was added.

[0225] On the second day, after transduction with a lentivirus, puromycin (1 μg / ml) was added to the bone marrow progenitor cell culture medium to select lentivirus-infected cells. The cells were treated for 4 days.

[0226] On the fourth day, fresh culture medium containing puromycin was added.

[0227] On the sixth day, the cells were rotated to precipitate, and fresh culture medium without puromycin was added.

[0228] On day 7, the cells were harvested for cell counting, and GFP flow was performed to determine the transduction efficiency. The remaining cells were given fresh bone marrow progenitor cell culture medium.

[0229] The bone marrow progenitor cells were cultured for an additional 3 days until day 10, and the culture medium was changed on day 9.

[0230] On the 10th day, cells were collected for counting and flow cytometry analysis was performed to determine the transduction efficiency.

[0231] [Table 17]

[0232] Bone marrow progenitor cell transplantation and treatment - Pre-treatment preparation: Prior to transplantation, mice were administered two doses of fludarabine (5 mg / kg for 2 days). On day 0 (2 days after fludarabine treatment), 36 mice were transplanted via IV with stem cells and progenitor cells transduced with a control vector, and 36 mice were transplanted with stem cells and progenitor cells transduced with a test vector. After transplantation, animals were checked once daily for morbidity and mortality. At the time of routine monitoring, animals were checked for any effects of the treatment on normal behavior such as motility, dullness of the eyes / coat, and any other abnormal effects, as well as visual estimations of feed and water consumption, and weight gain / loss (weight was measured twice a week). Deaths and observed clinical signs were recorded in detail for each animal in the comments section of the data sheet.

[0233] CTX treatment. Seven days after bone marrow transplantation, CTX (cyclophosphamide) treatment was initiated as intravenous (IP) administration at the following doses: 0, 10, 13, 16, 19, and 40 mg / kg once daily for 12 weeks. This treatment was not discontinued unless weight loss >15% occurred. Details of the experimental groups and treatment are shown in the table below.

[0234] [Table 18]

[0235] Sample collection. Seven days after the initial CTX administration, blood was collected by tail vein puncture. 100 μL of blood was collected for an immunophenotyping panel corresponding to LV vector expression and shRNA knockdown efficacy (see table below). Samples were analyzed by FACS. An additional 20 μL of blood was collected for complete CBC analysis. Collected samples were taken from each animal every seven days until the end of the study. Bone marrow was collected at the end of the study to evaluate complete grafts.

[0236] [Table 19]

[0237] End. Animals exhibiting unacceptable toxicity and / or severe weight loss (>20%), or those showing a continuous deterioration of their condition before reaching a coma, were humanely euthanized. Animals exhibiting severe suffering and / or obvious signs of pain were humanely euthanized by CO2 and subsequent neck dislocation. The study was terminated when a complete graft (over 90%) was observed in one group of animals. [Examples]

[0238] Human research Stem cell mobilization and retrieval. Three days prior to retrieval, the patient was CD34 + A 3- to 5-day G-CSF regimen was initiated to mobilize stem cells. Peripheral blood CD34 + The number of cells is 10.0 to 20.0 × 10⁶ per kg of body weight. 6 If the number of samples exceeded one, apheresis was performed. This material is CD34. + For stem cell isolation and transduction, the cells were transported to a GMP facility under controlled transport conditions at 2–8°C. Target size: 3.0–4.0 × 10⁶ 6 individual CD34 + Cells / kg, 2 to 3 × 10 6 Individual cells / kg were collected for reinfusion. Two days after collection (or 5 days before transplantation), the patient was given 15 mg / m². 2 The patient was treated with fludarabine for 5 days (until day 1 before transplantation). Alternatively, the patient was treated with 4 mg / kg busulfan on day 1 before transplantation. Then, on day 2 before transplantation, the patient received a single dose of 1000 mg / m². 2 The patient was treated with cyclophosphamide.

[0239] Cryopreservation of stem cells. Patient-derived cells (including hematopoietic stem cells) were, if applicable, centrifuged to obtain cell-rich pellets. A solution consisting of heparinized plasma light solution and 10% DMSO (dimethyl sulfoxide) was added to the plasma supernatant, in which the pelleted cells were resuspended. The cells were initially stored at -4°C, and then the samples were frozen at target temperatures ranging from -156°C (when stored in the vapor phase) to -196°C (when stored in the liquid phase). The cells were, if applicable, transported according to standard procedures.

[0240] CD34 + Transduction of cells by lentivir. Upon arrival at the GMP facility, CD34 + Cells were isolated by magnetic bead separation. Human CD3 via lentiviral vector. 4 + Cell transduction involves a 24-hour preliminary stimulation of cells in a medium supplemented with the cytokine stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), thrombopoietin (TPO), IL-6, IL-2, IL-3, fibronectin, or any combination thereof, followed by a 24-hour exposure to the vector (e.g., SEQ ID NO: 2 or SEQ ID NO: 4), both of which are present in serum-free X-Vivo 10 medium containing the cytokines SCF, FLT3L, and TPO (100 ng·ml each). -1 The procedure was carried out in the presence of [unspecified substance]. The cells were then cryopreserved and transported to the clinical setting.

[0241] Modified CD34 + Cell reinjection. Modified CD34 +After thawing the cells, a current standard washing protocol following the New York Blood Center protocol was used. This involved a two-step dilution of the thawed stem cell units with 2.5% human serum albumin and 5% dextran 40, followed by centrifugation at 10°C for 10 minutes. The supernatant was then removed, and the HSA and dextran solution was added again twice to a final DMSO concentration of less than 1.7%. The washed solution was infused into the patient as soon as possible. After a certain period following cell infusion, for example 7–45 days later, the patient began taking a low dose (50–200 mg or the non-myeloablative dose described herein) of oral cyclophosphamide once daily to facilitate the growth of the genetically modified bone marrow cell grafts. Modified CD34 + The patient has HIV at the time the cells are injected. + It is conceivable that in this case the cells are functioning to treat and / or cure HIV, or modified CD34 + The patient has HIV at the time the cells are injected. - It is conceivable that this could be the case, and in this instance, the cells are functioning to prevent future HIV infection. + Figures 9 and 10 show schematic diagrams of the patient's treatment, but the patient has HIV. - But it is understood that this is possible.

[0242] The above detailed description of embodiments of the Art is not intended to be exhaustive, nor is it intended to limit the Art to the exact forms disclosed above. Specific embodiments of the Art and examples relating to the Art are described above for illustrative purposes, but various equivalent modifications are possible within the scope of the Art, as will be apparent to those skilled in the art. The various embodiments described herein can also be combined to provide further embodiments.

[0243] From the foregoing description, it will be understood that specific embodiments of the Technology are described herein for illustrative purposes, but well-known structures and functions are not shown or described in detail in order to avoid unnecessarily obscuring the description of the embodiments of the Technology. Where the context allows, singular or plural terms may also include plural or singular terms, respectively.

[0244] While specific embodiments are described herein for illustrative purposes, it should be understood that various modifications can be made without departing from the Art. Furthermore, while advantages related to certain embodiments of the Art are described in the context of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments must necessarily demonstrate such advantages to be included within the scope of the Art. Accordingly, the disclosure and related Art may encompass other embodiments that are not expressly shown or described herein.

Claims

1. A method for performing a bone marrow transplant on a patient, Administer a population of cyclophosphamide-resistant modified cells and at least one dose of a non-myeloablative chemotherapeutic agent to the patient. The method comprising the above.

2. The method according to claim 1, wherein the population of cyclophosphamide-resistant modified cells comprises a heterologous gene encoding aldehyde dehydrogenase 1 (ALDH1).

3. The method according to claim 1, wherein a population of cyclophosphamide-resistant modified cells expresses ALDH1.

4. The method according to claim 1, wherein cyclophosphamide resistance in cyclophosphamide-resistant modified cells is conferred by the expression of aldehyde dehydrogenase 1 (ALDH1).

5. The method according to claim 1, wherein more than 50% of the patient's bone marrow is replaced with cyclophosphamide-resistant modified cells or cells derived therefrom within six months.

6. The method according to claim 1, wherein the patient has HIV.

7. The method according to claim 1, wherein the cyclophosphamide resistance of the modified cells is transient.

8. The method according to claim 1, wherein the cells are stem cells or immune cells.

9. The method according to claim 8, wherein the stem cells are embryonic stem cells, umbilical cord blood-derived stem cells, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), artificial PSCs (iPSCs), embryonic stem cells (ESCs), or cells derived from these, such as CD34+ cells, CD90+ cells, CD45+ cells, CD17+ cells, CD45RA- cells, CD38- cells, or any combination thereof.

10. The method according to claim 8, wherein the immune cell is a T cell.

11. The method according to claim 1, wherein the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

12. The method according to claim 1, further comprising contacting unmodified cells with an expression vector encoding ALDH1 expression to produce cyclophosphamide-resistant modified cells.

13. The method according to claim 12, wherein the expression vector is a viral vector or a non-viral vector.

14. The method according to claim 13, wherein the viral vector is a lentiviral vector or an adenovirus vector.

15. The method according to claim 12, wherein the expression vector is a retrovirus, a transposon, an episomal expression vector, a modified RNA, a plasmid, or any combination thereof.

16. At least one dose of a non-myeloablative chemotherapeutic agent is administered after the administration of modified cells. The method according to claim 1.

17. The method according to claim 1, wherein at least one non-myeloablative dose of the chemotherapeutic agent is a non-myeloablative dose of cyclophosphamide.

18. The method according to claim 17, wherein the non-myeloablative dose of cyclophosphamide is less than 2.5 mg / kg / day, ranging from about 0.16 mg / kg / day.

19. The method according to claim 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 0.41 mg / kg / day to approximately 1.63 mg / kg / day.

20. The method according to claim 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 0.81 mg / kg / day to approximately 1.46 mg / kg / day.

21. The method according to claim 17, wherein the non-myeloablative dose of cyclophosphamide is approximately 1.3 mg / kg / day.

22. The patient received 100 mg / m². ２ The method according to claim 17, wherein cyclophosphamide is not administered at a daily dose over a continuous period of 1 to 14 days.

23. The patient was 5-7 g / m² ２ The method according to claim 17, wherein the dose of cyclophosphamide is not administered over a period of 12 to 24 hours.

24. The method according to claim 1, wherein a non-myeloablative chemotherapeutic agent is administered daily for at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months.

25. The method according to claim 1, further comprising refraining from administering a non-myeloablative dose of a chemotherapeutic agent for a period of time between the administration of cyclophosphamide-resistant modified cells and the administration of at least one dose of a non-myeloablative chemotherapeutic agent.

26. The method according to claim 25, wherein the period is selected from the group consisting of approximately 3 days, approximately 7 days, approximately 10 days, and approximately 14 days.

27. The method according to claim 1, wherein more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the patient's bone marrow is replaced with modified cells.

28. The method according to claim 1, wherein the patient does not suffer myelolysis and / or immunodeficiency as a result of being administered at least one dose of a non-myeloablative chemotherapeutic agent.

29. The method according to claim 1, wherein the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, hypothrombocytopenia, hypoleukemia, hypoleukemia, or any combination thereof or related symptoms.

30. The method according to claim 1, wherein the pre-preparation step is performed before administering the cells.

31. The method according to claim 30, wherein the pre-adjustment step is a non-myeloablative chemotherapy pre-adjustment step.

32. The method according to claim 1, wherein the modified cells are resistant to HIV infection.

33. The method according to claim 32, wherein the modified cells heterologously express at least one mutation of an HIV coreceptor that is resistant to HIV infection, one or more mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of an HIV coreceptor, or any combination thereof.

34. The method according to claim 32, wherein the modified cells heterologously express shCCR5, shCXCR4, a C-peptide fusion inhibitor, or any combination thereof.

35. The method according to claim 32, wherein the modified cells do not express HIV coreceptors.

36. The method according to claim 32, wherein the modified cells either do not express CCR5, CXCR4, or express CCR5-Δ32, or a combination thereof.

37. ALDH1, and i) a heterologous nucleotide molecule encoding at least one HIV coreceptor mutant, one or more mutations in at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of an HIV coreceptor, or any combination thereof; and / or ii) one of the mutations or deletions of an endogenous HIV coreceptor. Cells containing heterologous nucleotide molecules that encode the expression of [a specific gene / substance].

38. Cells, i. Encoding molecules that reduce CCR5 expression; ii. Encoding molecules that reduce CXCR4 expression; iii. Encodes the expression of C-peptide fusion inhibitors; iv. Including sequences of 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof; v. Encode sequences of sequence numbers 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof; Or any combination of these. The cell according to claim 37, comprising heterogeneous nucleotide sequences.

39. The cell according to claim 37, expressing a C-peptide fusion inhibitor such as shCCR5, shCXCR4, and / or C44.

40. The cell according to claim 37, which has been modified to express a heterologous nucleotide sequence using a nonviral gene transfer system.

41. The cell according to claim 40, wherein the nonviral gene transfer system is a transposon gene transfer system.

42. The cell according to claim 37, wherein the transposon gene transfer system is the Sleeping Beauty gene transfer system or the PiggyBac transposon gene transfer system.

43. The cell according to claim 37, comprising the heterologous nucleic acid sequence of Sequence ID No.

4.

44. A composition comprising one or more cells as described in claim 37.

45. ALDH1, and i) One heteronucleotide molecule encoding at least one HIV coreceptor mutant, at least one mutation or multiple mutations in the HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of the HIV coreceptor, or any combination thereof. A nucleic acid molecule that codes for something.

46. Heterogeneous nucleotide sequences are, i. Reduce the expression of CCR5; ii. Reduce CXCR4 expression; iii. Encodes the expression of C-peptide fusion inhibitors; Or any combination of these. A nucleic acid molecule according to claim 45, which encodes a molecule.

47. The nucleic acid molecule according to claim 45, wherein the nucleic acid molecule encodes the expression of shCCR5, shCXCR4, and / or a C-peptide fusion inhibitor.

48. The nucleic acid molecule according to claim 45, wherein the molecule comprises a sequence of 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

49. The nucleic acid molecule according to claim 45, wherein the molecule comprises a nucleic acid molecule encoding sequence 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

50. A vector comprising the nucleic acid molecule described in claim 45.

51. The vector according to claim 50, which is a vector that can be used to produce lentiviruses.

52. The vector according to claim 50, which is a lentiviral vector.

53. The vector according to claim 50, comprising sequences of sequence numbers 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

54. The vector according to claim 50, comprising a nucleic acid molecule encoding sequence 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

55. ALDH1, and i) One heteronucleotide molecule encoding at least one HIV coreceptor mutant, at least one mutation or multiple mutations in the HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces the expression of the HIV coreceptor, or any combination thereof. A population of cells that heterologously express; and Non-myeloablative chemotherapy agent at least one dose A method of treating HIV in a subject, including administering a drug to that subject.

56. The method according to claim 55, wherein the cells express shCCR5, shCXCR4, and / or a C-peptide fusion inhibitor.

57. The method according to claim 55, wherein the cell comprises a nucleic acid molecule having a sequence of 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

58. The method according to claim 55, wherein the cell comprises a nucleic acid molecule encoding sequence 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

59. A method for expressing the target molecule in a subject, comprising administering ALDH1 and cells that heterologously express the target molecule to the subject, and administering a non-myeloablative dose of cyclophosphamide.

60. The method according to claim 59, wherein the cells are CD34+ and / or CD4+, or other as provided herein.

61. The method according to claim 59, wherein the target molecule is one that reduces the expression of CCR5; reduces the expression of CXCR4; encodes the expression of a C-peptide fusion inhibitor; or any combination thereof.

62. The method according to claim 61, wherein the molecule intended to reduce the expression of CCR5 is shCCR5.

63. The method according to claim 62, wherein the molecule comprises a nucleic acid molecule comprising or encoding SEQ ID NO: 6 and / or SEQ ID NO:

7.

64. The method according to claim 61, wherein the C-peptide fusion inhibitor comprises the sequence of SEQ ID NOs: 11, 15, 19, or any combination thereof.

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