Methods and compositions for non-myeloablative bone marrow reconstitution - Patent Application 20070122999

Abstract

To provide a pharmaceutical composition for use in a method that performs a bone marrow transplant in a patient.SOLUTION: The present invention provides a pharmaceutical composition comprising cyclophosphamide. The patient is administered a pharmaceutical composition containing at least one non-myeloablative dose of cyclophosphamide and a population of cyclophosphamide-resistant modified bone marrow cells, wherein the population of cyclophosphamide-resistant modified bone marrow cells includes a heterologous gene encoding aldehyde dehydrogenase 1 (ALDH1). The non-myeloablative dose of cyclophosphamide is about 0.16 mg / kg / day to less than 2.5 mg / kg / day.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO 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 The present disclosure provides methods and compositions for non-myeloablative bone marrow transplantation, including during the treatment of various diseases such as HIV, cancer (e.g., hematological cancer), and the like. In some embodiments, provided herein are modified stem cells that are engrafted into a patient's bone marrow and allow bone marrow reconstitution without the negative side effects experienced during traditional bone marrow transplantation. The cells can also be used to express proteins of interest that may be therapeutic in nature. [Background technology]

[0003] Bone marrow transplantation (BMT) is a procedure in which damaged or destroyed bone marrow is replaced 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 many other diseases, including severe aplastic anemia, lymphoma, multiple myeloma, immunodeficiency disorders, and some solid tumor cancers. Summary of the Invention [Problem to be solved by the invention]

[0004] Myeloablative BMT involves first treating a patient to kill cells in the bone marrow (both normal and abnormal), followed by the infusion of healthy bone marrow cells. In the first step, high-dose chemotherapy and / or radiation are required to kill the cells, followed by the introduction of allogeneic or autologous cells. This process of eradicating a patient's bone marrow is called myeloablative. 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 a sterile environment. Until the bone marrow is reconstituted, patients remain at high risk for infection, and the recovery period can last up to six months. During this period, patients are advised to remain near the hospital or clinic where they received the treatment if complications arise. In addition to acute toxicity, myeloablative chemotherapy is associated with numerous other side effects, including cataracts, growth retardation, cardiac toxicity, 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 fulfills these needs and more. [Means for solving the problem]

[0005] [Once the scope of claims is determined, all will be written] [Brief explanation of the drawings]

[0006] [Figure 1]

[0033] Figure 4 shows the percentage of live GFP (green fluorescent protein)-positive (GFP+) granulocytes in the peripheral blood of mice receiving 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 the indicated concentrations of intraperitoneal (ip) cyclophosphamide (CTX) once daily. On days 23, 35, and 42 of the study (corresponding to days 16, 28, and 35 of CTX administration, respectively), blood was collected by retro-orbital bleeding, and the percentage of live GFP+ granulocytes in the peripheral blood was assessed by flow cytometry. n = 3 in each of the groups without post-transplant CTX treatment, and n = 6 in all other groups. [Figure 2A] Figure 2 shows the percentage of GFP+ cells in the bone marrow, assessed by flow cytometry, for mice receiving bone marrow cells transduced with a control vector or test vector and subjected to a once-daily regimen of CTX at the indicated concentrations. n = 3 for each group without post-transplant CTX treatment, and n = 6 for all other groups. Figure 2A shows the % of total (dead and live) GFP+ cells. [Figure 2B] Figure 2B shows the percentage of GFP+ cells in the bone marrow, assessed by flow cytometry, for mice receiving bone marrow cells transduced with a control vector or test vector and subjected to a once-daily regimen of CTX at the indicated concentrations. n = 3 for each group without post-transplant CTX treatment, and n = 6 for all other groups. Figure 2B shows the % of live GFP+ granulocytes. [Figure 3]1 shows white blood cell (WBC) counts for mice receiving 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, n=3 in the group without post-transplant CTX treatment and n=5 in all other groups. [Figure 4] FIG. 1 shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 5] FIG. 1 shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 6] FIG. 1 shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 7] FIG. 1 shows a non-limiting schematic of the study design for dose ranging and lentiviral vector efficacy studies. [Figure 8] FIG. 1 shows a non-limiting schematic of the study design for lentiviral vector expression and efficacy of shRNA knockdown studies. [Figure 9] FIG. 1 shows a non-limiting schematic diagram of a study design for transplanting cells with multi-resistance to HIV and chemo-resistance to CTX into HIV+ patients according to one embodiment of the present disclosure. [Figure 10] 1 shows a non-limiting schematic diagram of a study design in which HIV+ patients are treated with CTX once a day after transplanting cells with multi-resistance to HIV and chemotherapy resistance to CTX into the patients. It is contemplated that the same study design can be performed on HIV- subjects to prevent HIV. [Figure 11] FIG. 1 shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. [Figure 12] FIG. 1 shows a non-limiting schematic diagram of a lentiviral vector according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0007] It is understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." As used herein, the term "about" means that numerical values ​​are approximations, and small variations do not significantly affect the practice of the disclosed embodiments.

[0008] As used in this specification and the appended claims, the singular forms "a," "an," and "the" should be understood to include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a "cell" includes a plurality of cells.

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

[0010] The term "about," when used before numerical designations, e.g., temperature, time, amount, concentration, and the like, including ranges, indicates approximations that may vary by (+) or (-) 20%, 10%, 5%, or 1%.

[0011] Similarly, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted by alternatives ("or").

[0012] The terms "administering," "administering," and the like refer to the introduction of an agent (e.g., cells) into a subject. Typically, an effective amount is administered, as determined by the treating physician or the like. Any route of administration can be used, including topical, subcutaneous, peritoneal, intravenous, intra-arterial, inhalation, vaginal, rectal, nasal, oral, buccal, introduction into the cerebrospinal fluid, or infusion into a body compartment. The terms and phrases "administering" and "administration of," when used in connection with compositions (and grammatical equivalents), refer to both direct administration, which can be administration to a patient by a medical professional or by the patient's self-administration, and / or indirect administration, which can be the act of prescribing a medication. For example, a physician who instructs a patient to self-administer an agent (e.g., cells) and / or provides a prescription for a medication to a patient is administering the agent to the patient. "Intermittent administration" or "regular administration" refers to multiple treatments administered on a daily, weekly, or monthly basis. Intermittent administration can also refer to administration of an agent once, twice, three times, or more 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 inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any process or composition using the transitional phrase "comprise" or "comprising" can also be said to be described with the transitional phrase "consisting of" or "consists."

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

[0015] As used herein, the term "contacting" means bringing two elements together in an in vitro system or in vivo system. For example, contacting a virus with a cell. "Contacting" a virus with an individual, patient, or cell includes administering a virus to an individual, such as a human, or patient, as well as introducing a compound into a sample containing, for example, a cell preparation or purified preparation containing the cell of interest.

[0016] The term "heterologous" when referring to a nucleic acid molecule, protein, vector, or expression cassette refers to a nucleic acid molecule, protein, vector, or expression cassette that is expressed in a cell through user manipulation and is not natural.For example, a heterologous gene refers to a gene that is expressed by a vector or other medium that is introduced into a cell, or a gene that is in a genome that has been modified through gene editing methods such as CRISPR or other recombinant techniques to replace genes in a cell.Those skilled in the art will understand that the term "heterologous" does not refer to a natural gene in the genome of an unmodified cell."Heterologous" can also be referred to as "exogenous".

[0017] The term "isolated," as used herein with respect to nucleic acids such as DNA or RNA, refers to molecules separated from other DNA or RNA molecules present in the natural source of the macromolecule, respectively. The term "isolated," as used herein, also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" includes nucleic acid fragments that do not naturally occur as fragments and are not found in their natural state. An "isolated cell," e.g., an isolated bone marrow cell, is a cell that is substantially free of other cellular material, tissue, and medium from the environment in which it is naturally found.

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

[0019] The term "nonmyeloablative" refers to treatment that produces no, minimal, or reversible cytopenias and is associated with little toxicity. A nonmyeloablative regimen is immunoablative. Examples of nonmyeloablative doses include, but are not limited to, approximately 1.3 mg / kg / day for a period of time that does not produce cumulative toxicity, or 1.0 to 1.5 mg / kg / day for 2 to 4 months (McKinley et al., Clin J Am Soc Nephrol. 2009, 4:1754-1760). Other nonmyeloablative doses are described throughout and are included in the definition of nonmyeloablative dose. An agent or dose of an agent that produces "cumulative toxicity" refers to a dose that produces toxicity in a patient over time. For example, cyclophosphamide, administered to humans at a dose of 2.5 mg / kg / day for several weeks, produces cumulative toxicity.

[0020] The terms "subject," "individual," or "patient" are used interchangeably herein to refer to a vertebrate, such as a primate, mammal, or preferably a human. Mammals include, but are not limited to, horses, dogs, cows, sheep, mice, rats, monkeys, and humans.

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

[0022] The term "variant," as used herein, refers to a nucleic acid or protein that differs from a reference nucleic acid or protein (i.e., calmodulin or a fragment thereof) but retains essential properties (i.e., biological activity). A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. A change in the nucleotide sequence may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. A nucleotide change can result in an amino acid substitution, addition, deletion, fusion, and / or truncation in the polypeptide encoded by the reference sequence.

[0023] The term "vector" is used herein to refer to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, the nucleic acid molecule of the vector. The vector may contain a sequence that directs autonomous replication in a cell or may contain a sequence sufficient to allow integration into cellular DNA. Vectors include, for example, plasmids (e.g., DNA 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, the vector has the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the vector includes 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 refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0025] Methods for performing bone marrow transplants Provided herein is a method for performing bone marrow transplantation in a patient in need thereof.Also provided is a method for replacing a subject's bone marrow cells with a cell population that expresses one heterologous nucleic acid molecule expression cassette or multiple heterologous expression cassettes, or with genome-edited cells that differ from the subject's genome.In some embodiments, these methods include administering one or more modified chemotherapy-resistant 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 amount.

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

[0027] The modified chemotherapy-resistant cells for use in the disclosed methods can be any suitable cells known to those skilled in the art. For example, the cells can be stem cells or immune cells. Non-limiting examples of stem cells include umbilical cord blood cells, fetal stem cells, and embryonic stem cells (ESCs). , hematopoietic stem cells (HSCs), hematopoietic progenitor cells, pluripotent stem cells (PSCs), induced 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, hematopoietic stem / progenitor cells from umbilical cord blood, stem / progenitor cells from umbilical cord tissue, iPSCs, HESCs, stem cells from fetal tissue, CD4+ cells, and the like. In some embodiments, the stem cells are CD34+.

[0028] Chemotherapy resistance The chemotherapy-resistant cells used in this method can be generated by 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 for performing bone marrow transplantation in a patient in need thereof is provided, comprising generating chemotherapy-resistant modified cells, administering an effective amount of chemotherapy-resistant modified cells to the patient, and administering at least one dose of chemotherapy agent.In certain embodiments, the dose is a non-myeloablative dose of chemotherapy agent.In some embodiments, the chemotherapy-resistant cells are resistant to cyclophosphamide.In some embodiments, the chemotherapy-resistant cells are resistant to non-myeloablative dose 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 can be a nucleic acid sequence encoding a cyclophosphamide resistance gene, a mutant thereof, or a portion thereof. In some embodiments, the cyclophosphamide resistance gene is aldehyde dehydrogenase 1 (ALDH1). In some embodiments, ALDH1 is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 1 or a mutant thereof. In some embodiments, ALDH1 is expressed in a lentiviral vector comprising the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a mutant thereof. Cells that are modified with ALDH1 but are resistant to cyclophosphamide can remain sensitive to other non-cyclophosphamide chemotherapeutic agents (i.e., the cells do not become multidrug resistant).

[0030] Any modified method known to those skilled in the art can be used to express an exogenous chemotherapy resistance gene, including viral vectors (e.g., adenoviruses, retroviruses such as replication-defective 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 a synthetic messenger RNA (mRNA). Synthetic mRNA provides the genetic information to make a protein of interest and can be chemically modified to avoid eliciting an immune response. Zangi et al. (2013) Nature Biotech 31:898-907. Because mRNA is not integrated into the host cell genome, the synthetic RNA acts for a period of time and then disappears when the cell divides. In some embodiments, the synthetic mRNA is modified, for example, with pseudouridine and / or 5-methyl-cytidine, to reduce the inherent antiviral response to single-stranded RNA. In some embodiments, the synthetic RNA encodes ALDH (e.g., ALDH1) and / or its respective equivalent.

[0031] In some embodiments, chemotherapy resistance, e.g., cyclophosphamide resistance, is transiently expressed by the modified cells. The sulfamide is expressed by the modified cells for a period of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, about 2 years, or about 3 years. Transient expression refers to the persistence of expression of the gene or protein that confers resistance. Transient means that resistance is not permanent.

[0032] In some embodiments, exogenous chemotherapy resistance gene, for example, cyclophosphamide resistance gene, is introduced into cells by any one of various well-known techniques, such as non-viral transfection of cells.The introduction into cells can be carried out by any non-viral 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, particle bombardment-mediated transfection (nanoparticles), cationic polymer-mediated transfection (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), and the like), or cell fusion.Other transfection methods include transfection reagents such as Lipofectamine™, Dojindo Highlymax™, Fugene™, jetPEI™, Effectene™, and DreamFect™.

[0033] Cell isolation and / or purification The chemotherapy-resistant modified cells for use in the present methods can be patient cells (i.e., autologous cells), donor cells (i.e., allogeneic cells), or any combination thereof, which have been modified to confer chemotherapy resistance. In certain embodiments, the methods provided herein further comprise isolating and / or purifying cells from the patient or donor. In certain of these embodiments, the methods further comprise modifying the 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 thereof, comprising isolating and / or purifying one or more cells from a patient or subject, modifying the 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 / lack of expression of certain markers, proliferation rate, and differentiation potential. In some embodiments, cells are isolated from a variety of markers, 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 In some embodiments, cells are isolated based on the presence of a particular marker or combination of markers, including TLR7, TLR9, and / or TRL1. In some embodiments, cells are isolated based on the absence of a particular marker, e.g., CD3, CD14, CD19, CD56, and / or CD66b. In other embodiments, negative selection is performed for markers, e.g., T cell, B cell, granulocyte, and / or myelomonocytic. In some embodiments, cells are isolated based on the presence of Thy-1, alone or in combination with any other marker. In some embodiments, HSCs are isolated based on the presence of Lin - Thy1 + Sca-1 + In some embodiments, the mouse HSCs are isolated based on the expression profile CD34 - , Sca1 + , c-kit + In some embodiments, human HSCs can be isolated based on their expression of CD34.

[0035] chemotherapy drugs In some embodiments, the methods provided herein include 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 can 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, epothilone, etoposide, fluorouracil, gemcitabine, hydroxybenzoates ... These include ciurea, idarubicin, imatinib, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nimustine (ACNU), oxaliplatin, paclitaxel, pemetrexed, temezolamide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, and vinorelbine.

[0036] Disease-specific modification In certain embodiments of the methods provided herein, the modified chemoresistant cells may contain one or more additional modifications not associated with chemoresistant. For example, in certain embodiments, the cells are further modified to express additional HIV / disease-specific modifications. Thus, in certain embodiments, the bone marrow transplantation methods provided herein further comprise incorporating one or more additional modifications, including one or more HIV / disease-specific modifications. For example, in certain embodiments, a method is provided for performing bone marrow transplantation in a patient in need thereof, comprising isolating and / or purifying one or more cells from a patient or subject, modifying one or more cells to be chemoresistant as described herein, incorporating one or more additional modifications into the one or more cells, administering an effective amount of one or more modified chemoresistant 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, the modified cells are further modified to be HIV-resistant. For example, the modified cells can be further modified to express at least one mutant HIV coreceptor that confers resistance to HIV infection, one or more mutations in at least one HIV coreceptor, expression of at least one HIV fusion inhibitor, or any combination thereof. In some embodiments, the cells are modified to express a molecule that inhibits or reduces expression of an HIV coreceptor. In some embodiments, the molecule is an antisense molecule. In some embodiments, the 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] The present disclosure provides cells that have been modified to have chemotherapy resistance, for example, cyclophosphamide resistance, and HIV resistance. In some embodiments, the cells can be modified to have cyclophosphamide resistance and HIV resistance. HIV resistance can be achieved by reducing the expression of at least one HIV co-receptor, inhibiting one of the at least one HIV co-receptor. The HIV resistance is conferred by a natural mutation or mutations, expression of at least one HIV fusion inhibitor, or any combination thereof. The HIV resistance is conferred by reduced expression of the CCR5 HIV coreceptor, reduced expression of the CXCR4 coreceptor, expression of a C-peptide fusion inhibitor (e.g., a C46 fusion inhibitor or a C34 fusion inhibitor), or any combination thereof.

[0039] The cells can also be modified to express any molecule of interest, as determined by the user or by the needs of a particular patient.

[0040] Administration to patients 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 would be apparent to those skilled in the art depending on the disease or condition being treated. Typical administration routes include intravenous, intraarterial, intramuscular, subcutaneous, intracranial, intranasal, intradermal, oral, or intraperitoneal routes.

[0041] In some embodiments, the subject's body surface area is 1 m 2 Approximately 1 x 10 8 to approximately 1 x 10 11 Cells can be administered to an individual on an absolute cell number basis, for example, the individual can receive from about 1000 cells / injection up to about 10 billion cells / injection, for example, about, at least about, or at most about 1 x 10 cells per injection. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (and so on) cells, or any range between any two of these values, inclusive, can be administered. In some embodiments, about 5 x 10 cells 6 From pcs / kg to about 10 x 10 6 1 / kg will be used for HSC transplantation.

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

[0043] In other embodiments, cells can be administered to such individuals on a relative number basis of cells, e.g., the individual can be administered from about 1000 cells per kilogram of the individual up to about 10 billion cells, e.g., about, at least about, or at most about 1 x 10 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 (etc.) cells, or any range between any two of these values, inclusive, 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 simultaneous with or sequential to 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 preconditioning step (also referred to herein as a "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, the preconditioning step (also referred to herein as a "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. The conditioning step is a preconditioning step with a non-myeloablative chemotherapy drug. In some embodiments, a preconditioning step is not performed before administering the cells. It is believed that the cells of the present disclosure will still be efficiently engrafted into the patient's bone marrow even if a preconditioning step (e.g., fludarabine) is not performed before administering the cells.

[0045] In some embodiments, at least one non-myeloablative dose of a chemotherapeutic agent for a human subject or patient is a non-myeloablative dose of cyclophosphamide, hi some embodiments, the non-myeloablative dose of cyclophosphamide is 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 about 0.15 mg / kg / day, about 0.2 mg / kg / day, about 0.25 mg / kg / day, about 0.3 mg / kg / day, about 0.35 mg / kg / day, about 0.4 mg / kg / day, about 0.45 mg / kg / day, about 0.5 mg / kg / day, about 0.55 mg / kg / day, about 0.6 mg / kg / day, about 0.65 mg / kg / day, about 0.7 mg / kg / day, about 0.75 mg / kg / day, about 0.8 mg / kg / day, about 0.85 mg / kg / day 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 is 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 is about 0.5 to about 2 mg / kg / day.

[0046] In some embodiments, the non-myeloablative dose of chemotherapeutic agent is administered daily for 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 more.

[0047] In some embodiments, the non-myeloablative dose is provided over a period of time that does not result in cumulative toxicity, 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 about 1 week, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days.

[0048] In some embodiments, there is at least one interruption period between the administration of cyclophosphamide-resistant modified cells and the administration of at least one dose of non-myeloablative chemotherapy.For example, the interruption period can 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 more.In some embodiments, the interruption period is about 3 days, about 7 days, about 10 days, and about 14 days.

[0049] In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about one year. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about six months. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about five months. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about four months. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about three months. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about two months. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about one month. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or 100% of the patient's bone marrow is replaced with modified cells within about two weeks. In some embodiments, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 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 become myeloablative and / or immunocompromised during the method, hi some embodiments, the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, low platelets, low white blood cells, low red blood cells, or any combination thereof, or related symptoms.

[0051] In another embodiment, upon treatment with the cells and chemotherapeutic agents of the present disclosure, a subject or group of subjects may exhibit one or more of the following results: (i) an increase in white blood cells of 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, or median % change) compared to a control; (ii) an increase in granulocytes of 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, or median % change) compared to a control; (iii) an increase in neutrophils of 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, or median % change) compared to a control; (iv) 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% compared to a control; is lymphocyte expansion of at least 99% (actual % change or median % change); (v) an increase in eosinophils of 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, or median % change) compared to a control; (vi) an increase in monocytes of 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, or median % change) compared to a control; (vii) an increase in basophils of 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, or median % change) compared to a control; (viii) an increase in red blood cells of 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, or median % change) compared to a control; (ix) an increase in all three blood cellular components (red blood cells, white blood cells, and platelets) of 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, or median % change) compared to a control; (x) freedom from recurrence for at least about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, about 40 years, about 45 years, about 50 years, about 55 years, about 60 years, or more; (xi) an increase in patient recurrence-free survival for at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, about 40 years, about 45 years, about 50 years, about 55 years, about 60 years, or more, compared to a control; (xii) an increase in patient survival compared to a control for at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, about 40 years, about 45 years, about 50 years, about 55 years, about 60 years, or more; (xiii) an increase in the intracellular lifespan of HIV of 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, or median % change) compared to a control; shortening; (xiv) an HIV reservoir reduction of 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, or median % change) compared to a control; and (xv) 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, or median % change) depletion of viral DNA compared to a control.

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

[0053] In some embodiments, the modified cells are administered to a subject for a period of time effective to reduce 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 can be a subject treated with a placebo, a baseline control, or a subject treated with unmodified cells.

[0054] Non-limiting symptoms include fever, headache, lack of energy, skin rash, skin pain, enlarged glands, 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, the modified cells are administered with at least one other HIV therapy. Suitable other HIV therapies include any HIV therapy known to those of skill 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 (NRTs), and ribovir-resistant HIV inhibitors (RIs). I) (e.g., 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 / lamivudine Drugs that are not approved include benzodiazepines (BENZY), ... 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®), doxorubicin (Denver®), fluconazole (Fenavir ... These include tegravir (Tivicay®) and elvitegravir (Vitekta®), chemokine co-receptor antagonists (CCR5 antagonists) (e.g., maraviroc (Selzentry®) or vicriviroc), cytochrome P4503A inhibitors, and immune-based therapies (e.g., hydroxychloroquine sulfate (Plaquenil). In some embodiments, the modified cells and at least one other HIV therapy are administered simultaneously. In other embodiments, the 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 specifically excluded, for example, in some embodiments, NRTIs are specifically excluded. In some embodiments, no other HIV therapy is administered other than the modified cells disclosed herein and at least one dose of a non-myeloablative chemotherapeutic agent (e.g., cyclophosphamide).

[0056] The cells, chemotherapeutic agents, and optionally other HIV therapies can be administered once to a patient with HIV, or multiple times during the course of therapy, for example, once every hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours, or once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more weeks, or any range between any two of these values, inclusive of the endpoints.

[0057] In some embodiments, a method of treating a patient with HIV is provided. In some embodiments, the method comprises: + This involves mobilizing stem cells from the bone marrow to the periphery. In some embodiments, the cells are mobilized by administering G-CSF (granulocyte colony-stimulating factor). G-CSF can be administered, for example, for one day, two days, three days, four days, or five days. In some embodiments, G-CSF is administered for three to five days. The mobilized cells can be captured using a methodology such as apheresis. In some embodiments, isolation of cells, for example by apheresis, involves the capture of CD34 + Cell counts range from 10.0 to 20.0 × 10 cells per kg of body weight 6 In some embodiments, the cell count is between 5.0 and 25.0 x 10 cells per kg of body weight. 6 Cd34+ cells are used as a marker to capture cells for transduction, although other cell markers, such as those described herein, can also be used. For example, specific markers or Cells that can be isolated based on the presence of a combination of markers include, for example, 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), and the like. low , CD45RA, CD123, ILT-7, MHC class II, MHC class II low , TLR7, and / or TRL9). In some embodiments, cells are isolated based on the absence of certain markers, e.g., CD3, CD14, CD19, CD56, and / or CD66b. In other embodiments, negative selection is performed for markers, e.g., T cell, B cell, granulocyte, and / or myelomonocytic. In some embodiments, cells are isolated based on the presence of Thy-1, alone or in combination with any other marker. In some embodiments, HSCs are isolated based on the presence of Lin - Thy1 + Sca-1 + In some embodiments, the mouse HSCs are isolated based on the expression profile CD34 - , Sca1 + , c-kit + In some embodiments, human HSCs can be isolated based on their expression of CD34. In some embodiments, the isolated cells are CD34+ or CD4+, or any combination thereof.

[0058] In some embodiments, the method includes centrifuging the cell harvest. This is done, for example, to develop a cell-enriched pellet. The cells are then resuspended in a cryopreservation solution and frozen. In some embodiments, the cryopreservation solution includes a solution of heparinized Plasmalyte and 10% DMSO (dimethyl sulfoxide). In some embodiments, the cells are initially stored at -4°C, and then the sample is frozen down to a target temperature of -156°C (if stored in vapor phase) to -196°C (if stored in liquid phase).

[0059] In some embodiments, the method includes transducing isolated cells to render them resistant to chemotherapeutic agents, such as cyclophosphamide. As described herein, chemotherapy resistance is achieved by expression of ALDH1. ALDH1 can be introduced into selected cells through the use of a vector (described throughout the specification), such as the use of a lentiviral vector. ALDH1 can be operably linked to a promoter, which can be cell-specific. 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 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 comprising the sequence of SEQ ID NO: 1. Due to the degenerate nature of the genetic code, the sequence of SEQ ID NO: 1 is provided as a non-limiting example, and other nucleic acid molecules can also be used to encode the expression of a protein comprising SEQ ID NO: 10. In some embodiments, ALDH1 comprises 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] The expression of ALDH1 in the vector can also be driven by an enhancer element. For example, the enhancer element can be a CD3E enhancer. In some embodiments, the CD3E enhancer comprises the sequence of SEQ ID NO: 9.

[0061] In some embodiments, CD34 + Cells can be isolated by magnetic bead separation. + Transduction of cells can be achieved by, for example, the use of cytokines such as stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT3L), and the like. The treatment may include a 24-hour pre-stimulation of cells in medium supplemented with ALDH1, thrombopoietin (TPO), IL-6, IL-2, IL-3, fibronectin, or any combination thereof. In some embodiments, the cells are then contacted (infected) with a lentivirus that expresses ALDH1. In some embodiments, the vector comprises the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5. The contacting may involve a 24-hour pre-stimulation of cells in medium supplemented with the cytokines SCF, FLT3L, and TPO (100 ng ml each) in serum-free X-Vivo 10 medium. -1 ) The cells may then optionally be frozen or not. In some embodiments, the cells are not contacted with an AAV or AV vector.

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

[0063] In some embodiments, after infusion of the modified cells, a non-myeloablative dose of chemotherapy, such as cyclophosphamide, is administered. In some embodiments, the dosage is 50-200 mg administered once daily. In some embodiments, the non-myeloablative dose of cyclophosphamide is about 0.15 mg / kg / day to less than 2.5 mg / kg / day, about 0.4 mg / kg / day to about 1.7 mg / kg / day, or about 0.8 mg / kg / day to about 1.5 mg / kg / day. In some embodiments, the non-myeloablative dose of cyclophosphamide is about 0.15 mg / kg / day, about 0.2 mg / kg / day, about 0.25 mg / kg / day, about 0.3 mg / kg / day, about 0.35 mg / kg / day, about 0.4 mg / kg / day, about 0.45 mg / kg / day, about 0.5 mg / kg / day, about 0.55 mg / kg / day, about 0.6 mg / kg / day, about 0.65 mg / kg / day, about 0.7 mg / kg / day, about 0.75 mg / kg / day, about 0.8 mg / kg / day, about 0.85 mg / kg / day 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 is 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 is about 0.5 to about 2 mg / kg / day. Doses can be administered as indicated herein. Without being bound by any particular theory, once-daily oral cyclophosphamide to facilitate grafting expands genetically modified bone marrow cells. Modified CD34 +The patient is HIV positive at the time the cells are infused. + In this case, the cells may function to treat and / or cure HIV, or may be modified CD34 + The patient is HIV positive at the time the cells are infused. - It is possible that, in this case, the cells are functioning to prevent future HIV infection. + A non-limiting schematic diagram of patient treatment is shown in Figures 9 and 10, where the patient is HIV - But it is understood that this is possible.

[0064] In some embodiments, the subject is also treated with fludarabine prior to infusion of the modified cells. In some embodiments, on day 2 after harvest (or day -5 pre-transplant), the patient is administered fludarabine (15 mg / m) for 5 days (until day -1 pre-transplant). 2 In some embodiments, instead of fludarabine, patients are treated with 4 In some embodiments, patients are treated with a single dose of 1000 mg / m² of cyclophosphamide on day -2 before transplantation. However, after the cells are infused, the subject is treated with a non-myeloablative dose of cyclophosphamide as described herein.

[0065] As described herein, the vector can also include other expression cassettes, including those expressing shCCR5 or fusion inhibitors, such as C44, C46, ​​or others described herein. The fusion inhibitor can 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 comprising 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 comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion inhibitor is membrane-anchored by a GPI anchor. In some embodiments, the anchor is encoded by a nucleic acid molecule comprising the sequence of SEQ ID NO: 16. In some embodiments, the anchor comprises the sequence of SEQ ID NO: 17. In some embodiments, the fusion-anchor protein comprises an IgG hinge region. In some embodiments, the IgG hinge region is IgG3. In some embodiments, the fusion inhibitor protein comprises the sequence of SEQ ID NO: 19. In some embodiments, the fusion inhibitor protein is encoded by a nucleic acid molecule comprising the sequence of SEQ ID NO: 18. In some embodiments, the anchor is GP41.

[0066] In some embodiments, the fusion inhibitor is placed under the control of a promoter different from that of the ALDH1 promoter.In some embodiments, the promoter is an EFS promoter.In some embodiments, the promoter is a 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 the hCCR5 shRNA of SEQ ID NO: 6, or its complement, and / or the antisense sequence of the hCCR5 shRNA of SEQ ID NO: 7, or its complement. In some embodiments, the sequence can also be reversed. In some embodiments, the vector comprises a mir30 expression cassette. In some embodiments, the mir30 expression cassette encodes the hCCR5 shRNA. In some embodiments, the mir30 construct comprises 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 can also be used.

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

[0069] In some embodiments, the disclosure provides a protein 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, a nucleic acid molecule is provided that comprises SEQ ID NO: 1 or a variant thereof, SEQ ID NO: 3 or a variant thereof, SEQ ID NO: 6 or a variant thereof, SEQ ID NO: 7 or a variant thereof, SEQ ID NO: 13 or a variant thereof, SEQ ID NO: 14 or a variant thereof, SEQ ID NO: 16 or a variant thereof, SEQ ID NO: 18 or a variant thereof, or any combination thereof. In some embodiments, the nucleic acid molecule is SEQ ID NO: 1 or a variant thereof, SEQ ID NO: 6 or a variant thereof, SEQ ID NO: 7 or a variant thereof, as well as SEQ ID NO: 3, SEQ ID NO: SEQ ID NO: 14, and one or more of SEQ ID NO: 18. In some embodiments, nucleic acid molecules are provided that encode proteins comprising SEQ ID NO: 2, and one or more of SEQ ID NO: 11, SEQ ID NO: 15, and SEQ ID NO: 19.

[0071] In some embodiments, the present disclosure provides nucleic acid molecules encoding ALDH1 or a variant thereof, shCCR5 molecules or variants thereof, and / or fusion inhibitors, including 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 the nucleic acid sequence provided herein. In some embodiments, a lentivirus is provided that comprises a single expression construct encoding each of ALDH1 or a mutant thereof, shCCR5 molecule or a mutant thereof, and / or 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 operably connecting the nucleic acid molecules encoding ALDH1, shCCR5, and fusion inhibitors (including immobilized fusion inhibitors) are not limiting, and other promoters and response elements can 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, nucleic acid sequences comprising SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.

[0074] In some embodiments, the nucleic acid molecule comprises a 5'LTR and a 3'LTR flanking the nucleic acid molecule encoding ALDH1, shCCR5, and / or a fusion inhibitor protein. For the avoidance of doubt, the shCCR5 sequence and the fusion inhibitor sequence can be replaced with other sequences of interest to be used for co-expression with ALDH1. Thus, in some embodiments, the nucleic acid molecule comprises a sequence encoding ALDH1 and a sequence of interest, which may be, for example, any other protein, antisense, miRNA, or other nucleic acid molecule desired to be expressed in bone marrow or the cell types provided herein.

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

[0076] In some embodiments, a method for treating HIV in a subject is provided, comprising administering to the subject a population of cells heterologously expressing ALDH1 and i) one of a heterologous nucleotide molecule encoding at least one HIV coreceptor mutant, at least one HIV coreceptor mutation or mutations, at least one HIV fusion inhibitor, a molecule that reduces HIV coreceptor expression, or 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 allogeneic to the subject. In some embodiments, the cells express shCCR5, shCXCR4, and / or a C-peptide fusion inhibitor. In some embodiments, the cells comprise a nucleic acid molecule comprising the sequence of SEQ ID NO: 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof. In some embodiments, the cells comprise a nucleic acid molecule encoding the sequence of SEQ ID NO: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof. In some embodiments, the cells are CD34+ and / or CD4+ or are CD34+ or CD4+ cells as provided herein. Others are provided.

[0077] In some embodiments, a method for expressing a molecule of interest in a subject is provided, comprising administering cells that heterologously express ALDH1 and a 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 ones 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 comprises a nucleic acid molecule that comprises or encodes SEQ ID NO:6 and / or SEQ ID NO:7.In some embodiments, the C-peptide fusion inhibitor comprises 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 is understood that various sequences are provided herein. In addition to exact sequences, sequences that are variants of the disclosed sequences are also provided. In some embodiments, sequences are provided that have at least, approximately, 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 to the sequences described. Those skilled in the art can easily understand how to determine the homology of two proteins or nucleic acids. For example, homology can be calculated after aligning the two sequences to ensure that the homology is at its highest level. In some embodiments, homology calculation can be performed using a published algorithm. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (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 in, for example, Zuker, M. Science 244:48-52, 1989; Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989; Jaeger et al., Methods Enzymol. 183:281-306, 1989, which are incorporated herein by reference for at least their material on nucleic acid alignment.For example, two sequences can be aligned using the website maintained by the National Center for Biotechnology Information using Blastn or BlastP using default settings.

[0079] For example, as used herein, a sequence that is said to have a certain homology percentage with another sequence refers to the sequence that has the said homology when calculated by any one or more of the above calculation methods.For example, if a first sequence is calculated to have 80% homology with a second sequence using Zucker's calculation method, even if the first sequence does not have 80% homology with the second sequence when calculated by any other calculation method, as defined herein, the first sequence has 80% homology with the second sequence.As another example, using both Zucker's calculation method and Pearson and Lipman's calculation method, If a first sequence is calculated to have 80 percent homology to a second sequence, the first sequence has 80 percent homology to the second sequence, as defined herein, even if the first sequence does not have 80 percent homology to the second sequence when calculated by either the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation method, or other calculation method. As yet another example, if a first sequence is calculated to have 80 percent homology to a second sequence using each of the calculation methods, the first sequence has 80 percent homology to the second sequence, as defined herein (although in practice, different calculation methods often result in different calculated percentages of homology).

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

[0081] Also provided herein is a composition, in certain embodiments, comprising the composition for use in the methods provided herein, comprising at least one chemotherapy-resistant modified cell provided herein.In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, carrier, or any combination thereof.

[0082] The compositions may contain pharmaceutically acceptable excipients, pharmaceutically acceptable salts, diluents, carriers, vehicles, and other such inactive agents well known to those skilled in the art. Vehicles and excipients commonly used 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, polyglycols, dimethyl sulfoxide, aliphatic alcohols, 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, polyglycols mixed with water, Ringer's solution, and the like. In one embodiment, a coloring agent is added to facilitate identification of the composition and accurate placement of the composition at the intended treatment site.

[0083] The composition may contain a preservative and / or stabilizer. 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 tonicity, the composition may contain a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg / ml. Other salts that may be present 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. Buffers are typically present 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, e.g., 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 which 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 in a subject (i.e., in vivo). For example, modified cells and / or non-myeloablative doses of chemotherapeutic agents are delivered to cells in vivo, or to a population of cells in vivo that form a tissue or organ in a subject, for the purpose of treating or preventing HIV or a 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 effect on specific types of cells.

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

[0089] References to sequences herein refer to sequences 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 manner and route.Non-limiting examples include internal, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intraarterial, intramuscular, intraperitoneal, intradermal, and subcutaneous routes.The composition is also suitable for transdermal delivery as part of a cream, gel, or patch.Other dosage forms include tablets; capsules; pills; powders; aerosols; suppositories; parenteral administration drugs; and oral liquids, including suspensions, solutions, and emulsions.Sustained-release dosage forms can also be used.Non-myeloablative doses of chemotherapy drugs 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 modifying cyclophosphamide resistance administering to the patient a population of selected cells and at least one dose of non-myeloablative cyclophosphamide.

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

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

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

[0097] 5. The method of any one of embodiments 1-4, 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 method of any one of embodiments 1-5, wherein the patient has HIV.

[0099] 7. The method of any one of embodiments 1-6, wherein the cyclophosphamide resistance of the modified cells is transient.

[0100] 8. The method of any one of embodiments 1 to 7, wherein the cell is a stem cell or an immune cell.

[0101] 9. The method of embodiment 8, wherein the stem cells are fetal stem cells, stem cells derived from umbilical cord blood, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), induced PSCs (iPSCs), embryonic stem cells (ESCs), or cells derived therefrom, 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 method of any one of embodiments 1-10, wherein the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

[0104] 12. The method of any one of embodiments 1-11, further comprising contacting the unmodified cells with an expression vector encoding expression of ALDH1 to produce modified cells that are cyclophosphamide resistant.

[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 adenoviral 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. At least one dose of non-myeloablative chemotherapy is administered after administration of modified cells 16. The method of any one of embodiments 1-15, wherein the patient is administered

[0109] 17. The method of any one of embodiments 1-16, 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 from about 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 from about 0.41 mg / kg / day to about 1.63 mg / kg / day.

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

[0113] 21. The method of embodiment 17, wherein the non-myeloablative dose of cyclophosphamide is about 1.3 mg / kg / day.

[0114] 22. The patient received 100 mg / m 2 22. The method of any one of embodiments 17-21, wherein the / day dose of cyclophosphamide is not administered for 1 to 14 consecutive days.

[0115] 23. Patients should receive 5-7g / m 2 23. The method of any one of embodiments 17-22, wherein the dose of cyclophosphamide is not administered over a period of 12 to 24 hours.

[0116] 24. The method according to any one of embodiments 1-23, wherein the non-myeloablative chemotherapy agent is administered daily for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months.

[0117] 25. The method according to any one of embodiments 1-24, further comprising administering no non-myeloablative dose of chemotherapy for a period of time between administration of the cyclophosphamide-resistant modified cells and administration of at least one dose of chemotherapy that is non-myeloablative.

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

[0119] 27. The method of any one of embodiments 1-26, wherein more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, or 100% of the patient's bone marrow is replaced with modified cells.

[0120] 28. The method of any one of embodiments 1-27, wherein the patient is not myeloablative and / or immunocompromised as a result of receiving at least one dose of a non-myeloablative chemotherapy agent.

[0121] 29. The method of any one of embodiments 1-28, wherein the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, low platelets, low white blood cells, or any combination thereof or related symptoms.

[0122] 30. The method of any one of embodiments 1 to 29, wherein the preconditioning step is performed before administering the cells.

[0123] 31. The method of embodiment 30, wherein the preconditioning step is a non-myeloablative chemotherapy preconditioning step.

[0124] 32. The method of 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 HIV co-receptor mutation that is resistant to HIV infection, one or more mutations in at least one HIV co-receptor, at least one HIV fusion inhibitor, a molecule that reduces expression of an HIV co-receptor, or any combination thereof.

[0126] 34. The method of embodiment 32, wherein the 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 an HIV co-receptor.

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

[0129] 37. ALDH1, and i) a heterologous nucleotide molecule encoding at least one HIV coreceptor mutant, a mutation or mutations in at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces expression of an HIV coreceptor, or any combination thereof; and / or ii) one of an endogenous HIV coreceptor mutation or deletion. A cell comprising a heterologous nucleotide molecule encoding expression of

[0130] 38. The cell of embodiment 37, wherein the cell comprises a heterologous nucleotide sequence that: i. encodes a molecule that reduces expression of CCR5; ii. encodes a molecule that reduces expression of CXCR4; iii. encodes expression of a C-peptide fusion inhibitor; iv. comprises a sequence of 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof; v. encodes a sequence of SEQ ID NO: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof; or any combination thereof.

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

[0132] 40. The cell of embodiment 37, which has been modified to express a heterologous nucleotide sequence using a non-viral gene transfer system.

[0133] 41. The cell of embodiment 40, wherein the non-viral gene transfer system is a transposon gene transfer system.

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

[0135] 43. The cell of any one of embodiments 37 to 42, comprising a heterologous nucleic acid sequence of SEQ ID NO: 4.

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

[0137] 45.ALDH1, and i) one of heterologous nucleotide molecules 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 expression of an HIV coreceptor, or any combination thereof; A nucleic acid molecule encoding

[0138] 46. ​​The nucleic acid molecule of embodiment 45, wherein the heterologous nucleotide sequence encodes a molecule that: i. reduces the expression of CCR5; ii. reduces the expression of CXCR4; encodes the expression of a C-peptide fusion inhibitor; or any combination thereof.

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

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

[0141] 49. The nucleic acid molecule of embodiment 45, wherein the molecule comprises a nucleic acid molecule encoding the sequence of SEQ ID NO: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

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

[0143] 51. The vector of embodiment 50, which is a vector that can be used to produce lentivirus.

[0144] 52. The vector of embodiment 50, which is a lentiviral vector.

[0145] 53. The vector of embodiment 50, comprising the sequence of SEQ ID NO: 1, 3, 6, 7, 8, 9, 12, 13, 14, 16, 18, or any combination thereof.

[0146] 54. The vector of embodiment 50, comprising a nucleic acid molecule encoding the sequence of SEQ ID NO: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

[0147] 55.ALDH1, and i) one of heterologous nucleotide molecules 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 expression of an HIV coreceptor, or any combination thereof; a population of cells heterologously expressing At least one nonmyeloablative dose of chemotherapy 20. A method of treating HIV in a subject comprising administering to the subject:

[0148] 56. The method of embodiment 55, wherein the cells express shCCR5, shCXCR4, and / or a C-peptide fusion inhibitor.

[0149] 57. The method of embodiment 55, wherein the cell comprises a nucleic acid molecule comprising a sequence 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 cell comprises a nucleic acid molecule encoding the sequence of SEQ ID NO: 10, 11, 6, 7, 13, 15, 17, 19, or any combination thereof.

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

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

[0153] 61. The method of embodiment 59, wherein the molecule of interest 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, wherein the molecule of interest that reduces the expression of CCR5 is shCCR5.

[0155] 63. The method of embodiment 62, wherein the molecule comprises a nucleic acid molecule comprising 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 NO: 11, 15, 19, or any combination thereof.

[0157] The following examples are illustrative, but not limiting, of 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. [Example]

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

[0159] Bone marrow transduction. Mouse bone marrow cells were transduced with a lentiviral vector expressing EGPF alone (i.e., pLV-Puro-EF1A-EGFP ("control vector")) or a lentiviral vector expressing EGFP and human ALDH1A (i.e., pLV-Puro-EF1A-EGFP-hALDH1A, FIG. 4, SEQ ID NO: 2 ("test vector")) at an MOI of 10. Forty-eight hours after transduction, the transduction efficiency was determined. The cells were then transplanted into irradiated mice as described below.

[0160] Transplantation. Syngeneic recipient mice (Balb / c) were pretreated with 0.5 mg / kg fludarabine on days -5 and -4, cyclophosphamide on day -2, and busulfan on day -1. On the day of transplantation (day 0), mice were anesthetized and 4 x 10 cells / mouse were injected in 100 μl of IMDM. 6 Bone marrow cells were transplanted by tail vein injection, and hematopoietic recovery of the transplants was monitored by FACS analysis of GFP.

[0161] Treatment with cyclophosphamide. Seven days after transplantation, mice were treated with different doses of CTX: 0 mg / kg ("0 mg / kg post-transplant CTX"), 16 mg / kg ("16 mg / kg post-transplant CTX"). In the control group, 100 mg / kg of CTX (post-CTX) or 30 mg / kg (post-implant CTX at 30 mg / kg) was injected intraperitoneally (ip) once daily. During this study, treatment included 5 weeks of CTX, 1 week without CTX, followed by 1 week of CTX treatment, i.e., a total of 6 weeks of CTX treatment.

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

[0163] Terminal analysis of bone marrow. Six weeks after CTX treatment, animals were sacrificed and GFP in the bone marrow was analyzed. + The percentage of cells was evaluated by flow cytometry. Granulocytes / neutrophils were identified using conventional forward scatter vs. side scatter variance, in which cells were plotted according to light reflection, size, and internal complexity (granularity), respectively. Each group without CTX treatment after transplantation had n=3, and all other groups had n=6. Six weeks later, the blood white blood cell count was determined for each mouse. The results showed the presence of outliers in each of the CTX-treated groups, and these data were removed in statistical analysis. Therefore, each group without CTX treatment after transplantation had n=3, and all other groups had n=5.

[0164] Results and Discussion When monitoring bone marrow repopulation after transplantation, peripheral blood can provide an indicator of transplant success. However, because bone marrow gives rise to a variety of immune cells with different lifespans, it is necessary to understand the cellular complexity of immune cells before transplantation. For example, the lifespan of lymphocytes can be as long as 180 days, while that of neutrophils is only 5–7 days. Furthermore, while monocytes can only survive for a few days in the periphery, they can also become tissue-resident and extend their lifespan to several months. Therefore, analyzing lymphocyte populations for donor bone marrow reconstitution will not provide an accurate reflection until all of the donor's pre-transplant lymphocytes have died, i.e., 6 months after the procedure. To provide a more "real-time" readout of the recipient's cellular bone marrow graft, the granulocyte population provides the best indicator. Because all of the donor's pre-transplant neutrophils die within 7 days, at 2 weeks post-transplant, these cells are the most direct reflection of the bone marrow environment and recipient engraftment success, which can also be measured by identifying the percentage of transduced neutrophils, which reflects the graft rate.

[0165] Following the method described above, blood was collected by retro-orbital bleeding on days 23, 35, and 42 of the study (days 16, 28, and 35 of CTX administration, respectively), and GFP was measured in the peripheral blood of each mouse. + The percentage of granulocytes was assessed by flow cytometry.

[0166] The results showed that all mouse groups were live GFP + Furthermore, on day 42 of the study, bone marrow cells transduced with the test vector (i.e., EGFP) were shown to contain granulocytes (Figure 1). + Mice receiving bone marrow cells transduced with the test vector and treated with 16 mg / kg / day of CTX had significantly more viable GFP-positive mice than mice receiving bone marrow cells transduced with the test vector and treated with 30 mg / kg / day of CTX. + showed similar percentages of granulocytes (compare Figure 1, "Test Vector, 16 mg / kg Post-Transplant CTX" and "Test Vector, 30 mg / kg Post-Transplant CTX").

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

[0168] White blood cells (WBCs) help fight infection caused by attacks from foreign agents that invade the body. WBC counts can be useful for 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 bone marrow cells transduced with either the control vector or the test vector and not treated with CTX had WBC counts within the normal range. There was one outlier in each CTX-treated group, which was removed in statistical analysis. The ranges for high, normal, and low WBCs follow the ranges known in the art, as 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. Notably, mice receiving test vector-transduced bone marrow cells and treated with 16 mg / kg / day CTX had significantly higher numbers of WBCs compared to mice receiving test vector-transduced bone marrow cells and treated with 30 mg / kg / day CTX (Figure 3, compare "Test Vector, 16 mg / kg pt Post-Transplant CTX" and "Test Vector, 30 mg / kg pt Post-Transplant CTX"). [Example]

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

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

[0171] Transduction of stem cells. CD34 from the same donor + Cells were cultured at 1 × 10 in fibronectin-coated 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-2, IL-3, IL-6, or any combination thereof, for 16 h at 37°C and 5% CO . 6 The priming medium was then removed and the cells were cultured at 1.35 × 10 cells / ml, corresponding to a multiplicity of infection (MOI) between MOI 2 and MOI 50. 8Fresh culture medium containing 100 tu / ml of 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. The 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 (as above, but without irradiation).

[0172] GFP analysis of peripheral blood and terminal analysis of spleen, blood, and bone marrow were performed as described above. A schematic of the study is shown in Figure 8, although this depiction is for illustrative purposes only and other study designs can be used. [Example]

[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 leukapheresis on days 5 and 6. CD34 + were purified by positive selection using immunomagnetic beads. 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 by staining for CD34 and staining for viability. The cells were then immediately transplanted into mice. Appropriate written informed consent in accordance with the Declaration of Helsinki protocols was obtained from the donors prior to collection.

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

[0175] Transduction of stem cells. CD34 from the same donor + Cells were cultured at 1 × 10 cells per well in fibronectin-coated 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 h at 37°C and 5% CO . 6 The priming medium was then removed and the cells were cultured at 1.35 × 10 cells / ml, corresponding to a multiplicity of infection (MOI) between MOI 2 and MOI 50. 8 ul / ml of vector ("control vector" = pLV-Puro-EF1A-EGFP Fresh culture medium was added, further containing a "test vector" (or "test vector"). Suitable "test vectors" included any one or combination of the following:

[0176] [Table 14]

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

[0178] GFP analysis of peripheral blood and terminal analysis of spleen, blood, and bone marrow were performed as described above. A schematic of the study is shown in Figure 8, although this depiction is for illustrative purposes only and other study designs can be used. [Example]

[0179] Lentiviral proof of concept studies Lentiviral vector expression of human CD34 + Cells were transduced with a lentiviral (LV) vector containing four expression cassettes. The expression levels of ALDH and C-peptide, shRNA-mediated CCR5 knockdown, and in some cases, shRNA-mediated CXCR4 knockdown were evaluated in transduced cells. The expression of cellular C-peptide was then correlated with the knockdown of CCR5.

[0180] Briefly, mobilized CSF, cryopreserved human CD34+ cells were thawed at 37°C until the contents of the vial were completely liquid. The cells were transferred to a sterile 15 mL conical tube (Corning, Tewksbury, MA). Prewarmed X-Vivo 10 serum-free medium (Lonza, Basel, Switzerland) was added dropwise and with gentle agitation to a volume of 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 a small aliquot was retained for counting and viability determination. The cells were then centrifuged, the supernatant was aspirated, and a 1.0 × 2.0 × 10 6 Resuspend to a density of 10 cells / ml and add 75 cm 3 The cells were transferred to tissue culture flasks (Corning, Tewksbury, MA).

[0181] The 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 cultured at 37°C in a 5% CO2 incubator for 24 hours. The cells were then removed from the incubator and centrifuged. The cells were cultured at 1.0 x 10 in X-Vivo 10 medium supplemented with 100 ng / mL of both SCF and Flt3L, 10 ng / mL of TPO, and 60 ng / mL of IL-3. 6 The cells were resuspended at a density of 10 cells / mL in 12 wells of tissue culture-untreated retronectin (5 μg / cm 2 ) Coated plate (Takarabai 1.0 × 10 per well (O Co., Ltd., Shiga Prefecture, Japan) 6 The cells were then seeded.

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

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

[0184] Flow cytometry staining and analysis proceeded immediately. If the LV construct contained a fluorescent marker, uptake was performed on a MacsQuant FACS Analyzer. If no fluorescent marker was contained in the vector, the target protein or HIV-1 A staining procedure for transcriptional activation of the LTR was performed.

[0185] The cells can be used for further characterization. If further characterization is not required, immediately after transduction (or expansion after transduction), the cells can be cultured in a control-rate freezer at 5x10 ng / ml in a solution consisting of 50% IMEM (Thermo Fisher Scientific, Carlsbad, CA), 45% human serum albumin (HAS) (Sigma-Aldrich, St. Louis, MO), and 5% 0.2 μm filtered DMSO. 6 Cells were cryopreserved at a density of 10 cells / mL. Cells can then be immediately transferred to liquid nitrogen or shipped on dry ice.

[0186] Chemical protection. Human CD34 transduced with LV vector (e.g., SEQ ID NO: 2 or SEQ ID NO: 5) exhibiting expression of ALDH. + Cells were treated with various doses of cyclophosphamide to demonstrate resistance to chemotherapy. Experiments can also be performed by treating mixed cultures of transduced and non-transduced cells with various doses of cyclophosphamide. The survival of both transduced and non-transduced cells was measured after treatment with each dose of cyclophosphamide.

[0187] Briefly, lyophilized mafosfamide was resuspended in sterile water to a working stock solution of 1 mg / mL. The transduced cells were then washed once with regular medium (RPMI + 10% FBS), and the transduced cells (both the test vector (e.g., SEQ ID NO: 4) and the control vector (e.g., SEQ ID NO: 5)) were cultured at 0.5 x 10 in MethoCult 3330 medium (StemCell Technologies, Vancouver, BC, Canada). 6The cells were resuspended to a density of 100 cells / mL in 0.8% methylcellulose in alpha medium 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] To each set of dishes (control and test dishes), mafosfamide was added to the following final concentrations: 0.15, 2.5, 5, 10, 15, and 20 μM. The plates were then incubated at 37°C, 5% CO2 for 14 days. After 14 days, colonies were scored on each plate using an inverted light microscope. A Mann-Whitney U test was then performed to determine the difference between cells expressing the test vector and cells expressing the control vector. It was determined whether there were any significant statistical differences in the number of colonies formed. [Example]

[0189] Fusion inhibition studies T cells isolated from the spleen and thymus of humanized mice were co-cultured with HIV strains as described above. The expression level of C44 was assessed using an anti-Gp41 antibody that recognizes the C-peptide, detected by flow cytometry. The expression level of C-peptide (positive percentage and median fluorescence intensity) was 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 the 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®); 2 M CaCl2; Alliance HIV-I p24 ELISA kit (PerkinElmer) or FlaQ assay reagent (Hayden et al., 2003); peripheral blood lymphocytes (PBLs); 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 recipe); Dulbecco's Modified Eagle's Medium (DMEM) culture medium (see recipe); Roswell Park Memorial Institute (RPMI) culture medium (see recipe); CCF2 loading solution (see recipe); stock solution of probenecid (250 mM) prepared in 250 mM NaOH (see recipe); growth medium (see recipe).

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

[0192] Preparation: HBSS (280 mM NaCl; 10 mM KCl; 1.5 mM NaHPO; 12 mM dextrose; 50 mM N-(2-hydroxyethylpiperazine)-N'-(2-ethanesulfonic acid) (HEPES) (pH 7.05); store at -20°C.

[0193] Flow cytometry staining buffer (1x phosphate-buffered saline (PBS) without Ca++ and Mg++; 0.5% HSA; store 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; 10% heat-inactivated fetal bovine serum; penicillin 100 U / ml; streptomycin 100 μg / ml).

[0196] CCF2 loading solution: CCF2-AM in dimethyl sulfoxide (CCF2 kit The cells were resuspended in solution A of the CCF2 kit to obtain a stock solution (1 mM CCF2-AM). The solution was aliquoted 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 Pluronic-F127 and 0.1% acetic acid (solution B from the CCF2 kit) by vortex mixing. 1 ml of CO2-independent medium was added and vortexed again. A stock solution of probenecid (250 mM) was prepared in 250 mM NaOH. The solution was aliquoted and stored at -20°C.

[0197] Growth medium (2.5 mM probenecid; 10% fetal bovine serum in CO2-independent medium).

[0198] Equipment: 175 cm2 culture flasks; 96-well V-bottom plates (Corning Incorporated); 5 ml, 10 ml, and 25 ml pipettes; 50 ml Falcon tubes; 0.22 μm pore size Steriflips (EMD Millipore); 2 ml Nalgene tubes (Thermo Fisher Scientific); clear ultracentrifuge tubes (BD); 37°C, 5% CO2 incubator; ultracentrifugation equipment with an SW28 rotor; and a flow cytometer. The fusion assay alone required a flow cytometer with violet laser excitation (405 nm) and two measurement parameters. A photomultiplier tube (PMT) with a 450 / 50 nm bandpass filter, commonly used for Pacific Blue detection, was used to detect the cleaved CCF2 substrate. Another PMT with a 515 / 20 nm bandpass filter, commonly used for Amcyan detection, was used to detect the uncleaved CCF2 substrate. Additional PMTs were required to measure the fluorochromes associated with the CD3 and CD4 antibodies. APC-Cy7 was excited by a 633 nm red laser and detected with a PMT equipped with a 755 long-pass filter. PE-Cy7 was excited by 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 x 10 7 293T cells were cultured in a T175cm culture medium with 20 ml of DMEM culture medium. 2The cells were seeded into tissue culture flasks and cultured overnight in a humidified incubator at 37°C with 5% CO2. 1.75 ml of H2O containing 60 μg of TN6-GFP proviral DNA, 20 μg of pCMV-BlaM-Vpr, and 10 μg of pAdVAntage vector was prepared. 2 ml of 2x HBSS was then slowly added and gently mixed by pipetting up and down. 250 μl of 2 M CaCl2 was added dropwise. DNA was precipitated by incubating at room temperature for 10 minutes. The 293T cell culture medium was replaced with 40 ml of fresh DMEM culture medium prewarmed to 37°C. 4 ml of DNA precipitate was then added and incubated at 37°C for 16 hours. The medium was then replaced with 40 ml of fresh DMEM culture medium prewarmed to 37°C and incubated at 37°C for 24 hours.

[0201] The supernatant of transfected 293T cells was collected into 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 the bucket of an SW28 rotor, balanced with DMEM culture medium as needed, and ultracentrifuged (72,000 × g, 90 minutes) at 4 °C without using the brake. The supernatant was removed, and the viral pellet was resuspended in 1 ml of DMEM, divided into 100 μl aliquots, and stored at -80 °C.

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

[0203] The PBLs were washed with RPMI culture medium, the PBLs were counted, and 2 × 10 7 The cell suspension was placed in a V-bottom 96-well plate, with 100 μl (2 × 10) of the cell suspension per condition to be tested. 6The cells were divided into aliquots of 1000 cells (1000 cells / 10 ...

[0204] 400ng of p24 Gag An amount of HIV-1 virions containing BlaM-Vpr equal to 1000 was added to all wells except the "uninfected control" and two compensation controls and incubated for 2 hours at 37°C.

[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 for 5 minutes at room temperature. The pellet was resuspended in 100 μl of CCF2-AM loading solution and incubated for 1 hour at room temperature in the dark; one well was 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, centrifuged for 5 minutes at room temperature, and the pellet was resuspended in 200 μl of growth medium. The cells were incubated at room temperature for 16 hours in the dark.

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

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

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

[0209] HIV challenge: Human CD4 transduced by lentiviral vector + T cells GFP reporter CD4 transduced by LV containing CCR5 shRNA / c-peptide / ALDH + T cells or T cells transduced with LV containing an empty control vector were cultured with R5, X4, or both experimental HIV strains. HIV infectivity was analyzed by reporter marker (when using a T cell reporter system) or by p24 ELISA. [Example]

[0210] Dose-ranging and lentiviral vector efficacy studies Sca1 transduced with lentivirus containing CCR5 shRNA / C-peptide / ALDH expression vector or control empty vector + / c-kit - Stem and progenitor cells were transplanted into pre-acclimated C57BL / 6 syngeneic mice.

[0211] Briefly, after 7 days, all mice began a daily regimen of cyclophosphamide (CTX) treatment. Both mouse groups were treated with various dosages of CTX (6 mice per group per dose): 0, 10, 13, 16, 19, and 40 mg / kg. One week after CTX treatment began, mice were bled weekly. A complete blood count (CBC) panel was performed to monitor any cytotoxic effects of chemotherapy (engraft efficacy: ALDH-mediated CTX resistance conferral), while flow cytometry analysis measured the expression of lineage-specific markers and C-peptide and CCR5 expression in these cells (expression efficiency: shRNA's efficacy in knocking down target genes). The study continued for at least 10 weeks of CTX treatment or until complete engraftment of the bone marrow occurred. 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 needle (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 controls) (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] Vector: 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 table below, the number of (donor) mice required to obtain the required number of cells for transduction was calculated.

[0215] [Table 15]

[0216] Isolation of mouse bone marrow. BALB / c mice (female, 20-25 g, 8-10 weeks old) were euthanized by CO2 asphyxiation. Subsequent experimental procedures were performed in a laminar airflow biosafety cabinet (BSC). Bone marrow cells were collected from femurs. Briefly, the contents of the bone marrow were flushed with 2 ml of HBSS using a 1 ml insulin syringe with a 27G × 1 / 2 needle. The contents were collected into a sterile 50 ml centrifuge tube. The collected BM cell suspension was then diluted with RPMI-1640 to a final volume of 7.5 ml. Any clusters in the bone marrow suspension were disaggregated by vigorous pipetting. The cells were then centrifuged, washed, and centrifuged again. The cell pellet from each femur was gently resuspended in 7.5 ml of RPMI-1640 to prepare a homogenous suspension. An aliquot of the cell suspension was removed for total cell number and viability using an NC-200 automated cell counter.

[0217] Isolation of progenitor cells. Transfer the isolated cells to a new tube, spin down, and resuspend in EasySep buffer (PBS + 2% FBS + 1 mM EDTA) in a volume range of 0.5-2 mL to a concentration of 1 x 10 8 The concentration was 100 cells / mL. Rat serum was then added to the sample at 50 μL / mL, and the sample was transferred to a 5 mL (12 x 75 mm) polystyrene round-bottom tube. EasySep Mouse Hematopoietic Progenitor Cell Isolation Cocktail was then added to the sample at 50 μL per mL of sample. The sample was mixed and incubated at 4°C for 15 minutes. Rapid spheres was vortexed for 30 seconds and then added to the sample at 75 μL / mL. The sample was 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 sorting.

[0218] Evaluation of isolated progenitor cells by flow cytometry. Cells were transferred to a new 96-well plate, spun down, 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 for 15 minutes at 4°C. The following panels and volumes were used:

[0219] [Table 16]

[0220] Controls included: Fluorescence Minus One (FMO) control for CD117, Ly6A / E, and lineage cocktail isotype control.

[0221] Each well was brought up to a total volume of 100 μL and incubated in the dark at 4°C for 30 minutes. Cells were then washed with 200 μL of flow cytometer buffer and resuspended in a volume. The flow cytometer was acquired on a MACS QuantCytometer. Purity was calculated by summing the percentage of Sca1- / c-kit+, Sca1+ / c-kit-, and Sca1+ / c-kit+ and multiplying by the percentage of live lineage cocktail-negative events.

[0222] Lentiviral transduction and progenitor cell expansion. On day 0, 12- or 24-well plates were coated with 0.3 ml of retronectin (100 ng / ml) and incubated for at least 2 hours at room temperature. The coating medium was then aspirated from the plates, and the plates were blocked with 2% BSA in 1x PBS at room temperature for at least 30 minutes, then washed three times with 1x PBS. The plates were now ready to use. Care should be taken not to let the plates dry out.

[0223] Freshly isolated bone marrow progenitor cells were cultured at approximately 0.2–0.4 × 10 cells per ml of bone marrow progenitor cell culture medium. 6The cells were seeded between 100 and 1000 cells / well. Lentivirus was added to the cells for 24 hours (MOI=3).

[0224] On day 1, the cells were spun down, the lentivirus-containing medium was removed, and fresh bone marrow progenitor culture medium was added.

[0225] On day 2, after lentiviral transduction, puromycin (1 μg / ml) was added to the bone marrow progenitor cell culture medium to select lentiviral-infected cells. Cells were treated for 4 days.

[0226] On day 4, fresh medium with puromycin was added.

[0227] On day 6, the cells were spun down and fed with fresh medium without puromycin.

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

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

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

[0231] [Table 17]

[0232] Bone marrow progenitor cell transplantation and treatment - Preconditioning: Prior to transplantation, mice received two doses of fludarabine (5 mg / kg for 2 days). On day 0 (2 days after treatment with fludarabine), 36 mice were transplanted via IV with stem and progenitor cells transduced with a control vector, and 36 mice were transplanted with stem and progenitor cells transduced with a test vector. After transplantation, animals were checked daily for morbidity and mortality. At routine monitoring times, animals were checked for any effects of the treatment on normal behavior, such as mobility, dull eyes / coat, and any other abnormal effects, as well as visual estimation of food and water consumption and weight gain / loss (weight was measured twice weekly). 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 at the following doses: 0, 10, 13, 16, 19, and 40 mg / kg, administered IP once daily for 12 weeks, and treatment was not discontinued unless there was a weight loss of >15%. Details of the experimental groups and treatments are shown in the table below.

[0234] [Table 18]

[0235] Sample collection. Seven days after the first CTX dose, 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. Collections were taken every 7 days from each animal until the end of the study. Bone marrow was collected at the end of the study to assess complete engraftment.

[0236] [Table 19]

[0237] Termination. Animals were humanely euthanized if there was unacceptable toxicity and / or severe weight loss (>20%), or if they showed continued deterioration before reaching coma. Animals showing obvious signs of severe distress and / or pain were humanely euthanized by CO2 followed by cervical dislocation. The study was terminated when complete grafting (>90%) was observed in one group of animals. [Example]

[0238] Human studies Stem cell mobilization and harvest. Three days prior to harvest, patients were transfected with CD34 + To mobilize stem cells, a 3- to 5-day G-CSF regimen was initiated. + The number of cells is 10.0 to 20.0 × 10 per kg of body weight. 6 When the number of cells exceeded 100, apheresis was performed. + Stem cells were transported to a GMP facility in a controlled transport at 2-8°C for isolation and transduction. The target was 3.0-4.0 x 10 6 CD34 + Cells / kg, 2 to 3 x 10 6 On day 2 after collection (or day -5 pre-transplant), patients were administered 15 mg / m 2 Patients were treated with 1000 mg / m fludarabine for 5 days (until pre-transplant day -1). Alternatively, on pre-transplant day -1, patients were treated with 4 mg / kg busulfan. Patients were then treated with a single dose of 1000 mg / m on pre-transplant day -2. 2 The patient was treated with cyclophosphamide.

[0239] Cryopreservation of stem cells. Patient-derived cells (containing hematopoietic stem cells) were optionally centrifuged to obtain a cell-rich pellet. A solution consisting of heparinized Plasmalyte solution and 10% DMSO (dimethyl sulfoxide) was added to the plasma supernatant, in which the pelleted cells were resuspended. Cells were initially stored at -4°C, and then samples were frozen down to a target temperature of -156°C (if stored in vapor phase) to -196°C (if stored in liquid phase). Cells were optionally shipped according to standard procedures.

[0240] CD34 + Lentiviral transduction of cells. Upon arrival at the GMP facility, CD34 + Cells were isolated by magnetic bead separation. Human CD3 4 + Transduction of cells involves 24 hours of pre-stimulation of cells in medium supplemented with the cytokines 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 24 hours of exposure to vector (e.g., SEQ ID NO: 2 or SEQ ID NO: 4), both of which are supplemented with the cytokines SCF, FLT3L, and TPO (100 ng ml each) in serum-free X-Vivo 10 medium. -1 The cells were then cryopreserved and shipped to the clinical site.

[0241] Modified CD34 + Re-infusion of cells. Modified CD34 +After thawing the cells, a current standard washing protocol was used, following the New York Blood Center protocol. This involved 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 twice again to a final DMSO concentration of less than 1.7%. The washed solution was infused into the patient as soon as possible. After a period of time following cell infusion, e.g., 7-45 days, the patient began taking a low dose (50-200 mg or a non-myeloablative dose as described herein) of oral cyclophosphamide once daily to facilitate the expansion of the gene-modified bone marrow cell graft. The modified CD34 + The patient is HIV positive at the time the cells are infused. + In this case, the cells may function to treat and / or cure HIV, or may contain modified CD34 + The patient is HIV positive at the time the cells are infused. - It is possible that, in this case, the cells are functioning to prevent future HIV infection. + A schematic diagram of patient treatment is shown in Figures 9 and 10, where the patient is HIV - But it is understood that this is possible.

[0242] The above detailed description of embodiments of the present technology is not intended to be exhaustive or to limit the present technology to the precise form disclosed above. Specific embodiments of the present technology and examples thereof have been described above for illustrative purposes, but those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present technology. The various embodiments described herein can also be combined to provide further embodiments.

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

[0244] While specific embodiments have been described herein for illustrative purposes, it will also be understood that various modifications can be made without departing from the present technology. Furthermore, while advantages associated with certain embodiments of the present technology have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily must exhibit such advantages to fall within the scope of the present technology. Thus, the disclosure and related technology may encompass other embodiments not expressly shown or described herein.

Claims

1. 1. A pharmaceutical composition comprising cyclophosphamide for use in a method of performing a bone marrow transplant in a patient, said patient being administered at least one dose of non-myeloablative cyclophosphamide and a pharmaceutical composition comprising a population of cyclophosphamide-resistant modified bone marrow cells; wherein the population of cyclophosphamide-resistant modified bone marrow cells comprises a heterologous gene encoding aldehyde dehydrogenase 1 (ALDH1); The pharmaceutical composition, wherein the non-myeloablative dose of cyclophosphamide is about 0.16 mg / kg / day to less than 2.5 mg / kg / day.

2. The pharmaceutical composition of claim 1 , wherein the population of cyclophosphamide-resistant modified cells expresses ALDH1.

3. The pharmaceutical composition of claim 1, wherein the cyclophosphamide resistance of the cyclophosphamide-resistant modified cells is conferred by expression of aldehyde dehydrogenase 1 (ALDH1).

4. The pharmaceutical composition of claim 1, wherein the patient has HIV.

5. The pharmaceutical composition of claim 1, wherein the bone marrow cells are bone marrow-derived progenitor cells or bone marrow-derived stem cells.

6. The pharmaceutical composition of claim 5 , wherein the bone marrow-derived stem cells are hematopoietic stem cells (HSCs).

7. The pharmaceutical composition of claim 5, wherein the bone marrow-derived stem cells are CD34+ cells.

8. 10. The pharmaceutical composition of claim 1, wherein the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

9. The method includes contacting unmodified cells with an expression vector encoding expression of ALDH1; 10. The pharmaceutical composition of claim 1, further comprising producing a population of modified cells that are cyclophosphamide resistant.

10. The pharmaceutical composition of claim 9 , wherein the expression vector is a viral vector or a non-viral vector.

11. The pharmaceutical composition of claim 10, wherein the viral vector is a lentiviral vector or an adenoviral vector.

12. 10. The pharmaceutical composition of claim 9, wherein the expression vector is a retrovirus, a transposon, an episomal expression vector, modified RNA, a plasmid, or any combination thereof.

13. 2. The pharmaceutical composition of claim 1, wherein the non-myeloablative dose of cyclophosphamide is about 1.3 mg / kg / day.

14. 2. The pharmaceutical composition of claim 1, wherein the population of cyclophosphamide-resistant modified cells does not cause the patient to experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, low platelets, low white blood cells, or any combination thereof or related symptoms.

15. 10. The pharmaceutical composition of claim 1, wherein the modified cells are resistant to HIV infection.

16. 16. The pharmaceutical composition of claim 15, wherein the modified cells heterologously express at least one HIV coreceptor mutation that is resistant to HIV infection, one or more mutations in at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces expression of an HIV coreceptor, or any combination thereof.

17. 16. The pharmaceutical composition of claim 15, wherein the modified cells heterologously express shCCR5, shCXCR4, a C-peptide fusion inhibitor, or any combination thereof.

18. 16. The pharmaceutical composition of claim 15, wherein the modified cells do not express an HIV co-receptor.

19. 16. The pharmaceutical composition of claim 15, wherein the modified cells do not express CCR5, CXCR4, or express CCR5-Δ32, or a combination thereof.

20. A pharmaceutical composition comprising cyclophosphamide for use in a method for expressing a molecule of interest in a subject undergoing bone marrow transplantation, the method comprising administering to the subject cells that heterologously express ALDH1 and the molecule of interest; and administering a non-myeloablative dose of cyclophosphamide, wherein the non-myeloablative dose of cyclophosphamide is from about 0.16 mg / kg / day to less than 2.5 mg / kg / day.

21. The pharmaceutical composition of claim 20, wherein the bone marrow cells are CD34+ cells.

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