CN115605084A - Compositions and methods for providing chemoprotection - Google Patents

Abstract

The present disclosure relates to compositions and methods for providing chemoprotection to target cells. In some embodiments, the methods comprise providing a heterologous glutamate-cysteine ligase (GCL) modified subunit (GCLM), GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function. In some embodiments, the heterologous gene/protein is GCLM.

Description

Compositions and methods for providing chemoprotection

technical field

The present disclosure relates to compositions and methods for providing chemoprotection to target cells.

Background technique

A huge hurdle to gene-modified stem cell therapy is the lack of enough transplants. One proposed solution is to make these cells chemoresistant to cytotoxic agents to select for them in vivo. However, it has been challenging to find a single resistance gene that confers sufficient resistance to agents that are especially toxic to hematopoietic stem/progenitor cells (HSPCs). Genes that confer multidrug resistance can yield promising results, but they come with safety concerns. Due to its high toxicity to HSPC, Busulfan is mainly used as a preconditioning regimen in HSPC transplantation. Therefore, improved chemoprotective compositions and methods are urgently needed.

Contents of the invention

Provided herein are methods and compositions for providing or improving chemoprotection in target cells. In some embodiments, the method comprises providing a heterologous glutamate-cysteine ligase (GCL) modifying subunit (GCLM), GCLC (GCL catalytic subunit), GCL (as a dimer or holoenzyme) Or any other polypeptide in the GSH synthetic pathway that provides an equivalent function. In some embodiments, the heterologous gene/protein is GCLM.

In some embodiments, methods for providing or improving chemoprotection in a patient in need thereof are provided. In some embodiments, the method comprises administering to the patient a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (GCL catalytic subunit), GCL (as a dimer or as a whole enzyme) Or any other polypeptide in the GSH synthetic pathway that provides an equivalent function. In some embodiments, the heterologous gene/protein is GCLM.

Additional embodiments provide glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (as a dimer or holoenzyme), or GSH in the synthetic pathway. Transgenic expression of any other polypeptide of equivalent function and allows in vivo selection after transplantation.

A further embodiment provides that the cells are stem cells or immune cells.

In some embodiments, the stem cells are fetal stem cells, cord blood-derived stem cells, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), induced PSCs (iPSCs), embryonic stem cells (ESCs), or cells derived therefrom, e.g. CD34+ cells, CD90+ cells, CD45+ cells, CD17+ cells, CD45RA- cells, or any combination thereof.

In some embodiments, the immune cells are T cells.

In some embodiments, the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

In some embodiments, the method further comprises subjecting the unmodified cell to a modified subunit encoding glutamate-cysteine ligase (GCL) GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or intact Enzymatic form) or expression vectors for the expression of other polypeptides in the GSH synthesis pathway that provide equivalent functions. In some embodiments, the expression vector encodes GCLM.

In some embodiments, the expression vector is a viral vector or a non-viral vector.

In some embodiments, the viral vector is a lentiviral or adenoviral vector.

In some embodiments, the expression vector is a retrovirus, transposon, episomal expression vector, modified RNA, plasmid, or any combination thereof.

In some embodiments, the patient is undergoing gene therapy, cell therapy, or CAR-T therapy.

In some embodiments, the chemical protection provided is against busulfan and/or naphthalene.

Description of drawings

Figure 1 shows the glutathione synthesis pathway and busulfan utilization.

Figure 2A shows lentiviral engineered constructs overexpressing human GCLM and GFP.

Figure 2B illustrates an embodiment provided herein.

Figures 3A and 3B show that the proliferation rate was unchanged and GSH was increased in GCLM-transduced cells. GSH levels changed by 2.1, 1.7 and 1.9 fold between non-transduced and transduced Jurkat, CEM and TF-1a cells, respectively. Untransduced CEM cells were found to be most sensitive to busulfan exposure. Overexpression of GCLM had no effect on cell viability and proliferation.

Figure 4 shows that GCLM-transduced CEM cells maintain high levels of GSH. Figure 4 shows that GSH levels increased 1.7-fold in GCLM-transduced cells and remained at least 3.5-fold higher in transduced cells at different doses and times 24 hours after busulfan exposure.

Figure 5 shows GCLM-transduced cells protected at low doses of busulfan. GCLM-transduced CEM cells showed at least a 3.5-fold higher proliferation rate/protection against lower doses of busulfan exposure.

Figure 6 shows GCLM-transduced cells protected at higher doses of busulfan. At higher doses of busulfan, non-transduced cells all died within 96 hours, while viable transduced cells expanded 5-fold and 3-fold at 10 and 20 μg/ml busulfan, respectively.

Figure 7 shows the proliferation of GCLM-transduced CEM cells following busulfan exposure for 1 or 2 hours. GCLM-transduced CEM cells were similarly protected in the face of 10 or 20 μg/ml and 1 or 2 hours of busulfan exposure and reached 100% after 96 hours from 20% of the initial transduced population.

detailed description

Although not always explicitly stated, it should be understood that all numerical designations begin with the term "about". As used herein, the term "about" means that the numerical values are approximate and that small variations do not significantly affect the practice of the disclosed embodiments.

It must be noted that, as used herein and in the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells.

definition

As used herein, the following terms have the following meanings.

The term "about" when used before a numerical designation such as temperature, time, amount, concentration, etc., includes ranges and means approximations that may vary (+) or (-) 20%, 10%, 5% or 1%.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when construed in an alternative ("or").

The terms "administering", "administer" and the like refer to introducing an agent (eg, a cell) into a subject. Generally, an effective amount is administered, which amount can be determined by the treating physician or the like. Any route of administration may be used, eg topical, subcutaneous, peritoneal, intravenous, intraarterial, inhalation, vaginal, rectal, nasal, oral, buccal, introduction into the cerebrospinal fluid or instillation into body compartments. When used in conjunction with a composition (and grammatical equivalents), the terms and phrases "administering" and "administration of" refer to direct administration, either by a medical professional to a patient or by a patient. Self-administration; and/or indirect administration, which may be the act of prescribing. For example, a physician instructing a patient to self-administer the agent (eg, cells) and/or providing a drug prescription to the patient administers the agent to the patient. "Regular administration" or "periodical administration" refers to multiple treatments per day, week or month. Regular administration can also refer to administering the agent once, twice, three times or more per day.

As used herein, the terms "comprising" (and any form of comprising, such as "comprise", "comprises" and "comprised"), "having" (and having any form of including, such as "have" and "has"), "including" (and any form of inclusion, such as "includes" and "includes") or " Containing" (and any form of containing, such as "contains" and "contains") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any step or combination using the transitional phrase "comprise" or "comprising" can also be said to be the same as described by the transitional phrase "consisting of" or "consists".

An "effective amount" is an amount of an agent or compound (eg, a cell or population of cells) sufficient to achieve a beneficial or desired result. An effective amount may be one or more administrations, applications or administrations. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks.

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 by the manipulation of the user and is not naturally occurring. For example, a heterologous gene refers to a gene expressed by a vector or other vehicle placed in a cell, or refers to a gene whose genome has been modified by gene editing methods (such as CRISPR) or other recombination techniques to replace a gene in a cell. Those skilled in the art will appreciate that the term "heterologous" does not refer to a gene that is naturally present in the genome of a cell that has not been modified. "Heterologous" may also be referred to as "exogenous". Thus, in some embodiments, a cell can express the same protein heterologously and from its own genome (endogenous).

The term "isolated" as used herein with reference to a nucleic acid such as DNA or RNA refers to a molecule that is separated from other DNA or RNA, respectively, present in the macromolecule's natural source. As used herein, the term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemical precursors when chemically synthesized. Substance nucleic acid or peptide. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur in nature as fragments and are not found in their natural state. An "isolated cell", such as an isolated bone marrow cell, is a cell that is substantially free of other cellular material, tissue, culture medium of its naturally occurring environment.

The term "myeloablative" means a treatment that causes long-term (usually irreversible) cytopenia of various types, kills cells in the bone marrow within 1 to 3 weeks after administration and does not allow autohematological 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 CTX per day or higher over a period leading to cumulative toxicity (McKinley et al., Clin J Am Soc Nephrol. 2009, 4:1754-1760).

The term "non-myeloablative" means a treatment that causes no, minimal or reversible cytopenias with minimal toxicity. The non-myeloablative regimen is immuno-ablation. Examples of nonmyeloablative doses include, but are not limited to: approximately 1.3 mg/kg per day for a period of time that does not result in cumulative toxicity; or 1.0 to 1.5 mg/kg per 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 included in the definition of nonmyeloablative dose. An agent or dose of an agent that results in "cumulative toxicity" is that dose that, over time, will result in toxicity in a patient. For example, cyclophosphamide administered to humans at a dose of 2.5 mg/kg per day over a period of several weeks resulted in cumulative toxicity. Other examples of non-myeloablative doses can be found, eg, in US Publication No. 2019/0269734, which is incorporated herein by reference in its entirety.

"Subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate such as a primate, mammal or preferably a human. Mammals include, but are not limited to, equines, canines, bovines, ovines, murines, rats, apes, and humans.

The term "sequence identity" in reference to protein or amino acid sequences (or DNA or RNA sequences) means that the sequences have been aligned and the After introducing gaps, the percentage of amino acid residues (or nucleotide residues) in the candidate sequence that are identical to the amino acid residues in a specific protein or amino acid sequence (or nucleotide residues in a specific DNA or RNA sequence) . Alignment can be achieved by any method known to those skilled in the art, for example by using publicly available programs such as BLAST and EMBOSS. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, but in some embodiments default parameters are used. These programs can be accessed, for example, at the National Center for Biotechnology Information.

As used herein, the term "variant" is a nucleic acid or protein that differs from a reference nucleic acid or protein but retains essential properties (ie, biological activity). A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and/or truncations in the polypeptide encoded by the reference sequence.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, for example, a carrier nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into cellular DNA. Vectors include, for example, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, bacterial or yeast artificial chromosomes, and viral vectors. Useful viral vectors include, for example, adenoviruses, retroviruses (such as, but not limited to, replication defective retroviruses), and lentiviruses.

The phrase "pharmaceutically acceptable" is used herein to refer to those substances which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications, and reasonable benefits. Compounds, materials, compositions and/or dosage forms with a commensurate risk/risk ratio.

As used herein, the term "GCLM" when referring to a protein form refers to a protein comprising the sequence of SEQ ID NO: 1 or a variant thereof.

MGTDSRAAKALLARARTLHLQTGNLLNWGRLRKKCPSTHSEELHDCIQKTLNEWSSQINPDLVREFPDVLECTVSHAVEKINPDEREEMKVSAKLFIVESNSSSSTRSAVDMACSVLGVAQLDSVIIASPPIEDGVNLSLEHLQPYWEELENLVQSKKIVAIGTSDLDKTQLEQLYQWAQVKPNSNQVNLASCCVMPPDLTAFAKQFDIQLLTHNDPKELLSEASFQEALQESIPDIQAHEWVPLWLLRYSVIVKSRGIIKSKGYILQAKRRGS(SEQ ID NO:1)

In some embodiments, the GCLM protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or substantially identical to SEQ ID NO:1. The sequence of this protein is also provided in Genbank Accession No. NM_002061.3 or NP_002052.1 (each incorporated herein by reference in its entirety). Genbank accession number NM_002061.3 provides an example of a nucleic acid molecule encoding SEQ ID NO:1.

As used herein, the term "GCLM" when referring to nucleotide form refers to a nucleic acid molecule encoding GCLM. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO:2 or a variant thereof, wherein such variant may be substantially identical to SEQ ID NO:2. Because of the degeneracy of the genetic code, SEQ ID NO: 2 is a non-limiting example, and other nucleotide sequences may be used to encode a GCLM protein or a protein identical or substantially identical thereto. SEQ ID NO: 2 is as follows:

ACCCGTCGCCACGCCCGCCGCAGGCCAAGGGCCAGTCACTTGCGGGCCGGCGTCCCGCAGCCCATTCGCGCCCCGCCCCTGCCCCGCCGCGGGATGAGTAACGGTTACGAAGCACTTTCTCGGCTACGATTTCTGCTTAGTCATTGTCTTCCAGGAAACAGCTCCCTCAGTTTGGAATCAGCTCTCCCGCTGCGGCCGCAGTAGCCGGAGCCGGAGCCGCAGCCACCGGTGCCTTCCTTTCCCGCCGCCGCCCAGCCGCCGTCCGGCCTCCCTCGGGCCCGAGCGCAGACCAGGCTCCAGCCGCGCGGCGCCGGCAGCCTCGCGCTCCCTCTCGGGTCTCTCTCGGGCCTCGGGCACCGCGTCCTGTGGGGCGGCCGCCTGCCTGCCCGCCCGCCCGCAGCCCCTTCGCTGCGCGGCCCCTGGGCGGCCGCTGCCATGGGCACCGACAGCCGCGCGGCCAAGGCGCTCCTGGCGCGGGCCCGCACCCTGCACCTGCAGACGGGGAACCTGCTGAACTGGGGCCGCCTGCGGAAGAAGTGCCCGTCCACGCACAGCGAGGAGCTTCATGATTGTATCCAAAAAACCTTGAATGAATGGAGTTCCCAAATCAACCCAGATTTGGTCAGGGAGTTTCCAGATGTCTTGGAATGCACTGTATCTCATGCAGTAGAAAAGATAAATCCTGATGAAAGAGAAGAAATGAAAGTTTCTGCAAAACTGTTCATTGTAGAATCAAACTCTTCATCATCAACTAGAAGTGCAGTTGACATGGCCTGTTCAGTCCTTGGAGTTGCACAGCTGGATTCTGTGATCATTGCTTCACCTCCTATTGAAGATGGAGTTAATCTTTCCTTGGAGCATTTACAGCCTTACTGGGAGGAATTAGAAAACTTAGTTCAGAGCAAAAAGATTGTTGCCATAGGTACCTCTGATCTAGACAAAACACAGTTGGAACAGCTGTATCAGTGGGCACAGGTAAAACCAAATAGTAACCAAGTTA ATCTTGCCTCCTGCTGTGTGATGCCACCAGATTTGACTGCATTTGCTAAACAATTTGACATACAGCTGTTGACTCACAATGATCCAAAAGAACTGCTTTCTGAAGCAAGTTTCCAAGAAGCTCTTCAGGAAAGCATTCCTGACATTCAAGCGCACGAGTGGGTGCCGCTGTGGCTACTGCGGTATTCGGTCATTGTGAAAAGTAGAGGAATTATCAAATCAAAAGGCTACATTTTACAAGCTAAAAGAAGGGGTTCTTAACTGACTTAGGAGCATAACTTACCTGTAATTTCCTTCAATATGAGAGAAAATTGAGATGTGTAAAAATCTAGTTACTGCCTGTAAATGGTGTCATTGAGGCAGATATTCTTTCGTCATATTTGACAGTATGTTGTCTGTCAAGTTTTAAATACTTATCTTGCCTCCATATCAATCCATTCTCATGAACCTCTGTATTGCTTTCCTTAAACTATTGTTTTCTAATTGAAATTGTCTATAAAGAAAATACTTGCAATATATTTTTCCTTTATTTTTATGACTAATATAAATCAAGAAAATTTGTTGTTAGATATATTTTGGCCTAGGTATCAGGGTAATGTATATACATATTTTTTATTTCCAAAAAAAATTCATTAATTGCTTCTTAACTCTTATTATAACCAAGCAATTTAATTACAATTGTTAAAACTGAAATACTGGAAGAAGATATTTTTCCTGTCATTGATGAGATATATCAGAGTAACTGGAGTAGCTGGGATTTACTAGTAGTGTAAATAAAATTCACTCTTCAATACATGAATGGAAACTTAAATTTTTTTTTATGTGTCCTTGCTTATAGTTTAGCTGTAATAATTTAACCTTGTATTCTTGTGCCATATTCTGTCTTTTTATTACTTATAAAGACAAACCAAAGTAAATCTGAAAGGAGACTAGAAGCTTTGAAATTATTGTTTGGGGGTTTTATAAAAGCAACTACTGTCACCTCCATCCAGATTCTTTTA AATTATTGATCCATCCATAGTATATATTGCTACTCATTCAAGAATCCTCAATAAGTATTGAGTATTTACCATATGTTGGGATACTGTGGGCTCTGGAGAGAGGAGGGGGCAATAGAGCTAGGAATTAAGAATCAGTTGAGTAAAATGTGTAATATTTATTCCCCATTAATAACTGACTAGGAAGGACTAAAAGCCAGAAAGGGGATGAAAAAAAAATCCTTAATTCAGGGCCGACATTATCTACTTAAACAACTTTGAGATATGGTCTTAATTATTTTAAAGCAGAATAATATAATTGAAAGTTTATAGCTAAAAGAGACTATATAGGTCATTTAGTATAATTCTTCATTAGTTTACGAACCACAAAATTGCAAATAAATAAGCTATGAACTTTGATGTACACTATAAATCTCCTTAATTCTATAAATTTGTGTCTGTAACCTGAATAGTTTGAAAACTTCTTTAAAAATCTCTTGTATTTCATCCGGGCGCAGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACGAGGTCAGGAGTTTGAGACCAGCCTGACCAACATGGTAAAACCCCATCTCTACTAAAATACAAAAATTGGCTGGGCGTGGTGGCACTCGCCTGTAATCTCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTACAGTGAGCCGAGATCACATCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCATCTCAAAAAAAAAAAAAAACTCTTGTATCTCAATATTTTTAAACCACAGGCCTAAATAAAACTAATTTTGCTCAAGTTTTCTCAACCTAGGGAAAAAGAACTATGGTTCCATATTCAAAATAAATATTATAGACCCTTTTCCTAAGTAGGATTTTGTGGTTTACTGATTGGGTAATTTGATCATTAAAATTATGTGAAATCTGCCCGGGCACACCTCATGCCTGTAATCCCAGCACTCTGGGA GGCCAAGGCAGATGATCACCTGAGGTCAGGAGTTCTAGACCAGCCTGGCTAACATGGTGAAACCCTGTATCTGCTAAAAATACAAAAATTAGCCAGGCGTGGTGGCGGGCTCCTGTAATCCCAGCTACTTTGGAGGCGAGGCACGAGAATCGCTTGAACCTGGGAGGCGGAGTTTGCAGTGAGCCGAGATCACGCCATTGCACTCCAGCCTGGGCGACAGAGCGAGACTGCGTCTCAAAAAAAAAAAAAAAAGAAAAATTATGTGAAATCATGTGATTTGCCTGGGAAAACTTGTTTAGATATTGAGCTACTTATGCCTTCTAGCCTTTATATTAATTGTATGTAATGTTATTAAATATATATATAGTTCATCTTTACATTTGGAAATGCCCAACATTTTTTTCATATAAGTCCTTAAACAAGCGTTCATTTTATTTTAAATCTATACAGTGAACTGGCCAAGATATTTTAAGAGGGAACTTTAATATCCCATTTATTGTTTTTATAACCCTGGACTTATAAAAATGGGTTGTTTGAAGGGTTATTTTGAAAGTGGGGGAAAAAAAAACTTAGTTGCTAATGTATCTAAACTTCAGCAGAGCTTTTTGGTGATCTCCTACCTGCACCCTCAACTCTTGACAAAGAAGCAAGACTATAGATTCATTTTCTGAAGGGGATCATGTATGGAATTTTTTGATGAGTTTTTACTTTTACCTCTCTACTCTTGATTTTCTATTATTGAATACTCTTTTAAAACACTGATTTTTAAGGCTTTATATATGTTTTCCAGGCTGATGTTCACATCTTTTTTTCATGAACTATCAGAATATAGTGAACACTTTTCAAATATTTAAGGACTTAATGTTTAAAAAGCCATAAAATAGAGAGTGGTAATACTACCAAATAATTACTTAAAACTGAAAGCTAAGTTATCAATAGTTTATATAAGAGATGTTTTCTGAGGAGATGTGCATCCAGTGAGACCAAGGTAGAAAGTTTA TATAATTGTTTTTTTTCCAGTAAATATGAAAAAAAAAGCTGTAGCTTGTTTATTACATGTCCAAAATACAGTGGAGCCTTACTTTAACACAATGTACTGTAACTTGGAATTTGTTCTGTTATGAGTCTATCTTGAATTCCCATCCATGAAACTGTAGTCACCAAAAGCAACAAGTATTTTCACATGATGTAAAAGACCATACTATGATGGCCATTGCTAGAAATTGAATCACAAATAATAGCTAATAATTTTTCATTTTTCAAAAAAGATCATTTGGATAGCAGCTATGTATAAAATGGAAAATAAAAAATTATTCTATTTTGCATGAATAGTTCAGACTTTCCCATACCACAGCCAAGCAGTAACTAAAATTAGGATCTTAATTTTCAATGATAAAAGGTCTAAGGTTCATTTAATTATGCTCCTTTAACACTGTCTTTCTAGATTTTTCACCCAGTATTTTCAAAATTTGGGAATGTAAACAATTGATATATTTATTGTATGTTGGCTAGCAGTTCATCCTTCTGCAAAATATGCATTCAGAGAAATGTGAAGCTTGTTTTAATGAAGACTTAAACCATTTGTGTCATTTGTGTTTTCATATTCAAATACACCAAATTAAAATTCTGAACCTATATTTTTCATCATTAACTTCCTAATATACCAGAACATATACCTTTTTCATGTAAAGTTGGCAATGGGATATGGCAGTTTTATTTTTGAAAAATATGTAACATGACTTTAATATTTTTATAGTTTTCAGAATTAGAAACATAGGAAGGGAAAATGTTTTAATTAGATAAGTCAACTTTTTATGTGTCTGTAGTGGTGTACTATAATAGCAAATTATAAAGCATTATTAAATGTTTATAATAATTTTTAATATTACCTACATTATGAATTTAACTAAAATAAAGTGTGAGTTGTATATTTTTTAATTGGGTTGTTTCAATAGCTGGAAGCATCCTGAAGCATTATATTGATTTTTGAACTATTTGAA CTCAAACTGAGTATGATTTGAAAATAAATTAATAATTTAAAAACATCCAAAAAAAAAAAAAAAAAA (SEQ ID NO: 2).

In some embodiments, the nucleotide sequence comprises the coding region of SEQ ID NO:2, ie, nucleotides 436 to 1260 of SEQ ID NO:2. In some embodiments, the nucleic acid molecule comprises only the coding region of SEQ ID NO: 2 or the sequence encoding the GCLM protein, or variants thereof. Variants as described herein may have substitutions or mutations. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the GCLM protein variant is substantially identical to SEQ ID NO:1.

Sequence identity, percent identity and related terms, as those terms are used herein, refer to the relatedness of two sequences, eg, two nucleic acid sequences or two amino acid or polypeptide sequences. In the context of amino acid sequences, the term "substantially identical" is used herein to mean that a first amino acid contains a sufficient or minimal number of amino acid residues that are i) identical to an aligned amino acid residue in a second amino acid sequence, or ii) Conservative substitutions are made such that the first and second amino acid sequences may have a common domain and/or a common functional activity. For example, an amino acid sequence that contains a common domain has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the context of nucleotide sequences, the term "substantially identical" is used herein to mean that a first nucleic acid sequence contains a sufficient or minimal number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first The first and second nucleotide sequences encode polypeptides having a common functional activity, or encode common structural polypeptide domains or common functional polypeptide activities. For example, a nucleotide sequence is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence, such as a sequence provided herein identity.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm using the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16 , 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6 determined, the algorithm has been incorporated into the GCG software package (available at http://www.gcg.com acquired) in the GAP program. In some embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weight 1, 2, 3, 4, 5 or 6 determined. A particularly preferred set of parameters (and parameters that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can be calculated using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), using a PAM120 weighted residual table, a gap length penalty of 12 and gap penalty 4 determined, this algorithm has been incorporated into the ALIGN program (version 2.0).

It is understood that the molecules and compounds of the embodiments of the present invention may have conservative or nonessential amino acid substitutions which do not substantially affect their function. The term "amino acid" is intended to cover all molecules, whether natural or synthetic, including amino functional groups and acid functional groups and capable of being included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners thereof; amino acid analogs having variable side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D-optical isomers or L-optical isomers and peptidomimetics. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine acid, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyramine acid, phenylalanine, tryptophan, histidine).

The cells used in the disclosed chemoprotection 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 cord blood cells, fetal stem cells, 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, cord blood-derived hematopoietic stem/progenitor cells, umbilical cord tissue-derived stem/progenitor cells, iPSCs, HESCs, fetal tissue-derived stem cells, CD4+ cells, and the like. In some embodiments, the stem cells are CD34+.

The compositions and methods described herein are useful in bone marrow transplant settings, such as those described in WO2020/018413.

For example, in some embodiments, provided herein are methods for performing bone marrow transplantation in a patient in need thereof. Also provided are methods for replacing bone marrow cells in a subject with a population of cells expressing a heterologous nucleic acid molecule expression cassette or cassettes, or with cells whose genome has been edited and differs from that of the subject. In some embodiments, the expressed heterologous protein is GCLM or a variant thereof. In some embodiments, the methods comprise administering to the patient one or more chemotherapy resistance modified cells and administering at least one dose of a chemotherapeutic agent. In certain embodiments, the dose is a non-myeloablative dose of a chemotherapeutic agent. In certain embodiments, the dose is a myeloablative dose of a chemotherapeutic agent. In some embodiments, the amount of cells is a therapeutically effective amount. In some embodiments, the chemotherapy administered is busulfan. In some embodiments, the chemotherapeutic agent is one or more of: actinomycin, all-trans retinoic acid, azacitidine, thiazole Azathioprine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine (BCNU), cisplatin (cisplatin), chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, multi doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin (idarubicin), imatinib, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, mercaptopurine ( mercaptopurine), methotrexate, mitoxantrone, nimustin (ACNU), oxaliplatin, paclitaxel, pemetrexed, Temezolamide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, valrubicin, Vemurafenib, vinblastine, vincristine, vindesine, and vinorelbine.

As provided herein, in some embodiments, the chemoresistance-modified cells used in the disclosed methods can be any suitable cells known to those of skill in the art. For example, the cells can be stem cells or immune cells. Non-limiting examples of stem cells include cord blood cells, fetal stem cells, 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, cord blood-derived hematopoietic stem/progenitor cells, umbilical cord tissue-derived stem/progenitor cells, iPSCs, HESCs, fetal tissue-derived stem cells, CD4+ cells, and the like. In some embodiments, the stem cells are CD34+.

The chemoresistance-modified cells used in the methods of the invention can be cells from the patient (i.e., autologous cells), cells from a donor (i.e., allogeneic cells), or any combination thereof, for which Cells are modified to confer chemotherapy resistance by expressing heterologous proteins such as GCLM or variants thereof. 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 method further comprises modifying the cell to be chemoresistant. For example, in certain embodiments, there is provided a method for performing bone marrow transplantation in a patient in need thereof, the method comprising isolating and/or purifying one or more cells from the patient or subject, as described herein modifying one or more cells to be chemoresistant, administering to a patient an effective amount of the one or more chemoresistant modified cells, and administering at least one dose of a chemotherapeutic agent.

Cells can be isolated by any method known to those skilled in the art, for example based on expression/lack of expression of certain markers, proliferation rate and differentiation potential. In some embodiments, cells are isolated based on the presence of specific markers or combinations of markers, including, for example, CD34, CD4, Sca-1 CD38, CD123, CD90, CD45, CD133, antigen presenting cell markers (CD8 , CD8α, CD11b, CD11c, CD103, CD205, CD24, CD115, CD117, CD135, CD11c ^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 specific markers, such as CD3, CD14, CD19, CD56, and/or CD66b. In other embodiments, negative selection is performed for markers such as T cells, B cells, granulocytes and/or myelomonocytes. 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 Lin ^Thy1+Sca-1+ expression profile. In some embodiments, mouse HSCs can be isolated by expression profile CD34 ⁻ , Scal ⁻ , c-kit ⁺ . In some embodiments, human HSCs can be isolated based on CD34 expression.

In some embodiments, the methods provided herein comprise administering to a patient one or more doses of a chemotherapeutic agent. In some embodiments, the dose is a non-myeloablative dose of a chemotherapeutic agent. In some embodiments, the dosage is myeloablative. The chemotherapeutic agent may be any suitable chemotherapeutic agent known to those skilled in the art. Non-limiting examples of chemotherapeutic agents include: actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin , carmustine (BCNU), cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin star, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, lomustine (CCNU), dichloromethyldiethylamine, melpha Lun, mercaptopurine, methotrexate, mitoxantrone, nimustine (ACNU) oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, thiotepa, thioguanine, Topotecan, trosulfan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, and vinorelbine.

In some embodiments, the methods provided herein comprise administering to a subject an effective amount of chemoresistance-modified cells and a non-myeloablative dose of a chemotherapeutic agent. The modified cells and chemotherapeutic agent may be administered by any suitable route, as will be apparent to the skilled artisan, depending on the disease or condition to be treated. Typical routes of administration include intravenous, intraarterial, intramuscular, subcutaneous, intracranial, intranasal, intradermal, oral or intraperitoneal.

In some embodiments, about 1 x ¹⁰⁸ to about ¹ x 1011 cells are administered to the subject per square meter of the subject's body surface area. Cells can be administered to an individual by the absolute number of cells, e.g., the individual can be administered from about 1000 cells/injection up to about 10 billion cells/injection, e.g., about, at least about, or at most about ¹ x 108 per injection , 1×10 ⁷ pieces, 5×10 ⁷ pieces, 1×10 ⁶ pieces, 5×10 ⁶ pieces, 1×10 ⁵ pieces, 5×10 ⁵ pieces, 1×10 ⁴ pieces, 5×10 ⁴ pieces, 1 x ¹⁰³ , 5 x ¹⁰³ (etc.) cells, or any range between any two numbers, inclusive. In some embodiments, about 5 x 10 ⁶ /kg to about 10 x 10 ⁶ /kg cells are used for HSC transplantation.

In other embodiments, the subject may be administered from about 1000 cells/injection/square meter up to about 10 billion cells/injection/square meter, for example about, at least about or at most about 1×10 ⁸ /m² per injection ² , 1×10 ⁷ /m ² , 5×10 ⁷ /m ² , 1×10 ⁶ /m ² , 5×10 ⁶ /m ² , 1×10 ⁵ /m ² , 5×10 ⁵ /m ² , 1×10 ⁴ /m ² , 5×10 ⁴ /m ² , 1×10 ³ /m ² , 5×10 ³ /m ² (etc.) cells, or any range between any two numbers, inclusive .

In other embodiments, the cells can be administered to such individuals by relative numbers of cells that can be administered to such individuals from about 1000 cells per kilogram of individual up to a maximum of about 10 billion cells, such as about, at least about, or at most about 1×10 ⁸ , 5×10 ⁷ , 1×10 ⁷ , 5×10 ⁶ , 1×10 ⁶ , 5×10 ⁵ , 1×10 ⁵ , 5×10 ⁴ , 1× 10 ⁴ , 5 x 10 ³ , 1 x 10 ³ (etc.) cells, or any range between any two numbers, inclusive.

In some embodiments, at least one non-myeloablative dose of a chemotherapeutic agent is administered to the patient. Administration of the chemotherapeutic agent may be simultaneous or sequential to administration of the modified cells. In some embodiments, at least one non-myeloablative dose of a chemotherapeutic agent is administered after administering the modified cells. In some embodiments, a preconditioning step (also referred to herein as a "pretreatment step") is performed prior to administration of the cells, wherein at least one dose of a chemotherapeutic agent, such as fludarabine, is administered to the patient prior to administration of the modified cells. ) or cyclophosphamide. In some embodiments, the preconditioning step is a non-myeloablative chemotherapeutic agent preconditioning step. In some embodiments, no preconditioning step is performed prior to administration of the cells. It is expected that even without a preconditioning step (eg, fludarabine) prior to administration of the cells, the cells of the present disclosure will still be able to effectively engraft into the patient's bone marrow.

In some embodiments, the at least one non-myeloablative dose of the chemotherapeutic agent for the human subject or patient is a non-myeloablative dose of busulfan. In some embodiments, the non-myeloablative dose of cyclophosphamide is about 0.1 mg/kg/d to about 8 mg/kg/d, about 0.1 mg/kg/d to about 7 mg/kg/d, about 0.1 mg/kg /d to about 6 mg/kg/d, about 0.1 mg/kg/d to about 5 mg/kg/d, about 0.1 mg/kg/d to about 4 mg/kg/d, about 0.1 mg/kg/d to about 3 mg /kg/d, about 0.1 mg/kg/d to about 2 mg/kg/d, about 0.1 mg/kg/d to about 1 mg/kg/d, about 0.5 mg/kg/d to about 8 mg/kg/d, About 0.5 mg/kg/d to about 7 mg/kg/d, about 0.5 mg/kg/d to about 6 mg/kg/d, about 0.5 mg/kg/d to about 5 mg/kg/d, about 0.5 mg/kg /d to about 4 mg/kg/d, about 0.5 mg/kg/d to about 3 mg/kg/d, about 0.5 mg/kg/d to about 2 mg/kg/d, about 0.5 mg/kg/d to about 1 mg /kg/d, about 1.0 mg/kg/d to about 8 mg/kg/d, about 1.0 mg/kg/d to about 7 mg/kg/d, about 1.0 mg/kg/d to about 6 mg/kg/d, About 1.0 mg/kg/d to about 5 mg/kg/d, about 1.0 mg/kg/d to about 4 mg/kg/d, about 1.0 mg/kg/d to about 3 mg/kg/d, about 1.0 mg/kg /d to about 2mg/kg/d, about 2mg/kg/d to about 8mg/kg/d, about 2mg/kg/d to about 7mg/kg/d, about 2mg/kg/d to about 6mg/kg/d d, about 2 mg/kg/d to about 5 mg/kg/d, about 2 mg/kg/d to about 4 mg/kg/d, about 2 mg/kg/d to about 3 mg/kg/d, about 3 mg/kg/d to about 8 mg/kg/d, about 3 mg/kg/d to about 7 mg/kg/d, about 3 mg/kg/d to about 6 mg/kg/d, about 3 mg/kg/d to about 5 mg/kg/d, About 3 mg/kg/d to about 4 mg/kg/d, about 4 mg/kg/d to about 8 mg/kg/d, about 4 mg/kg/d to about 7 mg/kg/d, about 4 mg/kg/d to about 6 mg/kg/d, about 4 mg/kg/d to about 5 mg/kg/d, about 5 mg/kg/d to about 8 mg/kg/d, about 5 mg/kg/d to about 7 mg/kg/d, about 5 mg /kg/d to about 6mg/kg/d, about 6mg/kg/d to about 8mg/kg/d, about 6mg/kg/d to about 7mg/kg/d, or about 7mg/kg/d to about 8mg/d kg/d. Although amounts and ranges are listed as a series, amounts that can be administered may be listed individually or as endpoints of a range. In some embodiments, the non-myeloablative dose is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg /kg, about 7mg/kg or about 8mg/kg. In some embodiments, the non-myeloablative dose is less than 8 mg/kg. Doses can be administered daily.

In some embodiments, a myeloablative dose is administered. In some embodiments, the myeloablative dose of busulfan is about 8 to about 12 mg/kg, which may be administered in one day.

In some embodiments, the non-myeloablative dose of the 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.

In some embodiments, a non-myeloablative dose is provided for a period of time that does not result in cumulative toxicity. For example, the period of time that does not result in cumulative toxicity is 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 For a period of 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.

In some embodiments, at least one interruption occurs within a time period between administration of the cyclophosphamide resistance-modified cells and at least one non-myeloablative dose of the chemotherapeutic agent. For example, the time 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 one month, about 2 months , about 3 months, about 6 months, about one year or more. In some embodiments, the period of time is about 3 days, about 7 days, about 10 days, and about 14 days.

In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about one year. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 6 months. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 5 months. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 4 months. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 3 months. In some embodiments, greater than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 2 months. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 1 month. In some embodiments, greater than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 2 weeks. In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells within about 1 week.

In some embodiments, the patient is not depleted of myeloid cells and/or immunocompromised during the method. In some embodiments, the patient is not experiencing clinically relevant anemia, neutropenia, thrombocytopenia, cytopenias, low platelets, low white blood cells, low red blood cells, or any combination thereof or associated symptoms.

In another embodiment, a subject or group of subjects may exhibit one or more of the following results following treatment with the cells and chemotherapeutics of the present disclosure:

(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%, compared to a control, 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 %);

(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% compared to a control , 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);

(iii) neutrophils increase by 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);

(iv) an increase in lymphocytes 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% compared to a control , 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);

(v) an increase in eosinophils compared to a control by 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);

(vi) increased monocytes by 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% compared to the control %, 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);

(vii) an increase in basophils compared to a control 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 %);

(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%, compared to a control, 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 %);

(ix) All three cellular components of blood (red blood cells, white blood cells, and platelets) are increased by 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);

(x) 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 without recurrence;

(xi) the recurrence-free survival time of the patient is increased by 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, compared to the control 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; or

(xii) the survival time of the patient is increased by 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.

In certain embodiments of the methods provided herein, the chemotherapy resistance modified cells may comprise one or more additional modifications that are not associated with chemotherapy resistance. For example, in certain embodiments, cells can be further modified to express additional HIV/disease-specific modifications. Accordingly, in certain embodiments, the methods of bone marrow transplantation provided herein further comprise the incorporation of one or more additional modifications, including one or more HIV/disease specific modifications. For example, in certain embodiments, there is provided a method for performing bone marrow transplantation in a patient in need thereof, the method comprising isolating and/or purifying one or more cells from the patient or subject, as described herein modifying one or more cells to be chemoresistant, incorporating one or more additional modifications into the one or more cells, administering to a patient an effective amount of the one or more chemoresistant modified cells , and administer at least one dose of a chemotherapeutic agent. In some embodiments, the dose is a non-myeloablative dose of a chemotherapeutic agent.

In some embodiments, the modified cells are further modified to be HIV resistant. For example, the modified cell can be further modified to express at least one mutant HIV coreceptor that confers resistance to HIV infection, a mutation or mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor expressions or any combination of them. In some embodiments, cells are modified to express molecules that inhibit or reduce HIV coreceptor expression. 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-Δ32 mutation. In some embodiments, both copies of the CCR5 gene in the cell are replaced by a CCR5-Δ32 mutation. In some embodiments, one copy of the CCR5 gene is replaced by a CCR5-Δ32 mutation.

The present disclosure provides cells modified to be resistant to chemotherapy, such as cyclophosphamide resistance and HIV resistance. In some embodiments, cells can be modified to be cyclophosphamide-resistant and HIV-resistant. HIV resistance may be conferred by reduced expression of at least one HIV coreceptor, mutation or mutations of at least one HIV coreceptor, expression of at least one HIV fusion inhibitor, or any combination thereof. HIV resistance can be conferred by reduced expression of the CCR5 HIV coreceptor, reduced expression of the CXCR4 coreceptor, expression of a C-peptide fusion inhibitor (eg, a C46 fusion inhibitor or a C34 fusion inhibitor), or any combination thereof.

Cells can also be modified to express any molecule of interest. Molecules of interest can be modified according to the needs of the user or a particular patient.

恩曲他滨/利匹韦林(rilpivirine)/富马酸替诺福韦二吡呋酯

埃替拉韦(elvitegravir)/可比司他(cobicistat)/恩曲他滨/富马酸替诺福韦二吡呋酯

和阿巴卡韦(abacavir)/多替拉韦(dolutegravir)/拉美夫定(lamivudine)

)、核苷/核苷酸逆转录酶抑制剂(NRTI)(例如，阿巴卡韦

依法韦伦/恩曲他滨(emtriacitabine)/富马酸替诺福韦二吡呋酯

拉美夫定/齐多夫定(zidovudine)

恩曲他滨/利匹韦林/富马酸替诺福韦二吡呋酯

恩曲他滨

拉美夫定

阿巴卡韦/拉美夫定

齐多夫定

阿巴卡韦/拉美夫定/齐多夫定(Trizivir)、恩曲他滨/富马酸替诺福韦二吡呋酯

去羟肌苷(didanosine)

去羟肌苷缓释剂(Videx

)、富马酸替诺福韦二吡呋酯

和司他夫定(stavudine)

)、非核苷逆转录酶抑制剂(NNRTI)、蛋白酶抑制剂(例如替拉那韦(tipranavir)

茚地那韦(indinavir)

阿扎那韦(atazanavir)/可比司他

沙奎那韦(saquinavir)

洛匹那韦(lopinavir)/利托那韦(ritonavir)

福沙那韦(fosamprenavir)

利托那韦

达芦那韦(darunavir)/可比司他

达芦那韦

阿扎那韦

奈非那韦(nelfinavir)

)、进入抑制剂(例如，恩夫韦肽(enfuvirtide)

)、整合酶抑制剂(例如，雷特格韦(raltegravir)

多替拉韦

和埃替拉韦

)、趋化因子辅助受体拮抗剂(CCR5拮抗剂)(例如，马拉维诺(maraviroc)

或维立韦罗(vicriviroc))、细胞色素P4503A抑制剂和基于免疫的疗法(例如，硫酸羟氯喹(hydroxychloroquine sulfate)(Plaquenil)。在一些实施方案中，修饰细胞和至少一种其它HIV疗法同时施用。在其它实施方案中，修饰细胞和至少一种其它HIV疗法依次施用。在一些实施方案中，明确排除至少一种上述其它HIV疗法的施用，例如，在一些实施方案中，明确排除NRTI。在一些实施方案中，除了本文公开的修饰细胞和至少一个非清髓剂量的化学治疗剂(例如，白消安)之外，不施用其它HIV疗法。In some embodiments, the modified cells are administered with at least one other HIV therapy. Suitable additional HIV therapies include any HIV therapy known to those skilled in the art. Non-limiting examples of other HIV therapies include combinations (e.g., efavirenz/emtricitabine/tenofovir disoproxil fumarate)

Emtricitabine/rilpivirine/tenofovir disoproxil fumarate

Elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate

and abacavir/dolutegravir/lamivudine

), nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) (eg, abacavir

Efavirenz/emtricitabine/tenofovir disoproxil fumarate

Lamevudine/zidovudine (zidovudine)

Emtricitabine/ Rilpivirine/ Tenofovir Disoproxil Fumarate

Emtricitabine

lamivudine

abacavir/lamivudine

zidovudine

Abacavir/lamevudine/zidovudine (Trizivir), emtricitabine/tenofovir disoproxil fumarate

Didanosine

Didanosine extended release (Videx

), tenofovir disoproxil fumarate

and stavudine

), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (such as tipranavir

Indinavir (indinavir)

Atazanavir/cobicistat

Saquinavir

Lopinavir/ritonavir

Fosamprenavir

Ritonavir

Darunavir/cobicistat

Darunavir

atazanavir

nelfinavir

), entry inhibitors (eg, enfuvirtide)

), integrase inhibitors (eg, raltegravir

Dolutegravir

and elvitegravir

), chemokine coreceptor antagonists (CCR5 antagonists) (eg, maraviroc

or vicriviroc), a cytochrome P4503A inhibitor, and an immune-based therapy (e.g., hydroxychloroquine sulfate (Plaquenil). In some embodiments, the modified cells are concomitant with at least one other HIV therapy Administration. 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 expressly excluded, eg, in some embodiments, NRTIs are expressly excluded. In some embodiments, no other HIV therapy is administered in addition to the modified cells disclosed herein and at least one non-myeloablative dose of a chemotherapeutic agent (eg, busulfan).

The cells, chemotherapeutics, and optionally other HIV therapies may be administered once to the HIV patient or may be administered multiple times, for example every 1 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 once, or Every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks Weeks, once in 10 weeks or more, or any range between any two numbers, inclusive.

In some embodiments, methods of treating HIV patients are provided. In some embodiments, the method comprises mobilizing the patient's CD34 ⁺ stem cells from the bone marrow into the periphery. In some embodiments, cells are mobilized by administering G-CSF (granulocyte colony stimulating factor). G-CSF can be administered, for example, as a 1-day, 2-day, 3-day, 4-day or 5-day regimen. In some embodiments, G-CSF is administered for 3-5 days. Mobilized cells can be captured using methods such as apheresis. In some embodiments, once the CD34 ⁺ cell count is at or above 10.0 to 20.0 x ¹⁰⁶ /kg body weight, the cells are isolated, eg, by apheresis. In some embodiments, the cell count is at or above 5.0 to 25.0 x ¹⁰⁶ /kg body weight. Although Cd34+ cells were used as a marker to capture cells for transduction, other cellular markers, such as those described herein, can also be used. For example, cells used are isolated based on the presence of specific markers or combinations of markers including, for example, CD34, CD4, Sca-1 CD38, CD123, CD90, CD45, CD133, antigen presenting cell markers (CD8, CD8α, CD11b, CD11c, CD103, CD205, CD24, CD115, CD117, CD135, CD11c ^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 specific markers, such as CD3, CD14, CD19, CD56, and/or CD66b. In other embodiments, negative selection is performed for markers such as T cells, B cells, granulocytes and/or myelomonocytes. 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 Lin ⁻ Thy1 ⁺ Sca-1 ⁺ expression profile. In some embodiments, mouse HSCs can be isolated by expression profile CD34 ⁻ , Sca1 ⁺ , c-kit ⁺ . In some embodiments, human HSCs can be isolated based on CD34 expression. In some embodiments, the isolated cells are CD34+ or CD4+ or any combination thereof.

In some embodiments, the method includes harvesting the cells by centrifugation. This is done, for example, to develop cell-rich clumps. Cells can then be resuspended in cryopreservation solution and frozen. In some embodiments, the cryopreservation solution comprises a solution of heparinized Plasmalyte and 10% DMSO (dimethyl sulfoxide). In some embodiments, cells are initially stored at -4°C, and samples are then frozen to a target temperature of -156°C (when stored in the gas phase) to -196°C (when stored in the liquid phase).

In some embodiments, the method comprises infusing the transduced cells into the subject. In some embodiments, the subject has HIV. In some embodiments, the subject does not have HIV, but is at high risk of HIV infection and thus desires to become HIV resistant.

In some embodiments, following infusion of the modified cells, a non-myeloablative dose of a chemotherapeutic agent, such as busulfan, is administered. Non-limiting examples of non-myeloablative doses are provided herein.

In some embodiments, there is provided a method of treating HIV in a subject, the method comprising administering to the subject a population of cells heterologously expressing ALDH1 and one of: i) encoding at least one Heterologous nucleotides of an HIV coreceptor mutant, a mutation or mutations of at least one HIV coreceptor, at least one HIV fusion inhibitor, a molecule that reduces HIV coreceptor expression, or any combination thereof molecular. In some embodiments, the method includes administering at least one non-myeloablative dose of a 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. The sequences of these proteins can be found eg in WO2020/018413, which is hereby incorporated by reference in its entirety.

In some embodiments, methods of treating cancer patients are provided. In some embodiments, the method comprises mobilizing the patient's CD34 ⁺ stem cells from the bone marrow into the periphery. In some embodiments, cells are mobilized by administering G-CSF (granulocyte colony stimulating factor). G-CSF can be administered, for example, as a 1-day, 2-day, 3-day, 4-day or 5-day regimen. In some embodiments, G-CSF is administered for 3-5 days. Mobilized cells can be captured using methods such as apheresis. In some embodiments, once the CD34 ⁺ cell count is at or above 10.0 to 20.0 x ¹⁰⁶ /kg body weight, the cells are isolated, eg, by apheresis. In some embodiments, the cell count is at or above 5.0 to 25.0 x ¹⁰⁶ /kg body weight. Although Cd34+ cells were used as a marker to capture cells for transduction, other cellular markers, such as those described herein, can also be used. For example, cells used are isolated based on the presence of specific markers or combinations of markers including, for example, CD34, CD4, Sca-1 CD38, CD123, CD90, CD45, CD133, antigen presenting cell markers (CD8, CD8α, CD11b, CD11c, CD103, CD205, CD24, CD115, CD117, CD135, CD11c ^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 specific markers, such as CD3, CD14, CD19, CD56, and/or CD66b. In other embodiments, negative selection is performed for markers such as T cells, B cells, granulocytes and/or myelomonocytes. 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 Lin ⁻ Thy1 ⁺ Sca-1 ⁺ expression profile. In some embodiments, mouse HSCs can be isolated by expression profile CD34 ⁻ , Sca1 ⁺ , c-kit ⁺ . In some embodiments, human HSCs can be isolated based on CD34 expression. In some embodiments, the isolated cells are CD34+ or CD4+ or any combination thereof.

In some embodiments, the method includes harvesting the cells by centrifugation. This is done, for example, to develop cell-rich clumps. Cells can then be resuspended in cryopreservation solution and frozen. In some embodiments, the cryopreservation solution comprises a solution of heparinized Plasmalyte and 10% DMSO (dimethyl sulfoxide). In some embodiments, cells are initially stored at -4°C, and samples are then frozen to a target temperature of -156°C (when stored in the gas phase) to -196°C (when stored in the liquid phase).

In some embodiments, the method includes transducing isolated cells to be resistant to chemotherapeutic agents such as busulfan. As described herein, chemotherapy resistance can be achieved through the expression of GCLM. GCLM can be introduced into selected cells by using vectors (as described throughout the specification), for example using lentiviral vectors. GCLM is operably linked to a cell-specific promoter. In some embodiments, the promoter is a CD34 promoter. In some embodiments, the promoter is the hCD34 promoter. In some embodiments, the promoter is the hCD4 promoter. In some embodiments, the sequence of GCLM is expressed as a protein as provided in SEQ ID NO:1. In some embodiments, the GCLM is encoded by a nucleic acid molecule comprising the sequence of SEQ ID NO: 2, or a coding region thereof. Due to the degenerate nature of the genetic code, the sequence of SEQ ID NO:2 is provided as a non-limiting example, and other nucleic acid molecules can be used to encode the expression of proteins comprising SEQ ID NO:1. In some embodiments, the GCLM comprises 1-10 conservative substitutions that do not alter the function of the GCLM. In some embodiments, the expressed GCLM is substantially identical to SEQ ID NO:1.

Expression of GCLM in vectors can also be driven by enhancer elements. For example, the enhancer element can be a CD3E enhancer.

In some embodiments, CD34 ⁺ cells can be isolated by magnetic bead separation. Lentiviral vector-mediated transduction of human CD34 ⁺ cells can include, for example, addition of 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, pre-stimulated the cells for 24 hours. In some embodiments, the cells are then contacted (infected) with a lentivirus expressing GCLM. Contacting can be performed in serum-free X-Vivo 10 medium in the presence of SCF, FLT3L and TPO cytokines (100 ng ml ⁻¹ each). The cells may then optionally be frozen or not. In some embodiments, the cells are not contacted with AAV or AV vectors.

In some embodiments, the method comprises infusing the transduced cells into the subject. In some embodiments, the subject has cancer. In some embodiments, following infusion of the modified cells, a non-myeloablative dose of a chemotherapeutic agent, such as busulfan, is administered. In some embodiments, the amount is a dose of 50-200 mg given daily. In some embodiments, the non-myeloablative dose of busulfan is about 0.15 mg/kg/d to less than 2.5 mg/kg/d, about 0.4 mg/kg/d to about 1.7 mg/kg/d, or about 0.8 mg /kg/d to about 1.5mg/kg/d. In some embodiments, the non-myeloablative dose of busulfan is about 0.15 mg/kg/d, about 0.2 mg/kg/d, about 0.25 mg/kg/d, about 0.3 mg/kg/d, about 0.35 mg /kg/d, about 0.4mg/kg/d, about 0.45mg/kg/d, about 0.5mg/kg/d, about 0.55mg/kg/d, about 0.6mg/kg/d, about 0.65mg/kg /d, about 0.7mg/kg/d, about 0.75mg/kg/d, about 0.8mg/kg/d, about 0.85mg/kg/d, about 0.9mg/kg/day, about 0.95mg/kg/d , about 1.0mg/kg/d, about 1.1mg/kg/d, about 1.2mg/kg/d, about 1.3mg/kg/d, about 1.4mg/kg/d, about 1.5mg/kg/d, about 1.6mg/kg/d, about 1.7mg/kg/d, about 1.8mg/kg/d, about 1.9mg/kg/d, about 2.0mg/kg/d, about 2.1mg/kg/d, about 2.2mg /kg/d, about 2.3mg/kg/d or about 2.4mg/kg/d. In some embodiments, the non-myeloablative dose of busulfan is about 1.3 mg/kg/d. In some embodiments, the non-myeloablative dose of busulfan is about 0.8 mg/kg/d to about 1.6 mg/kg/d, about 0.8 mg/kg/d, about 0.98 mg/kg/d, about 1.3 mg /kg/d, about 1.5mg/kg/d or about 1.6mg/kg/d. In some embodiments, the non-myeloablative dose of busulfan is about 0.5 to about 2 mg/kg/d. Doses can be administered as provided herein.

In some embodiments, the subject is also treated with fludarabine prior to infusion of the modified cells. In some embodiments, on day 2 post-harvest (or day -5 before transplant), the patient is treated with fludarabine (15 mg/m ² ) for 5 days (until day -1 before transplant). In some embodiments, instead of fludarabine, the patient may be treated with 4 mg/kg busulfan on day -1 prior to transplantation. In some embodiments, the patient is treated with a single dose of 1000 mg/m2 busulfan on day -2 prior to transplantation. However, following cell infusion, the subject is treated with the non-myeloablative doses of busulfan provided herein.

In some embodiments, the subject is treated with busulfan myeloablative.

In some embodiments, a single nucleic acid molecule, eg, a single vector, is used to encode or express each nucleic acid molecule or protein provided herein. In some embodiments, a single lentivirus comprises a nucleic acid sequence provided herein. In some embodiments, a lentivirus comprising a single expression construct encoding GCLM or a variant thereof is provided. The promoter and response element operably linked to the nucleic acid molecule encoding GCLM is non-limiting and other promoters and response elements may be used. Those skilled in the art will understand that the different promoters shown can be interchanged with each other.

In some embodiments, the nucleic acid molecule comprises 5'LTR and 3'LTR that flank the nucleic acid molecule encoding the GCLM protein. In some embodiments, the vector encodes another molecule of interest for expression in the same cell as the GCLM protein. Thus, in some embodiments, the nucleic acid molecule comprises a sequence encoding GCLM and a sequence of interest, which can be, for example, any other protein, antisense, miRNA or other nucleic acid molecule desired to be expressed in the bone marrow or the cell types provided herein.

Provided herein in certain embodiments are chemoresistance modified cells as described above with respect to the disclosed methods and the use of these cells in the disclosed methods. Also provided are methods of producing these cells by incorporating into suitable cells one or more modifications that confer chemoresistance, and optionally one or more additional modifications that are not associated with chemoresistance , such as additional disease-specific modifications.

In certain embodiments, also provided herein are compositions, including compositions for use in the methods provided herein, comprising at least one chemoresistant modified cell as provided herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, carrier, or any combination thereof.

The compositions may contain pharmaceutically acceptable excipients, pharmaceutically acceptable salts, diluents, carriers, vehicles and such other inactive agents well known to those skilled in the art. Vehicles and excipients commonly used in pharmaceutical formulations include, for example, talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, and the like. The solution can be prepared using water or a physiologically compatible organic solvent such as ethanol, 1,2-propylene glycol, polyethylene glycol, dimethyl sulfoxide, fatty alcohol, triglyceride, partial glycerol, and the like. Compositions can be prepared using conventional techniques, including sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propanediol, polyethylene glycol mixed with water, Ringer's solution Wait. In one aspect, coloring agents are added to aid in positioning and proper placement of the composition at the intended treatment site.

The compositions may include preservatives and/or stabilizers. Non-limiting examples of preservatives include methylparaben, ethylparaben, propylparaben, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride ( benzalkoniumchloride), benzyl alcohol, thimerosal, phenylmercurate, chlorhexidine, phenol, 3-cresol, quaternary ammonium compound (QAC), chlorobutanol, 2-ethoxyethanol, and imidazolidinyl urea ( imidurea).

To control tonicity, the composition may contain physiological salts, such as sodium salts. Sodium chloride (NaCl) is preferred and may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, anhydrous disodium phosphate, magnesium chloride and calcium chloride.

Compositions may include one or more buffering agents. Typical buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer; The concentration of the buffer is usually in the range of 5-20 mM. The pH of the composition is usually between 5 and 8, more usually between 6 and 8, for example between 6.5 and 7.5, or between 7.0 and 7.8.

In some embodiments, the composition can include a cryoprotectant. Non-limiting examples of cryoprotectants include glycols (eg, ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combination thereof.

In some embodiments, the cells are part of a population of cells in culture (ie, in vitro). In another embodiment, the cells are part of a cell population in a subject (ie, in vivo). For example, to treat or prevent cancer or a disease of interest, modified cells and/or non-myeloablative doses of chemotherapeutic agents can be delivered to in vivo cells or populations of in vivo cells that form tissues or organs in a subject. Alternatively, modified cells and/or non-myeloablative or myeloablative doses of chemotherapeutic agents may be delivered to cultured cells or populations of cultured cells for experiments to study their effects on specific types of cells.

Compositions can be included in implantable devices. Suitable implantable devices contemplated by the present invention include intravascular stents (eg, self-expanding stents, balloon-expandable stents, and stent-grafts), scaffolds, grafts, and the like. Such implantable devices may be coated or impregnated on at least one surface with a composition capable of treating or preventing cancer or other disease.

Compositions can be administered to a subject by any suitable means and route. Non-limiting examples include internal, pulmonary, rectal, nasal, vaginal, lingual, intravenous, intraarterial, intramuscular, intraperitoneal, intradermal, and subcutaneous routes.

Non-myeloablative or myeloablative doses of chemotherapeutic agents may be administered orally, parenterally, intravenously, or otherwise as provided herein.

As provided herein, in some embodiments, there is provided a cell, such as but not limited to, a stem cell or an immune cell, comprising a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC ( GCL catalytic subunit), GCL (as a dimer or holoenzyme), or any other polypeptide in the GSH synthetic pathway that serves equivalent function. In some embodiments, the cells comprise heterologous GCLM. In some embodiments, the cells further comprise at least one additional heterologously expressed molecule of interest. In some embodiments, the stem cells are fetal stem cells, cord blood-derived stem cells, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), induced PSCs (iPSCs), embryonic stem cells (ESCs), or cells derived therefrom, e.g. CD34+ cells, CD90+ cells, CD45+ cells, CD17+ cells, CD45RA- cells, or any combination thereof. In some embodiments, the immune cells are T cells.

In some embodiments, the molecule of interest is a chimeric antigen receptor. In some embodiments, a T cell comprising a heterologous GCLM or other protein in the GSH pathway as described above further comprises a chimeric antigen receptor (CAR). CAR can be any CAR. Cells comprising a heterologous CAR and a heterologous GCLM or related GSH pathway gene product can be used to select CAR cells once administered to a patient. In some embodiments, the heterologous GCLM protein comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, or a sequence at least 90% identical to or substantially identical to the amino acid sequence of SEQ ID NO: 1 . In some embodiments, the cells do not heterologously express any other proteins that confer chemotherapy resistance. In some embodiments, the cells do not express ALDH heterologously. In some embodiments, the cell heterologously expresses GCLM and expresses GCLM endogenously.

In some embodiments, there is provided a cell comprising a glutamate-cysteine ligase (GCL) modifying subunit encoding GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or Enzyme form) or an expressed heterologous nucleotide molecule of any other polypeptide in the GSH synthetic pathway that provides an equivalent function and one of: i) encoding at least one HIV coreceptor mutant, at least one A heterologous nucleotide molecule of HIV coreceptor mutation or mutations, at least one HIV fusion inhibitor, a molecule that reduces HIV coreceptor expression, or any combination thereof; and/or ii) endogenous HIV Coreceptor mutation or deletion. In some embodiments, the cell comprises a heterologous nucleotide molecule encoding GCLM. In some embodiments, the cell comprises a heterologous nucleotide sequence that encodes a molecule that reduces expression of CCR5; encodes a molecule that reduces expression of CXCR4; encodes expression of a C-peptide fusion inhibitor; or any combination of . In some embodiments, the cells express shCCR5, shCXCR4, and/or a C-peptide fusion inhibitor, eg, C44.

In some embodiments, cells are modified to express a heterologous nucleotide sequence provided herein using a non-viral gene transfer system. In some embodiments, the non-viral gene transfer system is a transposon gene transfer system. In some embodiments, the transposon gene transfer system is the Sleeping Beauty gene transfer system or the PiggyBac transposon gene transfer system. In some embodiments, cells are modified with a CRISPR system to heterologously express GCLM and other molecules of interest provided herein.

In some embodiments, nucleic acid molecules are provided that encode heterologous glutamate-cysteine ligase (GCL) modifying subunits GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or whole enzyme form) or any other polypeptide in the GSH synthetic pathway that provides an equivalent function and one of: i) encoding at least one HIV coreceptor mutant, a mutation of at least one HIV coreceptor or multiple mutations, at least one HIV fusion inhibitor, a molecule that reduces HIV coreceptor expression, or any combination thereof. In some embodiments, the heterologous nucleotide sequence encodes a molecule that reduces expression of CCR5; a molecule that reduces expression of CXCR4; a molecule that encodes expression of a C-peptide fusion inhibitor; or any combination thereof. In some embodiments, the nucleic acid molecule encodes the expression of shCCR5, shCXCR4, and/or a C-peptide fusion inhibitor.

In some embodiments, there is provided a vector as described herein comprising a nucleic acid molecule provided herein. In some embodiments, the vector is one that can be used to generate lentiviruses. In some embodiments, the vector is a lentiviral vector, as described herein.

In some embodiments, also provided herein are compositions comprising one or more cells as provided herein. In some embodiments, the composition is a pharmaceutical composition. Non-limiting examples of pharmaceutical compositions are provided herein. As described in this article,

80. A pharmaceutical composition comprising the cell of any one of claims 70-79 and a pharmaceutically acceptable buffer or excipient.

The cells provided herein can be used in a variety of methods for which chemoprotection is suitable. This can be used, for example, to administer chemotherapy-resistant genetically modified cells to a patient and then use chemotherapy to kill the non-chemoresistant cells, while protecting the chemotherapy-resistant cells and allowing them to continue to multiply. This can be used as an option to express heterologous proteins such as glutamate-cysteine ligase (GCL) modifying subunits GCLM, GCLC (catalytic subunit of GCL), GCL (as dimer or intact enzyme) Or any other polypeptide and molecule of interest in the GSH synthetic pathway that provides an equivalent function in a manner that modifies the cell. In addition, when the cells are used to heterologously express the glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (as a dimer or holoenzyme form) or any other polypeptide in the GSH synthesis pathway that provides an equivalent function when a normal or non-diseased cell replaces a diseased cell of the same cell type, it can be produced in the absence of glutamate-cysteine ligase (GCL) Molecules of interest other than the modified subunits GCLM, GCLC (GCL catalytic subunit), GCL (in dimer or holoenzyme form) or any other polypeptide in the GSH synthetic pathway that provide an equivalent function. This can be done, for example, during a bone marrow transplant or when cells express a defective or mutated form of a protein that causes disease. Examples of disorders are provided herein, including but not limited to lysosomal storage diseases.

Accordingly, in some embodiments, methods for providing or improving chemoprotection in target cells are provided. In some embodiments, the method comprises providing to the target cell a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (a catalytic subunit of GCL), GCL (as a dimer or holoenzyme) ) or any other polypeptide in the GSH synthetic pathway that provides an equivalent function. Cells can be administered, for example, to a patient in need of such target cells. In some embodiments, the heterologous protein is GCLM.

In some embodiments, methods for providing or improving chemoprotection in a patient in need thereof are provided. In some embodiments, the method comprises administering to said patient a protein expressing a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or intact enzymatic form) or any other polypeptide in the GSH synthesis pathway that provides an equivalent function. In some embodiments, the heterologous protein is GCLM. In some embodiments, the cells also comprise a heterologous molecule of interest, such as a protein or chimeric antigen receptor. Non-limiting examples of such proteins of interest are provided herein. However, these are non-limiting examples and any molecule of interest can be used in the cells and methods provided herein.

In some embodiments, glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (GCL catalytic subunit), GCL (as a dimer or holoenzyme), or GSH in the synthetic pathway provided Heterologous expression of any other polypeptide of equivalent function allows in vivo selection after transplantation.

In some embodiments, methods of conferring chemotherapy resistant cells in a patient are provided. In some embodiments, the method comprises administering to the patient a protein expressing a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or as a whole enzyme) ) or any other polypeptide in the GSH synthesis pathway that provides an equivalent function. In some embodiments, the cells heterologously express GCLM.

In some embodiments, methods of treating cancer patients are provided. In some embodiments, the method comprises administering to the patient a protein comprising an enzyme expressing a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or intact enzyme) form) or any other polypeptide in the GSH synthesis pathway that provides equivalent function in a pharmaceutical composition of cells with a non-myeloablative or myeloablative dose of a chemotherapeutic agent. In some embodiments, the cells express a heterologous GCLM. Nonmyeloablative or myeloablative doses of chemotherapeutic agents can be expressed in heterologous glutamate-cysteine ligase (GCL) modifying subunits GCLM, GCLC (catalytic subunit of GCL), GCL (as dimers or intact enzymatic form) or any other polypeptide in the GSH synthesis pathway that provides an equivalent function, such as GCLM before, after or concurrently with the cells.

In some embodiments, the method further comprises isolating stem cells or immune cells from the subject. In some embodiments, the isolated stem or immune cells are modified to express a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (a catalytic subunit of GCL), GCL (in the form of a di polymer or holoenzyme form) or any other polypeptide in the GSH synthetic pathway that provides an equivalent function, such as GCLM. In some embodiments, the isolated cells are synthesized by contacting the isolated cells with enzymes comprising GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or whole enzyme) encoding glutamate-cysteine ligase (GCL) modifying subunit. form) or any other polypeptide nucleic acid molecule in the GSH synthesis pathway that provides equivalent functions, the cells are modified to express heterologous glutamic acid-cysteine ligase (GCL) modified subunits GCLM, GCLC (GCL catalytic subunit), GCL (as a dimer or holoenzyme), or any other polypeptide in the GSH synthetic pathway that provides equivalent function.

In some embodiments, the vector is a viral vector and a non-viral vector. In some embodiments, the vector is a lentiviral vector, an adenoviral vector, a plasmid, a retrovirus, a transposon, an episomal expression vector, a modified RNA, or any combination thereof.

As provided herein, the cells may be stem cells or immune cells. In some embodiments, the stem cells are, but are not limited to, fetal stem cells, cord blood-derived stem cells, hematopoietic stem cells (HSCs), pluripotent stem cells (PSCs), induced PSCs (iPSCs), embryonic stem cells (ESCs), or stem cells derived therefrom. Cells, such as CD34+ cells, CD90+ cells, CD45+ cells, CD17+ cells, CD45RA- cells, or any combination thereof. In some embodiments, the immune cells are T cells. In some embodiments, the T cells further comprise a chimeric antigen receptor.

The modified cells can be autologous to the patient, allogeneic to the patient, or a combination thereof.

In some embodiments, the patient is undergoing gene therapy, cell therapy, or CAR-T therapy.

As provided herein, the chemical protection provided may be against busulfan and/or naphthalene. In some embodiments, the methods provided herein comprise administering to a patient (subject) a myeloablative dose or a non-myeloablative dose of busulfan and/or naphthalene.

In some embodiments, methods of performing bone marrow transplantation in a patient are provided. In some embodiments, the method comprises administering to the patient a population of busulfan-resistant modified cells and at least one non-myeloablative or myeloablative dose of busulfan. In some embodiments, the busulfan-resistant modified cell population comprises a glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or intact enzyme form) or any other heterologous gene in the GSH synthesis pathway that provides equivalent function. In some embodiments, the heterologous gene encodes GCLM. In some embodiments, the busulfan-resistant modified cell population expresses GCLM. In some embodiments, busulfan-resistant modified cells are conferred by expression of GCLM. In some embodiments, greater than 50% of the patient's bone marrow is replaced with busulfan-resistant modified cells or cells derived therefrom within 6 months. In some embodiments, the patient has HIV, cancer, A1AT deficiency, WAS, Hurler Syndrome, Hunter Syndrome, Pompe Disease, Fabry Fabry Disease, Mucopolysaccharidosis, MPS1H/S (Hurler/Scheie syndrome), MPS I H (Hurler's disease), MPS II- (Hunter syndrome) , MPS III A, B, C and D (Sanfillipo syndrome), MPS I S (Scheiesyndrome), MPS IV A and B (Morquio syndrome) ), MPS IX (hyaluronidase deficiency), MPS VII (Sly syndrome), MPS VI (Maroteaux-Lamy syndrome), lysosomal storage disease, and childhood onset Cerebral adrenoleukodystrophy (cALD). In some embodiments, the cancer is hematological cancer or malignancy. In some embodiments, the blood cancer is, but is not limited to, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lymphoma, AIDS-related lymphoma, Cutaneous T-cell lymphoma, Hodgkin's lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary syndrome, T-cell lymphoma, Waldenstrom's macroglobulinemia, chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, or myelodysplastic/myeloproliferative neoplasm.

The busulfan resistance provided herein and throughout may be transient, allowing heterologous expression of gene products such as GCLM to be transient.

In some embodiments, the method comprises subjecting unmodified cells to enzymes encoding glutamate-cysteine ligase (GCL) modifying subunits GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or holoenzyme) form) or any other polypeptide in the GSH synthetic pathway that provides equivalent function to the expression vector to generate busulfan-resistant modified cells. In some embodiments, the cells will express GCLM. In some embodiments, the expression vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is a lentiviral or adenoviral vector. In some embodiments, the expression vector is a retrovirus, transposon, episomal expression vector, modified RNA, plasmid, or any combination thereof.

In some embodiments, at least one non-myeloablative or myeloablative dose of a chemotherapeutic agent is administered after administration of the modified cells. In some embodiments, the at least one non-myeloablative or myeloablative dose of the chemotherapeutic agent is a non-myeloablative or myeloablative dose of busulfan. In some embodiments, the nonmyeloablative chemotherapeutic 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. In some embodiments, the method further comprises not administering the non-myeloablative dose of the chemotherapeutic agent for a period of time between administering the busulfan-resistant modified cells and the at least one non-myeloablative dose of the chemotherapeutic agent. In some embodiments, the period of time is selected from the group consisting of about 3 days, about 7 days, about 10 days, and about 14 days.

In some embodiments, more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells.

In some embodiments, the patient is not myeloablative and/or immunocompromised from administration of at least one non-myeloablative dose of a chemotherapeutic agent. In some embodiments, the patient is not experiencing clinically relevant anemia, neutropenia, thrombocytopenia, cytopenias, low platelets, low white blood cells, or any combination or related symptoms thereof.

In some embodiments, the modified cells are resistant to HIV infection. In some embodiments, the modified cell heterologously expresses at least one HIV coreceptor mutation, at least one HIV coreceptor mutation or mutations, at least one HIV A fusion inhibitor, a molecule that reduces HIV coreceptor expression, or any combination thereof. These may also be referred to as glutamate-cysteine ligase (GCL) modifying subunits GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or holoenzyme), or those provided in the GSH synthetic pathway. Molecules of interest other than any other polypeptide of equivalent function. In some embodiments, the modified cell heterologously expresses shCCR5, shCXCR4, a C-peptide fusion inhibitor, or any combination thereof. In some embodiments, the modified cells do not express HIV coreceptors. In some embodiments, the modified cell does not express CCR5, CXCR4, or expresses CCR5-Δ32, or a combination thereof.

In some embodiments, methods of treating HIV in a subject are provided. In some embodiments, the method comprises administering to the subject a heterologously expressing glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or whole enzyme form) or any other polypeptide in the GSH synthetic pathway that provides an equivalent function and one of: i) a cell population encoding at least one HIV coreceptor mutant, at least one HIV coreceptor a heterologous nucleotide molecule with a mutation or mutations, at least one HIV fusion inhibitor, a molecule that reduces HIV coreceptor expression, or any combination thereof; and at least one non-myeloablative dose of a chemotherapeutic agent. In some embodiments, the cells heterologously express GCLM. In some embodiments, the cells heterologously express shCCR5, shCXCR4, and/or a C-peptide fusion inhibitor. In some embodiments, the chemotherapeutic agent is busulfan.

In some embodiments, methods of expressing a molecule of interest in a subject are provided. In some embodiments, the method comprises administering to the subject expressing a heterologous heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL (as a dimer or whole enzyme form) or any other polypeptide and molecule of interest in the GSH synthesis pathway that provides equivalent function; and administering a non-myeloablative dose of busulfan. In some embodiments, the cells heterologously express GCLM. In some embodiments, the cells are CD34+ and/or CD4+, or other aspects as provided herein, eg, immune cells or stem cells as provided herein. In some embodiments, the molecule of interest is a molecule that reduces the expression of CCR5; reduces the expression of CXCR4; 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 of interest is in A1AT deficiency, WAS, Huerle syndrome, Hunter syndrome, Pompe disease, Fabry disease, mucopolysaccharidosis, MPS 1 H/S (Huerle syndrome), MPS I H (Hurler's disease), MPS II- (Hunter syndrome), MPS IIIA, B, C and D (Sandifer syndrome), MPS I S (Hurter syndrome), Yarrow syndrome), MPS IV A and B (Mercue syndrome), MPS IX (hyaluronidase deficiency), MPS VII (Sree syndrome), MPS VI (Marine-La syndrome), lysate Wild-type protein deficient in enzymatic storage diseases and childhood cerebral adrenoleukodystrophy (cALD). In some embodiments, the molecule of interest is a chimeric antigen receptor.

In some embodiments, methods of treating cancer in a patient are provided. In some embodiments, the method comprises administering busulfan to a patient, wherein the patient has been administered expression of a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (catalytic subunit of GCL), GCL ( Cells in dimer or holoenzyme form) or any other polypeptide in the GSH synthesis pathway that provides an equivalent function. In some embodiments, the cells heterologously express GCLM. In some embodiments, the method includes administering the cells to the patient prior to administering busulfan. In some embodiments, busulfan is administered in a non-myeloablative dose. In some embodiments, busulfan is administered in a myeloablative dose. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lymphoma, AIDS-related lymphoma, skin t-cell Lymphoma, Hodgkin's Lymphoma, Hodgkin's Lymphoma, Mycosis Fungoides, Non-Hodgkin's Lymphoma, Primary Central Nervous System Lymphoma, Sezary Syndrome, T-Cell Lymphoma, Macroglobulin Proteinemia, chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, or myelodysplastic/myeloproliferative neoplasm. In some embodiments, the cancer is osteosarcoma.

The following examples illustrate, not limit, the compounds, compositions and methods described herein. Other suitable modifications and alterations known to those skilled in the art are within the scope of the following embodiments.

Example 1

In certain embodiments, using methods and compositions utilizing glutamate-cysteine ligase (GCL), an enzyme that functions in the first step of the glutathione (GSH) synthesis pathway, Contributes to the clearance of busulfan. Chemoprotection of the glutamate-cysteine ligase (GCL) enzyme was analyzed by overexpressing its modifying subunit GCLM. Notably, any suitable form can be used, including GCLM (GCL modifying subunit), GCLC (GCL catalytic subunit), GCL (in dimer or holoenzyme form) and GSH synthetic pathways that serve the same function. other enzymes. (See Franklin et al., Mol. Aspects Med. 2009;30(1-2):86-98.)

method:

Jurkat, CEM and Tf-1a cell lines were transduced with lentiviral vectors expressing GCLM and green fluorescent protein (GFP) genes. By incubating a population of non-transduced cells or a mixed population of (20% transduced, 80% non-transduced) cells with busulfan at concentrations ranging from 0 to 200 μg/ml for 1 hour, 2 hours or 4 hours Chemical protection experiments. Cells were then incubated in cultures without busulfan for 24 hours, 48 hours, 72 hours and 96 hours. The area under the concentration-time curve (AUC) was estimated from different times and incubation concentrations by using the trapezoidal rule. Pre- and post-exposure GSH levels were measured using fluorometry. Cell survival and proliferation at different time points were measured by Nucleocounter NC-200 and flow cytometry using PI and 7-AAD staining protocols. Chemical selection of protected GFP+ cells was assessed by flow cytometry. Baseline GSH levels and GCLM expression were also measured by fluorescence and RT-PCR assays in primary human HSPCs harvested from 3 different donors.

Cells were incubated with busulfan (0, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, and 200 μg/ml) in serum-free medium (RPMI) for 1 hour, 2 hours and 4 hours. It was then washed with ice-cold buffer (PBS+0.5% HSA+0.1% glucose). Washed cells were cultured in RPMI+20% FBS. Measurements were taken at 24 hr, 48 hr, 72 hr and 96 hr time points.

result

GSH levels changed by 2.1, 1.7 and 1.9 fold between non-transduced and transduced Jurkat, CEM and TF-1a cells, respectively. Untransduced CEM cells were found to be most sensitive to busulfan exposure. Cell death and proliferation in all cell cultures proceeded in an AUC-dependent manner. 1.7-fold GSH activity in CEM cells conferred at least 3.5-fold protection against busulfan. After 72 hours of culture, the purity of transduced CEM cells increased from 20% to 88%, and the number of transduced viable cells increased from 30×10 ³ /ml to 84× after exposure to 10ug/ml busulfan for 1 hour 10 ³ /ml. At doses above 10 ug/ml or exposure times above 1 hour, transduced cells were 100% culture after 72 hours. At 72 hours, the fold expansion of transduced live cells was similar to untreated transduced controls and untransduced controls (2.8, 3.2 and 3.3). GSH levels in transduced cells remained stable throughout the experiment, whereas GSH in non-transduced cells was depleted after busulfan exposure. GSH levels in primary human HSPCs were similar to untransduced Tf-1a cells. Furthermore, RT-PCR analysis indicated that GCLM was not abundantly expressed in these cells.

Example 2:

The lack of sufficient engraftments for gene-modified hematopoietic stem/progenitor cell (HSPC) therapy has been the biggest challenge. It has been proposed to select these cells in vivo using chemotherapy resistance genes to cytotoxic agents. Busulfan, a cytotoxic chemotherapeutic agent commonly used to regulate regimens in HSPC transplantation, is cleared by glutathione (GSH) conjugation. Elevated levels of GSH in the cytoplasm mediate busulfan toxicity. The first step and rate-limiting enzyme of the GSH synthetic pathway involves glutamate-cysteine ligase (GCL) (Figure 1). The results presented herein demonstrate that increasing GCL enzyme activity by overexpressing the modified subunit GCLM confers protection against busulfan toxicity in vitro.

Jurkat, CEM, and Tf-1a cell lines were transduced with lentiviral vectors expressing GCLM and green fluorescent protein (GFP) genes (Figure 2A). By combining a population of untransduced cells or a mixed population of (20% transduced, 80% untransduced) cells with white Incubate with sulfan for 1 hour or 2 hours for chemical protection experiments. Cells were then incubated and viable cell numbers were recorded at 24 hr, 48 hr, 72 hr and 96 hr time points in cultures without busulfan (Figure 2B). GSH levels before and after transduction and after exposure were measured fluorometrically by FACS analysis. Cell survival and proliferation at different time points were measured daily by Nucleocounter NC-200 and flow cytometry using PI and 7-AAD staining protocols. Chemical selection of protected GFP-positive cells was assessed by flow cytometry.

Experiments showed that GSH levels were increased in transduced Jurkat, CEM and TF-1a cells (Fig. 3A). Overexpression of GCLM had no effect on cell viability and proliferation (Fig. 3B). GSH levels remained stable in transduced cells throughout the experiment, whereas GSH in non-transduced cells was depleted after busulfan exposure (Figure 4). Increased GSH activity in GLCM-transduced CEM cells provided at least 3.5-fold protection against busulfan at lower doses (Figure 5). It also exhibited protection and proliferation of transduced cell populations at higher doses (10 or 20 ug/ml) of busulfan exposure (Figure 6). Exposure to moderate levels of busulfan achieved selection of chemoprotected cells and 100% of the transduced cell population within 72-96 hours (Figure 7).

These results suggest that increased expression of individual GCL modifying subunits confers significant chemoprotection against busulfan at multiple doses and exposure times. This demonstrates that GCLM transgene expression can be used for busulfan protection, thereby selecting genetically modified cells. As discussed herein, one of the biggest obstacles to genetically modified autologous HSPC transplantation is the lack of sufficient engraftment. Busulfan is an agent with high toxicity to bone marrow stem cells and limited toxicity to other organs. In genetically modified autologous nonmyeloablative bone marrow transplantation, the GCLM gene can also be used to increase the engraftment rate of genetically modified stem cells using busulfan as the selection agent in vivo. These results were surprising and unexpected.

in conclusion:

Thus, the embodiments provided herein demonstrate that increased expression of a single heterologous GCL modifying subunit can confer significant chemoprotection against busulfan. This demonstrates that GCLM transgene expression can be used for post-transplantation in vivo selection in genetically modified autologous HSPC transplantation.

standard method

Standard methods in molecular biology are described in: Sambrook, Fritsch and Maniatis (2nd edition 1982 and 1989, 3rd edition 2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Volume 217, Academic Press, San Diego, CA). Standard methods also appear in: Ausbel et al. (2001) Current Protocols in Molecular Biology, Volumes 1-4, John Wiley and Sons Company, New York, NY, describes cloning and DNA mutagenesis in bacterial cells (Part 1 vol.), cloning in mammalian cells and yeast (vol. 2), glycoconjugates and protein expression (vol. 3), and bioinformatics (vol. 4).

Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see e.g. Coligan, et al. (2000) Current Protocols in Protein Science, Volume 2, John Wiley and Sons Company, New York ; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich (2001) Products for Life Science Research, St . Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons Company, New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see eg Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley Company, New York).

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (such as a Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference and In general. Pursuant to 37 C.F.R. §1.57(b)(1), applicant intends that this statement incorporated by reference be unrelated to each individual publication, database entry (such as a Genbank sequence or GeneID entry), patent application, or patent related, each is clearly identified pursuant to 37 C.F.R. §1.57(b)(2), even if such reference is not immediately adjacent to the specific statement incorporated by reference. The inclusion in the specification of a specific statement incorporated by reference does not in any way detract from the general statement incorporated by reference, if any. Citation of a reference herein does not constitute an admission that such reference is pertinent prior art, nor does it constitute any admission as to the content or date of such publication or document.

Embodiments of the invention are not limited in scope by the specific embodiments described herein. Various modifications to the embodiments, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and any accompanying drawings. Such modifications are also within the scope of the appended claims.

sequence listing

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Claims (90)

1. A method for providing or improving chemoprotection in a target cell, the method comprising providing to the target cell a heterologous glutamate-cysteine ligase (GCL) modification subunit, GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides an equivalent function.

2. A method for providing or improving chemoprotection in a patient in need thereof, the method comprising administering to the patient a cell expressing heterologous glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function.

3. The method of claim 1 or 2, wherein transgenic expression of the glutamate-cysteine ligase (GCL) modified subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function allows for in vivo selection following transplantation.

4. A method of conferring chemotherapy-resistant cells to a patient, the method comprising administering to the patient cells expressing heterologous glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function.

5. The method of claim 4, wherein the cell expresses heterologous GCLM.

6. A method of treating a cancer patient, the method comprising administering to the patient a pharmaceutical composition comprising cells expressing heterologous glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form) or any other polypeptide in the GSH synthesis pathway that provides equivalent function, together with a non-myeloablative or myeloablative dose of a chemotherapeutic agent.

7. The method of claim 6, wherein the cell heterologously expresses GCLM.

8. The method of claim 6 or 7, further comprising administering to the patient the cell expressing a heterologous glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function, before, after, or concurrently with the chemotherapeutic agent.

9. The method of any one of claims 4-8, further comprising isolating stem cells or immune cells from the patient.

10. The method of claim 9, wherein the isolated stem cell or immune cell is modified to express heterologous glutamate-cysteine ligase (GCL) modified subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function.

11. The method of claim 10, wherein the isolated stem cell or immune cell is modified to express a heterologous GCLM.

12. The method of claim 10 or 11, wherein the cell is modified by contacting the isolated cell with an expression vector comprising a nucleic acid molecule encoding GCLM or a glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function.

13. The method of claim 12, wherein the vector is a viral vector, a non-viral vector.

14. The method of claim 12, wherein the vector is a lentiviral vector, an adenoviral vector, a plasmid, a retrovirus, a transposon, an episomal expression vector, a modified RNA, or any combination thereof.

15. The method of any one of claims 1-14, wherein the cell is a stem cell or an immune cell.

16. The method of claim 15, wherein the stem cell is a fetal stem cell, a cord blood-derived stem cell, a Hematopoietic Stem Cell (HSC), a Pluripotent Stem Cell (PSC), an Induced PSC (iPSC), an Embryonic Stem Cell (ESC), or a cell derived therefrom, such as a CD34+ cell, a CD90+ cell, a CD45+ cell, a CD17+ cell, a CD45RA "cell, or any combination thereof.

17. The method of claim 15, wherein the immune cell is a T cell.

18. The method of any one of claims 1-16, wherein the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

19. The method of any one of claims 1-18, wherein the patient is undergoing cell therapy of gene therapy, CAR-T therapy.

20. The method of any one of claims 1-19, wherein the chemical protection provided is against busulfan and/or naphthalene.

21. The method of any one of claims 1-20, further comprising administering to the patient a myeloablative dose or a non-myeloablative dose of busulfan and/or naphthalene.

22. A method for performing a bone marrow transplant in a patient, the method comprising:

administering to the patient a population of anti-busulfan modified cells and at least one non-myeloablative dose of busulfan.

23. The method of claim 22, wherein the population of anti-busulfan modified cells comprises a heterologous gene encoding a glutamate-cysteine ligase (GCL) modified subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function.

24. The method of claim 22, wherein the heterologous gene encodes GCLM.

25. The method of claim 22, wherein the population of anti-busulfan modified cells express GCLM.

26. The method of claim 22, wherein the anti-busulfan resistance of the modified anti-busulfan cell is conferred by expression of GCLM.

27. The method of claim 22, wherein more than 50% of the patient's bone marrow is replaced by the anti-busulfan modified cells or cells derived therefrom within 6 months.

28. The method of claim 22, wherein the patient has HIV, cancer, A1AT deficiency, WAS, huler 'S syndrome, hunter syndrome, pompe disease, fabry disease, mucopolysaccharidosis, MPS1H/S (hudi' S syndrome), MPSIH (huler 'S disease), MPS II- (hunter' S syndrome), MPS III a, B, C and D (sanddy 'S syndrome), MPS I S (seekers' syndrome), MPS IV a and B (morsels syndrome), MPS IX (hyaluronidase deficiency), MPS VII (sirie 'S syndrome), MPS VI (marylar' S syndrome), lysosomal storage disease, and childhood cerebral adrenoleukodystrophy (cALD).

29. The method of claim 22, wherein the anti-busulfan activity of the modified cell is transient.

30. The method of claim 22, wherein the cell is a stem cell or an immune cell.

31. The method of claim 30, wherein the stem cell is a fetal stem cell, a cord blood-derived stem cell, a Hematopoietic Stem Cell (HSC), a Pluripotent Stem Cell (PSC), an Induced PSC (iPSC), an Embryonic Stem Cell (ESC), or a cell derived therefrom, such as a CD34+ cell, a CD90+ cell, a CD45+ cell, a CD17+ cell, a CD45RA "cell, a CD 38", or any combination thereof.

32. The method of claim 30, wherein the immune cell is a T cell.

33. The method of claim 22, wherein the modified cells are autologous to the patient, allogeneic to the patient, or a combination thereof.

34. The method of claim 22, further comprising contacting an unmodified cell with an expression vector encoding for expression of a glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function, to produce the anti-busulfan modified cell.

35. The method of claim 34, wherein the expression vector is a viral vector or a non-viral vector.

36. The method of claim 35, wherein the viral vector is a lentiviral or adenoviral vector.

37. The method of claim 34, wherein the expression vector is a retrovirus, a transposon, an episomal expression vector, a modified RNA, a plasmid, or any combination thereof.

38. The method of claim 22, wherein the at least one non-myeloablative dose of a chemotherapeutic agent is administered after administration of the modified cells.

39. The method of claim 22, wherein the at least one non-myeloablative dose of a chemotherapeutic agent is a non-myeloablative dose of busulfan.

40. The method of claim 22, wherein the non-myeloablative chemotherapeutic 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.

41. The method of claim 22, further comprising not administering the non-myeloablative dose of the chemotherapeutic agent for a period of time between administering the anti-busulfan modified cells and at least one non-myeloablative dose of the chemotherapeutic agent.

42. The method of claim 41, 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.

43. The method of claim 22, wherein more than about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of the patient's bone marrow is replaced by the modified cells.

44. The method of claim 22, wherein the patient is not depleted of bone marrow cells and/or immunocompromised by administration of the at least one non-myeloablative dose of a chemotherapeutic agent.

45. The method of claim 22, wherein the patient does not experience clinically relevant anemia, neutropenia, thrombocytopenia, pancytopenia, low platelets, low leukocytes, or any combination thereof or related symptoms.

46. The method of claim 22, wherein a preconditioning step is performed prior to administering the cells.

47. The method of claim 46, wherein the preconditioning step is a non-myeloablative chemotherapy preconditioning step.

48. The method of claim 22, wherein the modified cell is resistant to HIV infection.

49. The method of claim 48, wherein the modified cell heterologously expresses a mutation of at least one HIV co-receptor, a mutation or mutations of 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 that is resistant to HIV infection.

50. The method of claim 48, wherein the modified cell heterologously expresses shCCR5, shCXCR4, a C-peptide fusion inhibitor, or any combination thereof.

51. The method of claim 48, wherein the modified cell does not express an HIV co-receptor.

52. The method of claim 48, wherein the modified cell does not express CCR5, CXCR4, or expresses CCR5- Δ 32, or a combination thereof.

53. A cell comprising a heterologous nucleotide molecule encoding for expression of a glutamate-cysteine ligase (GCL) modified subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function, and one of: i) A heterologous nucleotide molecule encoding at least one HIV co-receptor mutant, a mutation or mutations of 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; and/or ii) endogenous HIV co-receptor mutations or deletions.

54. The cell of claim 53, wherein the cell comprises a heterologous nucleotide sequence that:

i. encoding a molecule that reduces expression of CCR 5;

encoding for reducing expression of CXCR 4;

expression of a gene encoding a C-peptide fusion inhibitor;

or any combination thereof.

55. The cell of claim 53, wherein the cell expresses shCCR5, shCXCR4, and/or a C-peptide fusion inhibitor, such as C44.

56. The cell of claim 53, wherein the cell is modified with a non-viral gene transfer system to express the heterologous nucleotide sequence.

57. The cell of claim 56, wherein the non-viral gene transfer system is a transposon gene transfer system.

58. The cell of claim 57, wherein the transposon gene transfer system is a sleeping beauty gene transfer system or a PiggyBac transposon gene transfer system.

59. A composition comprising one or more cells of any one of claims 53-58.

60. A nucleic acid molecule encoding a heterologous glutamate-cysteine ligase (GCL) modified subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form) or any other polypeptide in the GSH synthesis pathway that provides equivalent function and one of: i) A heterologous nucleotide molecule encoding at least one HIV co-receptor mutant, a mutation or mutations of 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.

61. The nucleic acid molecule of claim 60, wherein the heterologous nucleotide sequence encodes:

i. a molecule that reduces the expression of CCR 5;

a molecule that reduces the expression of said CXCR 4;

a molecule encoding expression of a C-peptide fusion inhibitor;

or any combination thereof.

62. The nucleic acid molecule of claim 60, wherein the nucleic acid molecule encodes expression of shCCR5, shCXCR4 and/or a C-peptide fusion inhibitor.

63. A vector comprising the nucleic acid molecule of claim 60.

64. The vector of claim 63, wherein the vector is a vector that can be used to produce a lentivirus.

65. The vector of claim 63, wherein the vector is a lentiviral vector.

66. A method of treating HIV in a subject, the method comprising administering to the subject a population of cells that heterologously express the glutamate-cysteine ligase (GCL) modifying subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides equivalent function and one of: i) A heterologous nucleotide molecule encoding at least one HIV co-receptor mutant, a mutation or mutations of 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;

and at least one non-myeloablative dose of a chemotherapeutic agent.

67. The method of claim 66, wherein the cells express shCCR5, shCXCR4, and/or a C-peptide fusion inhibitor.

68. The method of claim 66 or 67, wherein the cell heterologously expresses GCLM.

69. A method of expressing a molecule of interest in a subject, the method comprising administering to the subject a cell that heterologously expresses a heterologous glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form) or any other polypeptide in the GSH synthesis pathway that provides equivalent function and the molecule of interest; and administering a non-myeloablative dose of busulfan.

70. The method of claim 69, wherein the cell is an immune cell, a stem cell, or a CD34+ and/or CD4+ cell, or as otherwise provided herein.

71. The method of claim 69, wherein the molecule of interest is a molecule that reduces expression of CCR 5; a molecule that reduces expression of CXCR 4; a molecule encoding the expression of a C-peptide fusion inhibitor; or any combination thereof.

72. The method of claim 71, wherein the molecule of interest that reduces expression of CCR5 is shCCR5.

73. The method of claim 69, wherein the molecule of interest is a wild-type protein deficient in A1AT deficiency, WAS, hull 'S syndrome, hunter' S syndrome, pompe disease, fabry 'S disease, mucopolysaccharidosis, MPS1H/S (Hull-shot syndrome), MPS IH (Hull' S disease), MPSII- (Hunter syndrome), MPSIIIA, B, C and D (Mordenver syndrome), MPS IS (Siyeri syndrome), MPS IV A and B (Morsella syndrome), MPS IX (hyaluronidase deficiency), MPS VII (Ski syndrome), MPS VI (Mare-Lar syndrome), lysosomal storage disease, and childhood cerebral adrenoleukodystrophy (cALD).

74. An isolated stem cell or immune cell comprising a heterologous glutamate-cysteine ligase (GCL) modification subunit GCLM, GCLC (GCL catalytic subunit), GCL (in dimeric or intact enzyme form), or any other polypeptide in the GSH synthesis pathway that provides an equivalent function.

75. The cell of claim 74, wherein the cell comprises a heterologous GCLM.

76. The cell of claim 74 or 75, wherein said cell further comprises at least one additional heterologously expressed molecule of interest.

77. The cell of any one of claims 74-76, wherein the stem cell is a fetal stem cell, a cord blood-derived stem cell, a Hematopoietic Stem Cell (HSC), a Pluripotent Stem Cell (PSC), an Induced PSC (iPSC), an Embryonic Stem Cell (ESC), or a cell derived therefrom, such as a CD34+ cell, a CD90+ cell, a CD45+ cell, a CD17+ cell, a CD45 RA-cell, or any combination thereof.

78. The cell of any one of claims 74-76, wherein the immune cell is a T cell.

79. The cell of claim 78, wherein the T cell further comprises a Chimeric Antigen Receptor (CAR).

80. The cell of any one of claims 74-79, wherein the heterologous GCLM protein comprises the amino acid sequence of SEQ ID NO 1 or a variant thereof, or a sequence that is at least 90% identical to SEQ ID NO 1 or substantially identical to the amino acid sequence of SEQ ID NO 1.

81. The cell of any one of claims 74-80, wherein the cell does not heterologously express any other protein that confers resistance to chemotherapy.

82. The cell of any one of claims 74-81, wherein the cell does not heterologously express ALDH.

83. The cell of any one of claims 74-81, wherein the cell heterologously expresses GCLM and endogenously expresses GCLM.

84. A pharmaceutical composition comprising the cell of any one of claims 74-83 and a pharmaceutically acceptable buffer or excipient.

85. A method of treating cancer in a patient, the method comprising administering busulfan to a patient, wherein the patient has been administered the cells of claim 79.

86. The method of claim 81, wherein the method comprises administering the cells of claim 79 to the patient prior to administering the busulfan.

87. The method of claim 85 or 86, wherein busulfan is administered in a non-myeloablative dose.

88. The method of claim 85 or 86, wherein busulfan is administered in a myeloablative dose.

89. The method of any one of claims 85-88, wherein the cancer is a hematological cancer or osteosarcoma.

90. The method of claim 89, wherein the hematologic cancer is leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, sezary syndrome, T-cell lymphoma, waldenstrom's macroglobulinemia, chronic myeloproliferative neoplasm, langerhans' cell histiocytosis, multiple myeloma, myelodysplastic syndrome, or myelodysplastic/myeloproliferative tumors.

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