Compositions and methods for modifying eukaryotic cells

Abstract

This invention provides improved compositions and methods for enhancing genetic recombination in eukaryotic cells. [Solution] A method for transducing eukaryotic cells to express an introduced gene is provided, wherein the method comprises contacting the cells with (i) a viral vector encoding the introduced gene and (ii) a poloxamer, wherein the concentration of the poloxamer is less than 10 μg / mL when in contact with the cells. Using the composition and method of this disclosure, the target gene can be expressed by contacting cells with a viral vector such as a lentiviral vector and a poloxamer.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy, created on June 5, 2020, is named 51139-018WO2_Sequence_Listing_6.5.20_ST25 and has a size of 2,314 bytes.

[0002] This disclosure relates to compositions and methods for modifying eukaryotic cells, for example, by genetically modifying eukaryotic cells to express a target transgene, and in addition, for promoting cell proliferation and survival. [Background technology]

[0003] Genetic diseases associated with protein defects and loss-of-function mutations represent a challenging class of conditions that have historically remained difficult to treat. Cell-based therapies offer a promising pathway for the future, enabling the functional expression of target genes in patients in a stable manner. Preparing cells for this form of therapy often requires the cells to be genetically modified to express the desired gene. Improved methods are needed to enhance the genetic modification of eukaryotic cells. [Overview of the Initiative]

[0004] This disclosure relates to compositions and methods for modifying eukaryotic cells, such as pluripotent cells including pluripotent hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Using the compositions and methods described herein, cells, for example, can be genetically modified to promote the expression of a target transgene within an cell. For example, using the compositions and methods of this disclosure, a population of pluripotent cells, such as a population of HSCs and / or HPCs, can be contacted with a viral vector encoding a target transgene to transduce the cells and express the desired gene. The viral vector may be a retrovirus, such as a lentivirus. To stimulate viral transduction of target cells, the cells can be contacted with the viral vector, in addition to a poloxamer, for example, a poloxamer having an excess molar mass of 10,000 g / mol, a molar mass of more than 2,000 g / mol of polyoxypropylene subunits, and / or an ethylene oxide content of more than 40% by mass. Because cells prepared according to the procedures described herein can be provided to subjects (e.g., mammalian subjects such as human patients) with a disease associated with an endogenous deficiency in the gene of interest, the compositions and methods of this disclosure offer a range of important medical benefits. By administering modified cells to a subject, the subject may experience restoration of the expression of the deficiency gene. This therapeutic approach, though not limited by mechanism, provides a means by which subjects with genetic diseases can be treated, as well as a methodology for alleviating the symptoms of the disease.

[0005] The compositions and methods of this disclosure are, in part, based on a series of surprising discoveries. For example, poloxamers, including those previously shown to be ineffective in improving viral transduction efficiency, have been found to actually promote viral transduction when in contact with target cells at concentrations below 10 μg / mL (for example, concentrations of about 10 ng / mL to about 9 μg / mL, such as 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, or 1 μg / mL, among other concentrations described herein). This unexpected observation yields various benefits. For example, the transduction-enhancing activity of poloxamers at low concentrations allows for the use of small amounts of poloxamers to transduce target cells while still maintaining robust genetic recombination.

[0006] In a first aspect, the disclosure is characterized by a method of transducing eukaryotic cells to express a transgene by contacting the cells with (i) a viral vector encoding a transgene and (ii) a poloxamer.

[0007] In a further embodiment, the present disclosure is characterized by a method for expressing a transgene in eukaryotic cells by contacting cells with (i) a viral vector encoding the transgene and (ii) a poloxamer.

[0008] In a further embodiment, the present disclosure features a method for promoting the translocation of a viral vector encoding a transgene into the nucleus of a eukaryotic cell by contacting cells with (i) a viral vector and (ii) a poloxamer.

[0009] In some embodiments of any of the three aforementioned aspects of the present disclosure, the method further comprises contacting cells with a substance that reduces the activity and / or expression of protein kinase C (PKC).

[0010] In a further embodiment, the present disclosure is characterized by a method of transducing eukaryotic cells to express a transgene by contacting the cells with (i) a viral vector encoding a transgene, (ii) a substance that reduces the activity and / or expression of PKC, and (iii) a poloxamer.

[0011] In a further embodiment, the present disclosure is characterized by a method for expressing a transgene in eukaryotic cells by contacting cells with (i) a viral vector encoding a transgene, (ii) a substance that reduces the activity and / or expression of PKC, and (iii) a poloxamer.

[0012] In a further embodiment, the present disclosure is characterized by a method for promoting the translocation of a viral vector encoding a transgene into the nucleus of a eukaryotic cell by contacting cells with (i) a viral vector, (ii) a substance that reduces the activity and / or expression of PKC, and (iii) a poloxamer.

[0013] In yet another aspect, the disclosure features a method for promoting actin depolymerization in eukaryotic cells by contacting cells with (i) a substance that reduces the activity and / or expression of PKC, and (ii) poloxamer.

[0014] In a further embodiment, the present disclosure is characterized by a method of inhibiting the phosphorylation of cofilin in eukaryotic cells by contacting cells with (i) a substance that reduces the activity and / or expression of PKC, and (ii) poloxamer.

[0015] In a further embodiment, the present disclosure is a method for increasing the concentration of dephosphorylated cofilin in eukaryotic cells, the method comprising contacting the cells with (i) a substance that reduces the activity and / or expression of PKC, and (ii) poloxamer.

[0016] Methods for measuring actin depolymerization, cofilin phosphorylation, and the amount of dephosphorylated cofilin in eukaryotic cells are known in the art, for example, those described in Yoder et al., Cell 134:782-792 (2008), the entire disclosure of which is incorporated by reference in this invention.

[0017] In a further embodiment, the present disclosure is a method for promoting the survival and / or proliferation of eukaryotic cells, wherein the method comprises contacting the cells with (i) a substance that reduces the activity and / or expression of PKC, and (ii) poloxamer.

[0018] In some embodiments of any of the three aforementioned aspects of the present disclosure, the method further includes a method for transducing cells to express a transgene by contacting the cells with a viral vector encoding the transgene.

[0019] In some embodiments of any of the above aspects of this disclosure, the cells are mammalian cells, such as human cells. In some embodiments, the cells are pluripotent cells. The cells may be CD34+ cells. In some embodiments, the cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the cells are HSCs or HPCs.

[0020] In some embodiments, substances that reduce PKC activity and / or expression activate Akt signaling. Substances that reduce PKC activity and / or expression may be PKC inhibitors or agents that reduce the translation of ribonucleic acid (RNA) transcripts encoding PKC (i.e., messenger RNA transcripts encoding PKC).

[0021] In some embodiments, the substance that reduces the activity and / or expression of PKC is an agent that reduces the translation of the RNA transcript encoding PKC. In some embodiments, the agent contains nucleic acid. The nucleic acid may contain interfering RNA, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the nucleic acid contains antisense oligonucleotide.

[0022] In some embodiments, the nucleic acid anneals to an endogenous RNA transcript encoding PKC. The nucleic acid may be at least 85% complementary to the region of the endogenous RNA transcript encoding PKC (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary).

[0023] In some embodiments, the substance that reduces the activity and / or expression of PKC is a PKC inhibitor. The PKC inhibitor may be a staurosporine or a variant thereof. For example, the PKC inhibitor may be a compound represented by formula (I).

[0024] [ka] [In the formula, R1 is H, OH, optionally substituted alkoxide, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amide, halogen, optionally substituted C] 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6is alkynyl, or optionally substituted acyl, R a and R b are each independently H, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl, or R a and R b together with the atoms to which they are attached form an optionally substituted and optionally fused heterocycloalkyl ring, R c is O, NR d or S, R d is H, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 alkynyl, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl.

[0025] [ka] This represents an arbitrary combination, n is an integer from 0 to 4. m is an integer between 0 and 4. or a salt thereof.

[0026] In some embodiments, the PKC inhibitor is a compound represented by formula (II).

[0027] [ka] [In the formula, R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amide, halogen, oxo, or thiocarbonyl, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkinyl, or optionally substituted acyl, R a and R b These atoms bond with the atoms they bind to, forming optionally substituted and optionally condensed heterocycloalkyl rings. R c is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. or its salt.

[0028] In some embodiments, the PKC inhibitor is a compound represented by formula (III).

[0029] [ka] [In the formula, R1 is H, OH, oxo, or thiocarbonyl, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkinyl, or optionally substituted acyl, Ring A is an optionally substituted and optionally condensed heterocycloalkyl ring, R c is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. or its salt.

[0030] In some embodiments, the PKC inhibitor is a compound represented by formula (IV).

[0031] [ka] [In the formula, R1 is H, OH, or oxo, Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring. R c is either O or S, W is O, NH, or S. Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. or its salt.

[0032] In some embodiments, the PKC inhibitor is a compound represented by formula (V).

[0033] [ka] [In the formula, R1 is H, OH, or oxo, R c is either O or S, W is O, NH, or S. Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. p is either 0 or 1. or its salt.

[0034] In some embodiments, the PKC inhibitor is a compound represented by formula (VI).

[0035] [ka] [In the formula, R1 is H, OH, or oxo, Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. s is an integer between 0 and 8. or its salt.

[0036] In some embodiments, the PKC inhibitor is a compound represented by formula (VII).

[0037] [ka] [In the formula, R1 is H, OH, or oxo, R2 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R3 is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. or its salt.

[0038] In some embodiments, the PKC inhibitor is a compound represented by formula (VIII).

[0039] [ka] [In the formula, R1 is H, OH, or oxo, R2 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R3 is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. or its salt.

[0040] In some embodiments, the PKC inhibitor is a compound represented by formula (IX).

[0041] [ka] [In the formula, each X independently represents a halogen, an optionally substituted haloalkyl, cyano, an optionally substituted amino, hydroxyl, thiol, an optionally substituted alkoxy, an optionally substituted alkylthio, an optionally substituted acyloxy, an optionally substituted alkoxycarbonyl, an optionally substituted carboxy, ureido, an optionally substituted alkylsulfonyl, an optionally substituted arylsulfonyl, an optionally substituted heteroarylsulfonyl, an optionally substituted cycloalkylsulfonyl, an optionally substituted heterocycloalkylsulfonyl, an optionally substituted alkylsulfanyl, an optionally substituted arylsulfanyl, or an optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. or its salt.

[0042] In some embodiments, the PKC inhibitor is a compound represented by formula (1).

[0043] [ka] or its salt.

[0044] In some embodiments, the PKC inhibitor is staurosporine, i.e., (2S,3R,4R,6R)-3-methoxy-2-methyl-4-(methylamino)-29-oxa-1,7,17-triazaoctactacyclo[12.12.2.12,6.07,28.08,13.015,19.020,27.021,26]nonacosa-8,10,12,14,19,21,23,25,27-nonaen-16-one, represented by formula (2).

[0045] [ka] or its salt.

[0046] In some embodiments, the PKC inhibitor is a compound represented by formula (X).

[0047] [ka] [In the formula, R1 is H, OH, or oxo, Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. t is an integer between 0 and 6. or its salt.

[0048] In some embodiments, the PKC inhibitor is a compound represented by formula (XI).

[0049] [ka] [In the formula, R1 is H, OH, or oxo, R4 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. or its salt.

[0050] In some embodiments, the PKC inhibitor is a compound represented by formula (XII).

[0051] [ka] [In the formula, R1 is H, OH, or oxo, R4 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. or its salt.

[0052] In some embodiments, the PKC inhibitor is a compound represented by formula (XIII).

[0053] [ka] [In the formula, each X independently represents a halogen, an optionally substituted haloalkyl, cyano, an optionally substituted amino, hydroxyl, thiol, an optionally substituted alkoxy, an optionally substituted alkylthio, an optionally substituted acyloxy, an optionally substituted alkoxycarbonyl, an optionally substituted carboxy, ureido, an optionally substituted alkylsulfonyl, an optionally substituted arylsulfonyl, an optionally substituted heteroarylsulfonyl, an optionally substituted cycloalkylsulfonyl, an optionally substituted heterocycloalkylsulfonyl, an optionally substituted alkylsulfanyl, an optionally substituted arylsulfanyl, or an optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. or its salt.

[0054] In some embodiments, the PKC inhibitor is a compound represented by formula (3).

[0055] [ka] or its salt.

[0056] In some embodiments, the PKC inhibitor is a compound represented by formula (4).

[0057] [ka] or its salt.

[0058] In some embodiments, the PKC inhibitor is

[0059] [ka] It is a compound selected from either or its salts.

[0060] In some embodiments, the PKC inhibitor is a compound represented by formula (XIV).

[0061] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0062] In some embodiments, the PKC inhibitor is a compound represented by formula (XV).

[0063] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0064] In some embodiments, the PKC inhibitor is

[0065] [ka] It is a compound selected from either or its salts.

[0066] In some embodiments, the PKC inhibitor is a compound represented by formula (XVI).

[0067] [ka] [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] Alternatively, it is its salt, or a quaternized variant.

[0068] In some embodiments, the PKC inhibitor is a compound represented by formula (XVII).

[0069] [ka] [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] Alternatively, it is its salt, or a quaternized variant.

[0070] In some embodiments, the PKC inhibitor is

[0071] [ka] The compound is selected from TIFF2026108645000026.tif181170TIFF2026108645000027.tif231170TIFF2026108645000028.tif229170TIFF2026108645000029.tif231170TIFF2026108645000030.tif173170 or a salt thereof.

[0072] In some embodiments, the PKC inhibitor is a compound represented by formula (XVIII).

[0073] [ka] [In the formula, R is H, OH, C 1-6 It is an alkoxy or oxo, R2 is

[0074] [ka] And optionally, the sugar moiety in the formula is derived from D-glucose, D-galactose, or D-mannose. R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R4 is OH, C 1-6 Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 Alkoxycarbonylamino, C 2-20 These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 It is substituted with an alkoxy, R5 is H or C 1-6 It is alkyl, R6 is either free or aliphatic C 2-22 It is a hydroxyl esterified with a carboxylic acid, or C 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, free or aliphatic C 2-22amino acids acylated with carboxylic acids, C 1-6 These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R7 is either free or aliphatic C 2-22 OH, C esterified with a carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, azide, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 These are alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 It is substituted with an alkoxycarbonyl group. or its salt.

[0075] In some embodiments, the PKC inhibitor is a compound represented by formula (XIX).

[0076] [ka] [In the formula, R is H, OH, C 1-6 It is an alkoxy or oxo, R2 is

[0077] [ka] and; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R4 is OH, C 1-6 Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 Alkoxycarbonylamino, C 2-20 These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 It is substituted with an alkoxy, R5 is H or C 1-6 It is alkyl, R6 is either free or aliphatic C 2-22 It is a hydroxyl esterified with a carboxylic acid, or C 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R7 is either free or aliphatic C 2-22 OH, C esterified with a carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6Alkylsulfonyloxy, azido, free or amino acylated with aliphatic C 2-22 carboxylic acid, C 1-6 alkylamino, di-C 1-6 alkylamino, C 1-6 alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which is substituted in the phenyl moiety with halogen, hydroxyl, trifluoromethyl, C 1-6 alkyl, C 1-6 alkoxy, or C 1-6 alkoxycarbonyl.] Or a salt thereof.

[0078] In some embodiments, the PKC inhibitor is N-(1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(2-N-acetyl-muramyl)staurosporine, N-(6-O-mesyl-1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(6-azido-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-6-deoxy-2-N-acetylmyl)staurosporine N-(6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(2-N-acetyl-demethylmuramyl)staurosporine, N-(1-α-O-benzyl-2-N-acetylhomomuramyl)staurosporine, N-(1-α-O-benzyl-2-N-acetyl-L-homomuramyl)staurosporine, 1-α-anomer of N-(2-N-acetyl-L-homomuramyl)staurosporine, N-(1-α-O-benzyl-4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α-O -benzyl-4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-2-N-acetylmuramyl)staurosporine, 1-α-anomer of N-(4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, 1-α-anomer of N-(4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4,6-O-triacetyl The compound is selected from N-(1-deoxy-6-O-acetyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-truolsulfonyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-azido-2-N-acetylmuramyl)staurosporine, and N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, or a salt thereof.

[0079] In some embodiments, the PKC inhibitor is a compound represented by formula (XX).

[0080] [ka] [In the formula, Z1 is H or OH, Z2 is either H or OH. R1 is H, a halogen, or an optionally substituted alkyl group. R2 is H or halogen, R is an OH group or an optionally substituted alkoxy. X is an optionally substituted alkyl or optionally substituted acyl, optionally where X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents the phenyl moiety. ] or a salt thereof.

[0081] In some embodiments, the PKC inhibitor is a compound represented by formula (XXI).

[0082] [ka] [In the formula, Z1 is H or OH, Z2 is either H or OH. R1 is H, a halogen, or an optionally substituted alkyl group. R2 is H or halogen, R is an OH group or an optionally substituted alkoxy. X is an optionally substituted alkyl or optionally substituted acyl, optionally where X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents the phenyl moiety. ] or a salt thereof.

[0083] In some embodiments, the PKC inhibitor is a compound represented by formula (XXII), (XXIII), (XXIV), or (XXV).

[0084] [ka] [In the formula, each R1 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl, Each R2 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, or an acyl having 30 or fewer carbon atoms. Each X is independently O, OH, and H, or a pair of hydrogen atoms. Each Q is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each n is an independent integer between 0 and 4. Each m is an independent integer between 0 and 4. or its salt.

[0085] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII).

[0086] [ka] [In the formula, each R1 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl, Each R2 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, or 30 or fewer carbon atoms. Each R8 is independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, Each R9 is independently an optionally substituted acyl, optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, carbonyl, carbonyldioxy, esterified carboxy, carbamoyl, N-mono- or N,N-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-di-substituted aminosulfonyl, Each R 10 is independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, Each X is independently O, OH, and H, or a pair of hydrogen atoms, Each n is independently an integer from 0 to 4, Each m is independently an integer from 0 to 4, Each n’ is independently an integer from 0 to 4, Each m’ is independently an integer from 0 to 4.] Or a salt thereof.

[0087] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVIII)

[0088]

Chemical formula

[0089] In some embodiments, the PKC inhibitor is a compound represented by formula (XXIX).

[0090] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0091] In some embodiments, the PKC inhibitor is a compound represented by formula (XXX).

[0092] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0093] In some embodiments, the PKC inhibitor is a compound represented by formula (XXXI).

[0094] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0095] In some embodiments, the PKC inhibitor is

[0096] [ka] Select from TIFF2026108645000044.tif230170, TIFF2026108645000045.tif235170, TIFF2026108645000046.tif240170, and TIFF2026108645000047.tif162170.

[0097] In some embodiments of any of the above aspects or embodiments of this disclosure, the concentration of the substance that reduces the activity and / or expression of PKC is about 100 μM to about 1 mM (for example, about 100 μM, 105 μM, 110 μM, 115 μM, 120 μM, 125 μM, 130 μM, 135 μM, 140 μM, 145 μM, 150 μM, 155 μM, 160 μM, 165 μM, 170 μM, 175 μM, 180 μM, 185 μM, 190 μM, 195 μM, 200 μM, 205 μM, 210 μM, 215 μM, 220 μM) when in contact with cells. μM, 225 μM, 230 μM, 235 μM, 240 μM, 245 μM, 250 μM, 255 μM, 260 μM, 265 μM, 2 70μM, 275μM, 280μM, 285μM, 290μM, 295μM, 300μM, 305μM, 310μM, 315μM, 320μM, 325μM, 330μM, 335μM, 340μM, 345μM, 350μM, 355μM, 360μM, 365μM , 370μM, 375μM, 380μM, 385μM, 390μM, 395μM, 400μM, 405μM, 410μM, 415μM M, 420μM, 425μM, 430μM, 435μM, 440μM, 445μM, 450μM, 455μM, 460μM, 46 5μM, 470μM, 475μM, 480μM, 485μM, 490μM, 495μM, 500μM, 505μM, 510μM, 5 15μM, 520μM, 525μM, 530μM, 535μM, 540μM, 545μM, 550μM, 555μM, 560μM, 565μM, 570μM, 575μM, 580μM, 585μM, 590μM, 595μM, 600μM, 605μM, 610μM , 615μM, 620μM, 625μM, 630μM, 635μM, 640μM, 645μM, 650μM, 655μM, 660 μM, 665 μM, 670 μM, 675 μM, 680 μM, 685 μM, 690 μM, 695 μM, 700 μM, 705 μM, 71 0μM, 715μM, 720μM, 725μM, 730μM, 735μM, 740μM, 745μM, 750μM, 755μM, 7 60μM, 765μM, 770μM, 775μM, 780μM, 785μM, 790μM, 795μM, 800μM, 805μM,These are 810μM, 815μM, 820μM, 825μM, 830μM, 835μM, 840μM, 845μM, 850μM, 855μM, 860μM, 865μM, 870μM, 875μM, 880μM, 885μM, 890μM, 895μM, 900μM, 905μM, 910μM, 915μM, 920μM, 925μM, 930μM, 935μM, 940μM, 945μM, 950μM, 955μM, 960μM, 965μM, 970μM, 975μM, 980μM, 985μM, 990μM, 995μM, or 1mM. In some embodiments, the concentration of the substance that reduces PKC activity and / or expression is approximately 200 μM to approximately 600 μM when in contact with cells (e.g., approximately 200 μM, 205 μM, 210 μM, 215 μM, 220 μM, 225 μM, 230 μM, 235 μM, 240 μM, 245 μM, 250 μM, 255 μM). , 260μM, 265μM, 270μM, 275μM, 280μM, 285μM, 290μM, 295μM, 300μM, 305μM, 310μM, 315 μM, 320 μM, 325 μM, 330 μM, 335 μM, 340 μM, 345 μM, 350 μM, 355 μM, 360 μM, 365 μM, 370 μM, 37 5μM, 380μM, 385μM, 390μM, 395μM, 400μM, 405μM, 410μM, 415μM, 420μM, 425μM, 430μM, 435μM, 440μM, 445μM, 450μM, 455μM, 460μM, 465μM, 470μM, 475μM, 480μM, 485μM, 490μM The concentrations are M, 495 μM, 500 μM, 505 μM, 510 μM, 515 μM, 520 μM, 525 μM, 530 μM, 535 μM, 540 μM, 545 μM, 550 μM, 555 μM, 560 μM, 565 μM, 570 μM, 575 μM, 580 μM, 585 μM, 590 μM, 595 μM, or 600 μM. In some embodiments, the concentration of the substance that reduces PKC activity and / or expression is about 400 μM when in contact with cells.

[0098] In some embodiments, the concentration of the poloxamer is from about 10 ng / mL to about 9.5 μg / mL (e.g., about 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, 285 ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL, 330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL,540ng / mL、545ng / mL、550ng / mL、555ng / mL、560ng / mL、565ng / mL、570ng / mL、575ng / mL、580ng / mL、585ng / mL、590ng / mL、595ng / mL、600ng / mL、605ng / mL、610ng / mL、615ng / mL、620ng / mL、625ng / mL、630ng / mL、635ng / mL、640ng / mL、645ng / mL、650ng / mL、655ng / mL、660ng / mL、665ng / mL、670ng / mL、675ng / mL、680ng / mL、685ng / mL、690ng / mL、695ng / mL、700ng / mL、705ng / mL、710ng / mL、715ng / mL、720ng / mL、725ng / mL、730ng / mL、735ng / mL、740ng / mL、745ng / mL、750ng / mL、755ng / mL、760ng / mL、765ng / mL、770ng / mL、775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / mL、895ng / mL、900ng / mL、905ng / mL、910ng / mL、915ng / mL、920ng / mL、925ng / mL、930ng / mL、935ng / mL、940ng / mL、945ng / mL、950ng / mL、955ng / mL、960ng / mL、965ng / mL、970ng / mL、975ng / mL、980ng / mL、985ng / mL、990ng / mL、995ng / mL、1μg / mL、1.1μg / mL、1.2μg / mL、1.3μg / mL、1.4μg / mL、1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4μg / mL、2.5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μ g / mL、3.8μg / mL、3.9μg / mL、4μg / mL、4.1μg / mL、4.2μg / mL、4.3μg / mL、4.4μg / mL、4.5μg / mL、 4.6μg / mL、4.7μg / mL、4.8μg / mL、4.9μg / mL、5μg / mL、5.1μg / mL、5.2μg / mL、5.3μg / mL、5.4μ g / mL、5.5μg / mL、5.6μg / mL、5.7μg / mL、5.8μg / mL、5.9μg / mL、6μg / mL、6.1μg / mL、6.2μg / mL、 6.3μg / mL、6.4μg / mL、6.5μg / mL、6.6μg / mL、6.7μg / mL、6.8μg / mL、6.9μg / mL、7μg / mL、7.1μ g / mL、7.2μg / mL、7.3μg / mL、7.4μg / mL、7.5μg / mL、7.6μg / mL、7.7μg / mL、7.8μg / mL、7.9μg / m L, 8μg / mL, 8.1μg / mL, 8.2μg / mL, 8.3μg / mL, 8.4μg / mL, 8.5μg / mL, 8.6μg / mL, 8.7μg / mL, 8.8μg / mL, 8.9μg / mL, 9μg / mL, 9.1μg / mL, 9.2μg / mL, 9.3μg / mL, 9.4μg / mL, or 9.5μg / mL).,

[0099] In some embodiments, the concentration of the poloxamer is from about 10 ng / mL to about 9 μg / mL (e.g., about 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, 285 ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL, 330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL,545ng / mL、550ng / mL、555ng / mL、560ng / mL、565ng / mL、570ng / mL、575ng / mL、580ng / mL、585ng / mL、590ng / mL、595ng / mL、600ng / mL、605ng / mL、610ng / mL、615ng / mL、620ng / mL、625ng / mL、630ng / mL、635ng / mL、640ng / mL、645ng / mL、650ng / mL、655ng / mL、660ng / mL、665ng / mL、670ng / mL、675ng / mL、680ng / mL、685ng / mL、690ng / mL、695ng / mL、700ng / mL、705ng / mL、710ng / mL、715ng / mL、720ng / mL、725ng / mL、730ng / mL、735ng / mL、740ng / mL、745ng / mL、750ng / mL、755ng / mL、760ng / mL、765ng / mL、770ng / mL、775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / mL、895ng / mL、900ng / mL、905ng / mL、910ng / mL、915ng / mL、920ng / mL、925ng / mL、930ng / mL、935ng / mL、940ng / mL、945ng / mL、950ng / mL、955ng / mL、960ng / mL、965ng / mL、970ng / mL、975ng / mL、980ng / mL、985ng / mL、990ng / mL、995ng / mL、1μg / mL、1.1μg / mL、1.2μg / mL、1.3μg / mL、1.4μg / mL、1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4μg / mL、2.5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μg / mL、3.8μg / mL、3.9μg / mL、4μg / mL、4.1μg / mL、4.2μg / mL、4.3μg / mL、4.4μg / mL、4.5μ g / mL、4.6μg / mL、4.7μg / mL、4.8μg / mL、4.9μg / mL、5μg / mL、5.1μg / mL、5.2μg / mL、5. 3μg / mL、5.4μg / mL、5.5μg / mL、5.6μg / mL、5.7μg / mL、5.8μg / mL、5.9μg / mL、6μg / mL、 6.1μg / mL, 6.2μg / mL, 6.3μg / mL, 6.4μg / mL, 6.5μg / mL, 6.6μg / mL, 6.7μg / mL, 6.8μg / mL, 6.9μg / mL, 7μg / mL, 7.1μg / mL, 7.2μg / mL, 7.3μg / mL, 7.4μg / mL, 7.5μg / mL, 7.6μg / mL, 7.7μg / mL, 7.8μg / mL, 7.9μg / mL, 8μg / mL, 8.1μg / mL, 8.2μg / mL, 8.3μg / mL, 8.4μg / mL, 8.5μg / mL, 8.6μg / mL, 8.7μg / mL, 8.8μg / mL, 8.9μg / mL, or 9μg / mL).,

[0100] In some embodiments, the concentration of the poloxamer is from about 50 ng / mL to about 8 μg / mL (e.g., about 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, 285 ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL, 330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL, 545 ng / mL, 550 ng / mL, 555 ng / mL, 560 ng / mL, 565 ng / mL, 570 ng / mL, 575 ng / mL,580ng / mL、585ng / mL、590ng / mL、595ng / mL、600ng / mL、605ng / mL、610ng / mL、615ng / mL、620ng / mL、625ng / mL、630ng / mL、635ng / mL、640ng / mL、645ng / mL、650ng / mL、655ng / mL、660ng / mL、665ng / mL、670ng / mL、675ng / mL、680ng / mL、685ng / mL、690ng / mL、695ng / mL、700ng / mL、705ng / mL、710ng / mL、715ng / mL、720ng / mL、725ng / mL、730ng / mL、735ng / mL、740ng / mL、745ng / mL、750ng / mL、755ng / mL、760ng / mL、765ng / mL、770ng / mL、775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / mL、895ng / mL、900ng / mL、905ng / mL、910ng / mL、915ng / mL、920ng / mL、925ng / mL、930ng / mL、935ng / mL、940ng / mL、945ng / mL、950ng / mL、955ng / mL、960ng / mL、965ng / mL、970ng / mL、975ng / mL、980ng / mL、985ng / mL、990ng / mL、995ng / mL、1μg / mL、1.1μg / mL、1.2μg / mL、1.3μg / mL、1.4μg / mL、1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4μg / mL、2.5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μg / mL、3.8μg / mL、3.9μg / mL、4μg / mL、4.1μg / mL、4.2μg / mL、4.3μg / mL、4.4μg / mL、4.5μg / mL、4.6μg / mL、4.7μg / mL、4. .8μg / mL、4.9μg / mL、5μg / mL、5.1μg / mL、5.2μg / mL、5.3μg / mL、5.4μg / mL、5.5μg / mL、5.6μg / mL、5.7μg / mL、5.8μg / mL、5.9μg / mL μg / mL, 6μg / mL, 6.1μg / mL, 6.2μg / mL, 6.3μg / mL, 6.4μg / mL, 6.5μg / mL, 6.6μg / mL, 6.7μg / mL, 6.8μg / mL, 6.9μg / mL, 7μg / mL, 7.1μg / mL, 7.2μg / mL, 7.3μg / mL, 7.4μg / mL, 7.5μg / mL, 7.6μg / mL, 7.7μg / mL, 7.8μg / mL, 7.9μg / mL, or 8μg / mL).,

[0101] In some embodiments, the concentration of the poloxamer is from about 100 ng / mL to about 7 μg / mL (e.g., 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 205 ng / mL, 210 ng / mL, 215 ng / mL, 220 ng / mL, 225 ng / mL, 230 ng / mL, 235 ng / mL, 240 ng / mL, 245 ng / mL, 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, twenty-eight five ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL, 330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL, 545 ng / mL, 550 ng / mL, 555 ng / mL, 560 ng / mL, 565 ng / mL, 570 ng / mL, 575 ng / mL, 580 ng / mL, 585 ng / mL, 590 ng / mL, 595 ng / mL, 600 ng / mL, 605 ng / mL, 610 ng / mL, 615 ng / mL, 620 ng / mL, when contacting the cells. It should be noted that there may be some inaccuracies in the original text where "twenty-eight five ng / mL" is an incorrect expression. It should probably be "285 ng / mL". The translation is done based on the provided text as accurately as possible.625ng / mL、630ng / mL、635ng / mL、640ng / mL、645ng / mL、650ng / mL、655ng / mL、660ng / mL、665ng / mL、670ng / mL、675ng / mL、680ng / mL、685ng / mL、690ng / mL、695ng / mL、700ng / mL、705ng / mL、710ng / mL、715ng / mL、720ng / mL、725ng / mL、730ng / mL、735ng / mL、740ng / mL、745ng / mL、750ng / mL、755ng / mL、760ng / mL、765ng / mL、770ng / mL、775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / mL、895ng / mL、900ng / mL、905ng / mL、910ng / mL、915ng / mL、920ng / mL、925ng / mL、930ng / mL、935ng / mL、940ng / mL、945ng / mL、950ng / mL、955ng / mL、960ng / mL、965ng / mL、970ng / mL、975ng / mL、980ng / mL、985ng / mL、990ng / mL、995ng / mL、1μg / mL、1.1μg / mL、1.2μg / mL、1.3μg / mL、1.4μg / mL、1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4μg / mL、2.5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μg / mL、3.8μg / mL、3.9μg / mL、4μg / mL、4.1μg / mL、4.2μg / mL、4.3μg / mL、4.4μg / mL、4.5μg / mL、4.6μg / mL、4.7μg / mL, 4.8μg / mL, 4.9μg / mL, 5μg / mL, 5.1μg / mL, 5.2μg / mL, 5.3μg / mL, 5.4μg / mL, 5.5μg / mL, 5.6μg / mL, 5.7μg / mL, 5.8μg / mL, 5.9μg / mL, 6μg / mL, 6.1μg / mL, 6.2μg / mL, 6.3μg / mL, 6.4μg / mL, 6.5μg / mL, 6.6μg / mL, 6.7μg / mL, 6.8μg / mL, 6.9μg / mL, or 7μg / mL).,

[0102] In some embodiments, the concentration of the poloxamer is from about 250 ng / mL to about 6 μg / mL (e.g., 250 ng / mL, 255 ng / mL, 260 ng / mL, 265 ng / mL, 270 ng / mL, 275 ng / mL, 280 ng / mL, 285 ng / mL, 290 ng / mL, 295 ng / mL, 300 ng / mL, 305 ng / mL, 310 ng / mL, 315 ng / mL, 320 ng / mL, 325 ng / mL, 330 ng / mL, 335 ng / mL, 340 ng / mL, 345 ng / mL, 350 ng / mL, 355 ng / mL, 360 ng / mL, 365 ng / mL, 370 ng / mL, 375 ng / mL, 380 ng / mL, 385 ng / mL, 390 ng / mL, 395 ng / mL, 400 ng / mL, 405 ng / mL, 410 ng / mL, 415 ng / mL, 420 ng / mL, 425 ng / mL, 430 ng / mL, 435 ng / mL, 440 ng / mL, 445 ng / mL, 450 ng / mL, 455 ng / mL, 460 ng / mL, 465 ng / mL, 470 ng / mL, 475 ng / mL, 480 ng / mL, 485 ng / mL, 490 ng / mL, 495 ng / mL, 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL, 545 ng / mL, 550 ng / mL, 555 ng / mL, 560 ng / mL, 565 ng / mL, 570 ng / mL, 575 ng / mL, 580 ng / mL, 585 ng / mL, 590 ng / mL, 595 ng / mL, 600 ng / mL, 605 ng / mL, 610 ng / mL, 615 ng / mL, 620 ng / mL, 625 ng / mL, 630 ng / mL, 635 ng / mL, 640 ng / mL, 645 ng / mL, 650 ng / mL, 655 ng / mL, 660 ng / mL, 665 ng / mL, 670 ng / mL, 675 ng / mL, 680 ng / mL, 685 ng / mL, 690 ng / mL, 695 ng / mL, 700 ng / mL, 705 ng / mL, 710 ng / mL, 715 ng / mL, 720 ng / mL, 725 ng / mL, 730 ng / mL, 735 ng / mL, 740 ng / mL, 745 ng / mL, 750 ng / mL, 755 ng / mL, 760 ng / mL, 765 ng / mL, 770 ng / mL) when contacting the cells.775ng / mL、780ng / mL、785ng / mL、790ng / mL、795ng / mL、800ng / mL、805ng / mL、810ng / mL、815ng / mL、820ng / mL、825ng / mL、830ng / mL 、835ng / mL、840ng / mL、845ng / mL、850ng / mL、855ng / mL、860ng / mL、865ng / mL、870ng / mL、875ng / mL、880ng / mL、885ng / mL、890ng / mL 、895ng / mL、900ng / mL、905ng / mL、910ng / mL、915ng / mL、920ng / mL、925ng / mL、930ng / mL、935ng / mL、940ng / mL、945ng / mL、950ng / mL L、955ng / mL、960ng / mL、965ng / mL、970ng / mL、975ng / mL、980ng / mL、985ng / mL、990ng / mL、995ng / mL、1μg / mL、1.1μg / mL、1.2μg / mL、 1.3μg / mL、1.4μg / mL、1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4μg / mL、2. .5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μg / mL μg / mL, 3.8μg / mL, 3.9μg / mL, 4μg / mL, 4.1μg / mL, 4.2μg / mL, 4.3μg / mL, 4.4μg / mL, 4.5μg / mL, 4.6μg / mL, 4.7μg / mL, 4.8μg / mL, 4.9μg / mL, 5μg / mL, 5.1μg / mL, 5.2μg / mL, 5.3μg / mL, 5.4μg / mL, 5.5μg / mL, 5.6μg / mL, 5.7μg / mL, 5.8μg / mL, 5.9μg / mL, or 6μg / mL).,

[0103] In some embodiments, the concentration of the poloxamer is from about 500 ng / mL to about 5 μg / mL (e.g., 500 ng / mL, 505 ng / mL, 510 ng / mL, 515 ng / mL, 520 ng / mL, 525 ng / mL, 530 ng / mL, 535 ng / mL, 540 ng / mL, 545 ng / mL, 550 ng / mL, 555 ng / mL, 560 ng / mL, 565 ng / mL, 570 ng / mL, 575 ng / mL, 580 ng / mL, 585 ng / mL, 590 ng / mL, 595 ng / mL, 600 ng / mL, 605 ng / mL, 610 ng / mL, 615 ng / mL, 620 ng / mL, 625 ng / mL, 630 ng / mL, 635 ng / mL, 640 ng / mL, 645 ng / mL, 650 ng / mL, 655 ng / mL, 660 ng / mL, 665 ng / mL, 670 ng / mL, 675 ng / mL, 680 ng / mL, 685 ng / mL, 690 ng / mL, 695 ng / mL, 700 ng / mL, 705 ng / mL, 710 ng / mL, 715 ng / mL, 720 ng / mL, 725 ng / mL, 730 ng / mL, 735 ng / mL, 740 ng / mL, 745 ng / mL, 750 ng / mL, 755 ng / mL, 760 ng / mL, 765 ng / mL, 770 ng / mL, 775 ng / mL, 780 ng / mL, 785 ng / mL, 790 ng / mL, 795 ng / mL, 800 ng / mL, 805 ng / mL, 810 ng / mL, 815 ng / mL, 820 ng / mL, 825 ng / mL, 830 ng / mL, 835 ng / mL, 840 ng / mL, 845 ng / mL, 850 ng / mL, 855 ng / mL, 860 ng / mL, 865 ng / mL, 870 ng / mL, 875 ng / mL, 880 ng / mL, 885 ng / mL, 890 ng / mL, 895 ng / mL, 900 ng / mL, 905 ng / mL, 910 ng / mL, 915 ng / mL, 920 ng / mL, 925 ng / mL, 930 ng / mL, 935 ng / mL, 940 ng / mL, 945 ng / mL, 950 ng / mL, 955 ng / mL, 960 ng / mL, 965 ng / mL, 970 ng / mL, 975 ng / mL, 980 ng / mL, 985 ng / mL, 990 ng / mL, 995 ng / mL, 1 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5μg / mL、1.6μg / mL、1.7μg / mL、1.8μg / mL、1.9μg / mL、2μg / mL、2.1μg / mL、2.2μg / mL、2.3μg / mL、2.4 μg / mL、2.5μg / mL、2.6μg / mL、2.7μg / mL、2.8μg / mL、2.9μg / mL、3μg / mL、3.1μg / mL、3.2μg / mL、3.3μg / mL、3.4μg / mL、3.5μg / mL、3.6μg / mL、3.7μg / mL、3.8μg / mL、3.9μg / mL、4μg / mL、4.1μg / mL、4.2μg / mL、4.3μg / mL、4.4μg / mL、4.5μg / mL、4.6μg / mL、4.7μg / mL、4.8μg / mL、4.9μg / mL、or 5μg / mL)。

[0104] In some embodiments, the poloxamer concentration, when in contact with cells, is approximately 750 ng / mL to approximately 3 μg / mL (e.g., 750 ng / mL, 755 ng / mL, 760 ng / mL, 765 ng / mL, 770 ng / mL, 775 ng / mL, 780 ng / mL, 785 ng / mL, 790 ng / mL, 795 ng / mL, 800 ng / mL, 805 ng / mL, 810 ng / mL, 8 15ng / mL, 820ng / mL, 825ng / mL, 830ng / mL, 835ng / mL, 840ng / mL, 845ng / mL, 850ng / mL, 855ng / mL, 860ng / m L, 865ng / mL, 870ng / mL, 875ng / mL, 880ng / mL, 885ng / mL, 890ng / mL, 895ng / mL, 900ng / mL, 905ng / mL, 910n g / mL, 915ng / mL, 920ng / mL, 925ng / mL, 930ng / mL, 935ng / mL, 940ng / mL, 945ng / mL, 950ng / mL, 955ng / mL, 9 60ng / mL, 965ng / mL, 970ng / mL, 975ng / mL, 980ng / mL, 985ng / mL, 990ng / mL, 995ng / mL, 1μg / mL, 1.1μg / mL, 1.2μg / mL, 1.3μg / mL, 1.4μg / mL, 1.5μg / mL, 1.6μg / mL, 1.7μg / mL, 1.8μg / mL, 1.9μg / mL, 2μg / mL, 2.1μg / mL , 2.2 μg / mL, 2.3 μg / mL, 2.4 μg / mL, 2.5 μg / mL, 2.6 μg / mL, 2.7 μg / mL, 2.8 μg / mL, 2.9 μg / mL, or 3 μg / mL).

[0105] In some embodiments, the poloxamer concentration, when in contact with cells, is approximately 800 ng / mL to approximately 2 μg / mL (e.g., 800 ng / mL, 805 ng / mL, 810 ng / mL, 815 ng / mL, 820 ng / mL, 825 ng / mL, 830 ng / mL, 835 ng / mL, 840 ng / mL, 845 ng / mL, 850 ng / mL, 855 ng / mL, 860 ng / mL, 865 ng / mL, 870 ng / mL, 875 ng / mL, 880 ng / mL, 885 ng / mL, 890 ng / mL, 895 ng / mL, 900 ng / mL, 905 ng / mL, 910 ng / mL). These values ​​are 1 μg / mL, 915 ng / mL, 920 ng / mL, 925 ng / mL, 930 ng / mL, 935 ng / mL, 940 ng / mL, 945 ng / mL, 950 ng / mL, 955 ng / mL, 960 ng / mL, 965 ng / mL, 970 ng / mL, 975 ng / mL, 980 ng / mL, 985 ng / mL, 990 ng / mL, 995 ng / mL, 1 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, or 2 μg / mL.

[0106] In some embodiments, the poloxamer concentration, when in contact with cells, is approximately 900 ng / mL to approximately 1.1 μg / mL (for example, 900 ng / mL, 905 ng / mL, 910 ng / mL, 915 ng / mL, 920 ng / mL, 925 ng / mL, 930 ng / mL, 935 ng / mL, 940 ng / mL, 945 ng / mL, 950 ng / mL, 955 ng / mL, 960 ng / mL, 965ng / mL, 970ng / mL, 975ng / mL, 980ng / mL, 985ng / mL, 990ng / mL, 995ng / mL, 1μg / mL, 1.01μg / mL, 1.02μg / m L, 1.03 μg / mL, 1.04 μg / mL, 1.05 μg / mL, 1.06 μg / mL, 1.07 μg / mL, 1.08 μg / mL, 1.09 μg / mL, or 1.1 μg / mL).

[0107] In some embodiments, the concentration of poloxamer is approximately 1 μg / mL when in contact with cells.

[0108] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,050 g / mol (e.g., approximately 2,055 g / mol, 2,060 g / mol, 2,075 g / mol, 2,080 g / mol, 2,085 g / mol, 2,090 g / mol, 2,095 g / mol, 2,100 g / mol, 2,200 g / mol, 2,300 g / mol, 2,400 g / mol, 2,500 g / mol, 2,600 g / mol, 2,700 g / mol, 2,800 g / mol, 2,900 g / mol, 3,000 g / mol, 3,100 g It has an average molar mass of polyoxypropylene subunits of 5,000 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol or more.

[0109] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,250 g / mol (e.g., about 2,300 g / mol, 2,400 g / mol, 2,500 g / mol, 2,600 g / mol, 2,700 g / mol, 2,800 g / mol, 2,900 g / mol, 3,000 g / mol, 3,100 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3 It has an average molar mass of polyoxypropylene subunits of 600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol or more.

[0110] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,750 g / mol (e.g., about 2,800 g / mol, 2,900 g / mol, 3,000 g / mol, 3,100 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3,800 g It has an average molar mass of polyoxypropylene subunits of 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol or more.

[0111] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,250 g / mol (for example, an average molar mass of polyoxypropylene subunits of about 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol or more).

[0112] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,625 g / mol (for example, an average molar mass of polyoxypropylene subunits of about 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol or more).

[0113] In some embodiments, the poloxamer has a molecular weight of from about 2,050 g / mol to about 4,000 g / mol (e.g., about 2,050 g / mol, 2,055 g / mol, 2,060 g / mol, 2,065 g / mol, 2,070 g / mol, 2,075 g / mol, 2,080 g / mol, 2,085 g / mol, 2,090 g / mol, 2,095 g / mol, 2,100 g / mol, 2,105 g / mol, 2,110 g / mol, 2,115 g / mol, 2,120 g / mol, 2,125 g / mol, 2,130 g / mol, 2,135 g / mol, 2,140 g / mol, 2,145 g / mol, 2,150 g / mol, 2,155 g / mol, 2,160 g / mol, 2,165 g / mol, 2,170 g / mol, 2,175 g / mol, 2,180 g / mol, 2,185 g / mol, 2,190 g / mol, 2,195 g / mol, 2,200 g / mol, 2,205 g / mol, 2,210 g / mol, 2,215 g / mol, 2,220 g / mol, 2,225 g / mol, 2,230 g / mol, 2,235 g / mol, 2,240 g / mol, 2,245 g / mol, 2,250 g / mol, 2,255 g / mol, 2,260 g / mol, 2,265 g / mol, 2,270 g / mol, 2,275 g / mol, 2,280 g / mol, 2,285 g / mol, 2,290 g / mol, 2,295 g / mol, 2,300 g / mol, 2,305 g / mol, 2,310 g / mol, 2,315 g / mol, 2,320 g / mol, 2,325 g / mol, 2,330 g / mol, 2,335 g / mol, 2,340 g / mol, 2,345 g / mol, 2,350 g / mol, 2,355 g / mol, 2,360 g / mol, 2,365 g / mol, 2,370 g / mol, 2,375 g / mol, 2,380 g / mol, 2,385 g / mol, 2,390 g / mol, 2,395 g / mol, 2,400 g / mol, 2,405 g / mol, 2,410 g / mol, 2,415 g / mol, 2,420 g / mol, 2,425 g / mol, 2,430 g / mol, 2,435 g / mol, 2,440 g / mol, 2,445 g / mol, 2,450 g / mol, 2,455 g / mol, 2,460 g / mol, 2,465 g / mol, 2,470 g / mol, 2,475 g / mol, 2,480g / mol、2,485g / mol、2,490g / mol、2,495g / mol、2,500g / mol、2,505g / mol、2,510g / mol、2,515g / mol、2,520g / mol、2,525g / mol、2,530g / mol、2,535g / mol、2,540g / mol、2,545g / mol、2,550g / mol、2,555g / mol、2,560g / mol、2,565g / mol、2,570g / mol、2,575g / mol、2,580g / mol、2,585g / mol、2,590g / mol、2,595g / mol、2,600g / mol、2,605g / mol、2,610g / mol、2,615g / mol、2,620g / mol、2,625g / mol、2,630g / mol、2,635g / mol、2,640g / mol、2,645g / mol、2,650g / mol、2,655g / mol、2,660g / mol、2,665g / mol、2,670g / mol、2,675g / mol、2,680g / mol、2,685g / mol、2,690g / mol、2,695g / mol、2,700g / mol、2,705g / mol、2,710g / mol、2,715g / mol、2,720g / mol、2,725g / mol、2,730g / mol、2,735g / mol、2,740g / mol、2,745g / mol、2,750g / mol、2,755g / mol、2,760g / mol、2,765g / mol、2,770g / mol、2,775g / mol、2,780g / mol、2,785g / mol、2,790g / mol、2,795g / mol、2,800g / mol、2,805g / mol、2,810g / mol、2,815g / mol、2,820g / mol、2,825g / mol、2,830g / mol、2,835g / mol、2,840g / mol、2,845g / mol、2,850g / mol、2,855g / mol、2,860g / mol、2,865g / mol、2,870g / mol、2,875g / mol、2,880g / mol、2,885g / mol、2,890g / mol、2,895g / mol、2,900g / mol、2,905g / mol、2,910g / mol、2,915g / mol、2,920g / mol、2,925g / mol、2,930g / mol、2,935g / mol、2,940g / mol、2,945g / mol、2,950g / mol、2,955g / mol、2,960g / mol、2,965g / mol、2,970g / mol、2,975g / mol、2,980g / mol、2,985g / mol、2,990g / mol、2,995g / mol、3,000g / mol、3,005g / mol、3,010g / mol、3,015g / mol、3,020g / mol、3,025g / mol、3,030g / mol、3,035g / mol、3,040g / mol、3,045g / mol、3,050g / mol、3,055g / mol、3,060g / mol、3,065g / mol、3,070g / mol、3,075g / mol、3,080g / mol、3,085g / mol、3,090g / mol、3,095g / mol、3,100g / mol、3,105g / mol、3,110g / mol、3,115g / mol、3,120g / mol、3,125g / mol、3,130g / mol、3,135g / mol、3,140g / mol、3,145g / mol、3,150g / mol、3,155g / mol、3,160g / mol、3,165g / mol、3,170g / mol、3,175g / mol、3,180g / mol、3,185g / mol、3,190g / mol、3,195g / mol、3,200g / mol、3,205g / mol、3,210g / mol、3,215g / mol、3,220g / mol、3,225g / mol、3,230g / mol、3,235g / mol、3,240g / mol、3,245g / mol、3,250g / mol、3,255g / mol、3,260g / mol、3,265g / mol、3,270g / mol、3,275g / mol、3,280g / mol、3,285g / mol、3,290g / mol、3,295g / mol、3,300g / mol、3,305g / mol、3,310g / mol、3,315g / mol、3,320g / mol、3,325g / mol、3,330g / mol、3,335g / mol、3,340g / mol、3,345g / mol、3,350g / mol、3,355g / mol、3,360g / mol、3,365g / mol、3,370g / mol、3,375g / mol、3,380g / mol、3,385g / mol、3,390g / mol、3,395g / mol、3,400g / mol、3,405g / mol、3,410g / mol、3,415g / mol、3,420g / mol、3,425g / mol、3,430g / mol、3,435g / mol、3,440g / mol、3,445g / mol、3,450g / mol、3,455g / mol、3,460g / mol、3,465g / mol、3,470g / mol、3,475g / mol、3,480g / mol、3,485g / mol、3,490g / mol、3,495g / mol、3,500g / mol、3,505g / mol、3,510g / mol、3,515g / mol、3,520g / mol、3,525g / mol、3,530g / mol、3,535g / mol、3,540g / mol、3,545g / mol、3,550g / mol、3,555g / mol、3,560g / mol、3,565g / mol、3,570g / mol、3,575g / mol、3,580g / mol、3,585g / mol、3,590g / mol、3,595g / mol、3,600g / mol、3,605g / mol、3,610g / mol、3,615g / mol、3,620g / mol、3,625g / mol、3,630g / mol、3,635g / mol、3,640g / mol、3,645g / mol、3,650g / mol、3,655g / mol、3,660g / mol、3,665g / mol、3,670g / mol、3,675g / mol、3,680g / mol、3,685g / mol、3,690g / mol、3,695g / mol、3,700g / mol、3,705g / mol、3,710g / mol、3,715g / mol、3,720g / mol、3,725g / mol、3,730g / mol、3,735g / mol、3,740g / mol、3,745g / mol、3,750g / mol、3,755g / mol、3,760g / mol、3,765g / mol、3,770g / mol、3,775g / mol、3,780g / mol、3,785g / mol、3,790g / mol、3,795g / mol、3,800g / mol、3,805g / mol、3,810g / mol、3,815g / mol、3,820g / mol、3,825g / mol、3,830g / mol、3,835g / mol、3,840g / mol、3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol l, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / mol, 3,925g / mol, 3,930g It has an average molar mass of polyoxypropylene subunits of 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0114] In some embodiments, the poloxamer has a molecular weight of from about 2,750 g / mol to about 4,000 g / mol (e.g., about 2,750 g / mol, 2,755 g / mol, 2,760 g / mol, 2,765 g / mol, 2,770 g / mol, 2,775 g / mol, 2,780 g / mol, 2,785 g / mol, 2,790 g / mol, 2,795 g / mol, 2,800 g / mol, 2,805 g / mol, 2,810 g / mol, 2,815 g / mol, 2,820 g / mol, 2,825 g / mol, 2,830 g / mol, 2,835 g / mol, 2,840 g / mol, 2,845 g / mol, 2,850 g / mol, 2,855 g / mol, 2,860 g / mol, 2,865 g / mol, 2,870 g / mol, 2,875 g / mol, 2,880 g / mol, 2,885 g / mol, 2,890 g / mol, 2,895 g / mol, 2,900 g / mol, 2,905 g / mol, 2,910 g / mol, 2,915 g / mol, 2,920 g / mol, 2,925 g / mol, 2,930 g / mol, 2,935 g / mol, 2,940 g / mol, 2,945 g / mol, 2,950 g / mol, 2,955 g / mol, 2,960 g / mol, 2,965 g / mol, 2,970 g / mol, 2,975 g / mol, 2,980 g / mol, 2,985 g / mol, 2,990 g / mol, 2,995 g / mol, 3,000 g / mol, 3,005 g / mol, 3,010 g / mol, 3,015 g / mol, 3,020 g / mol, 3,025 g / mol, 3,030 g / mol, 3,035 g / mol, 3,040 g / mol, 3,045 g / mol, 3,050 g / mol, 3,055 g / mol, 3,060 g / mol, 3,065 g / mol, 3,070 g / mol, 3,075 g / mol, 3,080 g / mol, 3,085 g / mol, 3,090 g / mol, 3,095 g / mol, 3,100 g / mol, 3,105 g / mol, 3,110 g / mol, 3,115 g / mol, 3,120 g / mol, 3,125 g / mol, 3,130 g / mol, 3,135 g / mol, 3,140 g / mol, 3,145 g / mol, 3,150 g / mol, 3,155 g / mol, 3,160 g / mol, 3,165 g / mol, 3,170 g / mol, 3,175 g / mol, 3,180g / mol、3,185g / mol、3,190g / mol、3,195g / mol、3,200g / mol、3,205g / mol、3,210g / mol、3,215g / mol、3,220g / mol、3,225g / mol、3,230g / mol、3,235g / mol、3,240g / mol、3,245g / mol、3,250g / mol、3,255g / mol、3,260g / mol、3,265g / mol、3,270g / mol、3,275g / mol、3,280g / mol、3,285g / mol、3,290g / mol、3,295g / mol、3,300g / mol、3,305g / mol、3,310g / mol、3,315g / mol、3,320g / mol、3,325g / mol、3,330g / mol、3,335g / mol、3,340g / mol、3,345g / mol、3,350g / mol、3,355g / mol、3,360g / mol、3,365g / mol、3,370g / mol、3,375g / mol、3,380g / mol、3,385g / mol、3,390g / mol、3,395g / mol、3,400g / mol、3,405g / mol、3,410g / mol、3,415g / mol、3,420g / mol、3,425g / mol、3,430g / mol、3,435g / mol、3,440g / mol、3,445g / mol、3,450g / mol、3,455g / mol、3,460g / mol、3,465g / mol、3,470g / mol、3,475g / mol、3,480g / mol、3,485g / mol、3,490g / mol、3,495g / mol、3,500g / mol、3,505g / mol、3,510g / mol、3,515g / mol、3,520g / mol、3,525g / mol、3,530g / mol、3,535g / mol、3,540g / mol、3,545g / mol、3,550g / mol、3,555g / mol、3,560g / mol、3,565g / mol、3,570g / mol、3,575g / mol、3,580g / mol、3,585g / mol、3,590g / mol、3,595g / mol、3,600g / mol、3,605g / mol、3,610g / mol、3,615g / mol、3,620g / mol、3,625g / mol、3,630g / mol、3,635g / mol, 3,640g / mol, 3,645g / mol, 3,650g / mol, 3,655g / mol, 3,660g / mol, 3,665g / mol, 3,670g / mol, 3,675g / mol, 3,680g / m ol, 3,685g / mol, 3,690g / mol, 3,695g / mol, 3,700g / mol, 3,705g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,73 0g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol, 3,760g / mol, 3,765g / mol, 3,770g / mol, 3,775g / mol , 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,810g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3 ,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / m It has an average molar mass of polyoxypropylene subunits of ol, 3,925 g / mol, 3,930 g / mol, 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0115] In some embodiments, the poloxamer has a molecular weight of from about 3,250 g / mol to about 4,000 g / mol (e.g., about 3,250 g / mol, 3,255 g / mol, 3,260 g / mol, 3,265 g / mol, 3,270 g / mol, 3,275 g / mol, 3,280 g / mol, 3,285 g / mol, 3,290 g / mol, 3,295 g / mol, 3,300 g / mol, 3,305 g / mol, 3,310 g / mol, 3,315 g / mol, 3,320 g / mol, 3,325 g / mol, 3,330 g / mol, 3,335 g / mol, 3,340 g / mol, 3,345 g / mol, 3,350 g / mol, 3,355 g / mol, 3,360 g / mol, 3,365 g / mol, 3,370 g / mol, 3,375 g / mol, 3,380 g / mol, 3,385 g / mol, 3,390 g / mol, 3,395 g / mol, 3,400 g / mol, 3,405 g / mol, 3,410 g / mol, 3,415 g / mol, 3,420 g / mol, 3,425 g / mol, 3,430 g / mol, 3,435 g / mol, 3,440 g / mol, 3,445 g / mol, 3,450 g / mol, 3,455 g / mol, 3,460 g / mol, 3,465 g / mol, 3,470 g / mol, 3,475 g / mol, 3,480 g / mol, 3,485 g / mol, 3,490 g / mol, 3,495 g / mol, 3,500 g / mol, 3,505 g / mol, 3,510 g / mol, 3,515 g / mol, 3,520 g / mol, 3,525 g / mol, 3,530 g / mol, 3,535 g / mol, 3,540 g / mol, 3,545 g / mol, 3,550 g / mol, 3,555 g / mol, 3,560 g / mol, 3,565 g / mol, 3,570 g / mol, 3,575 g / mol, 3,580 g / mol, 3,585 g / mol, 3,590 g / mol, 3,595 g / mol, 3,600 g / mol, 3,605 g / mol, 3,610 g / mol, 3,615 g / mol, 3,620 g / mol, 3,625 g / mol, 3,630 g / mol, 3,635 g / mol, 3,640 g / mol, 3,645 g / mol, 3,650 g / mol, 3,655 g / mol, 3,660 g / mol, 3,665 g / mol, 3,670 g / mol, 3,675 g / mol, 3,680g / mol, 3,685g / mol, 3,690g / mol, 3,695g / mol, 3,700g / mol, 3,705g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,730g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol, 3,760g / mol, 3 ,765g / mol, 3,770g / mol, 3,775g / mol, 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,810g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3 ,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / mol, 3,925g / mol, 3,930g / mol, It has an average molar mass of polyoxypropylene subunits of 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol.

[0116] In some embodiments, poloxamer is present in concentrations of approximately 3,625 g / mol to approximately 4,000 g / mol (for example, approximately 3,625 g / mol, 3,630 g / mol, 3,635 g / mol, 3,640 g / mol, 3,645 g / mol, 3,650 g / mol, 3,655 g / mol, 3,660 g / mol, 3,665 g / mol, 3,670 g / mol, 3,675 g / mol, 3,680 g / mol, 3,685 g / mol, 3,690 g / mol, 3,695 g / mol, 3,70700 g / mol, 3,680 g / mol, 3,685 g / mol, 3,690 g / mol, 3,695 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 5g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,730g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol l, 3,760g / mol, 3,765g / mol, 3,770g / mol, 3,775g / mol, 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,8 10g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3 It has an average molar mass of polyoxypropylene subunits of 915 g / mol, 3,920 g / mol, 3,925 g / mol, 3,930 g / mol, 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0117] In some embodiments, poloxamer is present in amounts exceeding 40% by mass (for example, about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%). It has an average ethylene oxide content of 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more.

[0118] In some embodiments, the poloxamer has an average ethylene oxide content of more than 50% by mass (for example, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0119] In some embodiments, the poloxamer has an average ethylene oxide content of more than 60% by mass (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0120] In some embodiments, the poloxamer has an average ethylene oxide content of more than 70% by mass (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0121] In some embodiments, the poloxamer has an average ethylene oxide content of about 40% to about 90% (for example, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%).

[0122] In some embodiments, the poloxamer has an average ethylene oxide content of about 50% to about 85% (for example, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%).

[0123] In some embodiments, the poloxamer has an average ethylene oxide content of about 60% to about 80% (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%).

[0124] In some embodiments, poloxamer is present in concentrations greater than 10,000 g / mol (e.g., approximately 10,100 g / mol, 10,200 g / mol, 10,300 g / mol, 10,400 g / mol, 10,500 g / mol, 10,600 g / mol, 10,700 g / mol, 10,800 g / mol, 10,900 g / mol, 11,000 g / mol, 11,100 g / mol). 0g / mol, 11,200g / mol, 11,300g / mol, 11,400g / mol, 11,500g / mol, 11,600g / mol, 11,700g / mol, 11, 800g / mol, 11,900g / mol, 12,000g / mol, 12,100g / mol, 12,200g / mol, 12,300g / mol, 12,400g / mol, 1 2,500g / mol, 12,600g / mol, 12,700g / mol, 12,800g / mol, 12,900g / mol, 13,000g / mol, 13,100g / mol l, 13,200g / mol, 13,300g / mol, 13,400g / mol, 13,500g / mol, 13,600g / mol, 13,700g / mol, 13,800g / It has an average molar mass of 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0125] In some embodiments, the poloxamer has an average molar mass greater than 11,000 g / mol (e.g., about 11,100 g / mol, 11,200 g / mol, 11,300 g / mol, 11,400 g / mol, 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol).

[0126] In some embodiments, the poloxamer has an average molar mass greater than 12,000 g / mol (e.g., about 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 十三,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,40 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol).

[0127] In some embodiments, the poloxamer has an average molar mass greater than 12,500 g / mol (e.g., about 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol).

[0128] In some embodiments, poloxamer is present in concentrations of approximately 10,000 g / mol to approximately 15,000 g / mol (for example, approximately 10,000 g / mol, 10,100 g / mol, 10,200 g / mol, 10,300 g / mol, 10,400 g / mol, 10,500 g / mol, 10,600 g / mol, 10,700 g / mol, 10,800 g / mol, 10,900 g / mol). l, 11,000g / mol, 11,100g / mol, 11,200g / mol, 11,300g / mol, 11,400g / mol, 11,500g / mol, 11,600g / mol , 11,700g / mol, 11,800g / mol, 11,900g / mol, 12,000g / mol, 12,100g / mol, 12,200g / mol, 12,300g / mol, 12,400g / mol, 12,500g / mol, 12,600g / mol, 12,700g / mol, 12,800g / mol, 12,900g / mol, 13,000g / mol, 1 3,100g / mol, 13,200g / mol, 13,300g / mol, 13,400g / mol, 13,500g / mol, 13,600g / mol, 13,700g / mol, 13 It has an average molar mass of 800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0129] In some embodiments, poloxamer is present in concentrations of approximately 11,000 g / mol to approximately 15,000 g / mol (for example, approximately 11,000 g / mol, 11,100 g / mol, 11,200 g / mol, 11,300 g / mol, 11,400 g / mol, 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol). It has an average molar mass of 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0130] In some embodiments, poloxamer is present in concentrations of approximately 11,500 g / mol to approximately 15,000 g / mol (for example, approximately 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol). It has an average molar mass of g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0131] In some embodiments, poloxamer is present in concentrations of approximately 12,000 g / mol to approximately 15,000 g / mol (for example, approximately 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, It has an average molar mass of 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0132] In some embodiments, poloxamer is present in concentrations of approximately 12,500 g / mol to approximately 15,000 g / mol (for example, approximately 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol). It has an average molar mass of g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0133] In some embodiments, the poloxamer is P407.

[0134] In some embodiments, the poloxamer is P338.

[0135] In some embodiments, the poloxamer is P288.

[0136] In some embodiments, the poloxamer is P188.

[0137] In some embodiments, the viral vector is selected from the group consisting of retroviridae family viruses, adeno-associated viruses, adenoviruses, parvoviruses, coronaviruses, rhabdoviruses, paramyxoviruses, picornaviruses, alphaviruses, herpesviruses, and poxviruses. The viral vector may be a retroviridae family viral vector, such as a lentiviral vector, an alpha-retrovirus vector, or a gamma-retrovirus vector. In some embodiments, the retroviridae family viral vector includes a central polypurine band, a Woodchuck hepatitis virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-autoinactivating LTR.

[0138] In some embodiments, the viral vector is a pseudotyped viral vector containing a viral genome derived from a certain type of virus and one or more viral capsids or envelope proteins derived from different species of viruses. Examples of pseudotyped viral vectors include vesicular stomatitis virus (VSV), RD114 virus, mouse leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan encephalitis virus (VEE), human formy virus (HFV), walleye dermatosarcoma virus (WDSV), Semlik Forest virus (SFV), rabies virus, avian leukemia virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), canine arthritis encephalitis virus (CAEV), and Synnoble It may contain one or more viral envelope proteins derived from viruses selected from viruses (SNV), Cherry twisted leaf virus (ChTLV), monkey T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), bird carcinoma virus (MH2), avian encephalomyelitis virus (AEV), alpha mosaic virus (AMV), bird sarcoma virus CT10, and equine infectious anemia virus (EIAV).

[0139] In some embodiments, contact of cells with one or more agents described above or herein occurs ex vivo. The cells may be freshly cultured before contact, or they may be cryopreserved and thawed before contact.

[0140] In some embodiments, before the cells come into contact with poloxamer, the cells first come into contact with a substance that reduces the activity and / or expression of PKC. For example, before the cells come into contact with poloxamer, the cells may first come into contact with a substance that reduces the activity and / or expression of PKC for about 30 minutes to about 6 hours (e.g., about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours before the cells come into contact). In some embodiments, before the cells come into contact with poloxamer, the cells first come into contact with a substance that reduces the activity and / or expression of PKC for about 1 hour to about 3 hours (e.g., about 1 hour, 2 hours, or 3 hours before the cells come into contact with poloxamer). In some embodiments, about 2 hours before the cells come into contact with poloxamer, the cells first come into contact with a substance that reduces the activity and / or expression of PKC.

[0141] In some embodiments, before the cells come into contact with poloxamers, the cells are washed to remove any substances that reduce the activity and / or expression of PKC, so that the cells first come into contact with the poloxamers.

[0142] In some embodiments, cells come into contact with a substance that reduces PKC activity and / or expression, as well as a poloxamer, simultaneously. For example, cells may come into contact with a substance that reduces PKC activity and / or expression, a poloxamer, and a viral vector simultaneously.

[0143] In some embodiments, cells are exposed to a substance that reduces PKC activity and / or expression before coming into contact with the viral vector. In these examples, cells may come into contact with the viral vector and poloxamer simultaneously. Alternatively, cells may come into contact with poloxamer before coming into contact with the viral vector. In some embodiments, cells come into contact with the viral vector before coming into contact with poloxamer.

[0144] Accordingly, in some embodiments of the present disclosure, cells first come into contact with a substance that reduces PKC activity and / or expression, then come into contact with a poloxamer, and subsequently come into contact with a viral vector.

[0145] In some embodiments, the cells further come into contact with cyclosporine, such as cyclosporine A (CsA) or cyclosporine H (CsH). The cells may come into contact with poloxamer and cyclosporine simultaneously. Alternatively, the cells may come into contact with poloxamer before coming into contact with staurosporine. In some embodiments, the cells come into contact with cyclosporine before coming into contact with poloxamer.

[0146] In some embodiments, the cyclosporine concentration, when in contact with cells, is approximately 1 μM to approximately 10 μM (e.g., approximately 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM). μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9μM, 4μM, 4.1μM, 4.2μM, 4.3μM, 4.4μM, 4.5μM, 4.6μM, 4.7μM, 4.8μM, 4.9μM, 5μM, 5.1μM , 5.2μM, 5.3μM, 5.4μM, 5.5μM, 5.6μM, 5.7μM, 5.8μM, 5.9μM, 6μM, 6.1μM, 6.2μM, 6.3μM, 6. 4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM, 7μM, 7.1μM, 7.2μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM The concentrations are 7.7 μM, 7.8 μM, 7.9 μM, 8 μM, 8.1 μM, 8.2 μM, 8.3 μM, 8.4 μM, 8.5 μM, 8.6 μM, 8.7 μM, 8.8 μM, 8.9 μM, 9 μM, 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM, 9.9 μM, or 10 μM. In some embodiments, cyclosporine is CsA, and the cyclosporine concentration is about 6 μM when in contact with cells. In some embodiments, cyclosporine is CsH, and the cyclosporine concentration is about 8 μM when in contact with cells.

[0147] In some embodiments, the cells further come into contact with the activator of prostaglandin E receptor signaling. The cells may come into contact with poloxamers and the activator of prostaglandin E receptor signaling simultaneously. Alternatively, the cells may come into contact with poloxamers before coming into contact with the activator of prostaglandin E receptor signaling. In some embodiments, the cells come into contact with the activator of prostaglandin E receptor signaling before coming into contact with poloxamers.

[0148] In some embodiments, the activator of prostaglandin E receptor signaling is a small molecule such as a compound described in WO2007 / 112084 or WO2010 / 108028, and the disclosures of each of these are incorporated herein by reference because they are related to prostaglandin E receptor signaling activators.

[0149] In some embodiments, the activator of prostaglandin E receptor signaling is a small organic molecule, prostaglandin, Wnt pathway agonist, cAMP / PI3K / AKT pathway agonist, Ca 2+ second messenger pathway agonist, a small molecule such as a nitric oxide (NO) / angiotensin signaling agonist, or another compound known to stimulate the prostaglandin signaling pathway, such as a compound selected from mebeverine, fludalanolide, atenolol, pindolol, gaboxadol, kynurenic acid, hydralazine, thiabendazole, vicrine, besamicol, perboside, imipramine, chlorpropamide, 1,5-pentamethylenetetrazole, 4-aminopyridine, diazoxide, benfotiamine, 12-methoxydodecenoic acid, N-formyl-Met-Leu-Phe, galamine, IAA94, chlorotrianisene, and / or derivatives of any of these compounds.

[0150] In some embodiments, the activator of prostaglandin E receptor signaling is a naturally occurring, synthetic chemical molecule or polypeptide that binds to and / or interacts with the prostaglandin E receptor and typically activates or increases one or more of the downstream signaling pathways associated with the prostaglandin E receptor.

[0151] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of prostaglandin (PG)A2 (PGA2), PGB2, PGD2, PGE1 (alprostadil), PGE2, PGF2, PGI2 (epoprostenol), PGH2, PGJ2, and derivatives and analogs thereof.

[0152] In some embodiments, the activators of prostaglandin E receptor signaling include 15d-PGJ2, delta-I2-PGJ2, 2-hydroxyheptadecatrienoic acid (HHT), thromboxanes (TXA2 and TXB2), PGI2 analogs (e.g., iloprost and treprostinil), PGF2 analogs (e.g., travoprost, carboprost tromethamine, tafluprost, latanoprost, bimatoprost, unoprostone isopropyl, cloprostenol, oestrophan, and superphan), PGE1 analogs (e.g., 11-deoxyPGE1, misoprostol, and porcine prost), and Furthermore, these are Corey alcohol-A ([3aa,4a,5,6aa]-(-)-[hexahydro-4-(hydroxymethyl)-2-oxo-2H-cyclopenta / b / furan-5-yl][1,1'-biphenyl]-4-carboxylate), Corey alcohol-B (2H-cyclopenta[b]furan-2-one,5-(benzoyloxy)hexahydro-4-(hydroxymethyl)[3aR-(3aa,4a,5,6aa)]), and Corey diol ((3aR,4S,5R,6aS)-hexahydro-5-hydroxy-4-(hydroxymethyl)-2H-cyclopenta[b]furan-2-one).

[0153] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin E receptor such as prostaglandin E2 (PGE2), or an analog or derivative thereof. Prostaglandins generally refer to hormone-like molecules derived from 20 carbon-carbon fatty acids containing a 5-carbocyclic ring, as described herein and known in the art. Examples of PGE2 "analogs" or "derivatives" include 16,16-dimethylPGE2, 16-16-dimethylPGE2p-(p-acetamidobenzamide)phenyl ester, II-deoxy-16,16-dimethylPGE2, 9-deoxy-9-methylene-16,16-dimethylPGE2, 9-deoxy-9-methylenePGE2, 9-ketofluprostenol, and 5-trans Examples include, but are not limited to, PGE2, 17-phenyl-omega-trinolic PGE2, PGE2 selinolamide, PGE2 methyl ester, 16-phenyltetranol PGE2, 15(S)-15-methyl PGE2, 15(R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE2, nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19(R) hydroxy PGE2.

[0154] In some embodiments, the activators of prostaglandin E receptor signaling are prostaglandin analogs or derivatives having a structure similar to PGE2 with a halogen substituted at position 9 (see, for example, WO2001 / 12596, which is incorporated herein by reference in whole), as well as 2-decarboxy-2-phosphinicoprostaglandin derivatives, such as those described in US2006 / 0247214, which is incorporated herein by reference in whole.

[0155] In some embodiments, the activator of prostaglandin E receptor signaling is a non-PGE2-based ligand. In some embodiments, the activator of prostaglandin E receptor signaling is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Further examples of non-PGE2-based EP2 agonists include carbazole and fluorene, disclosed in WO2007 / 071456, which is incorporated herein by reference with respect to the disclosure of such agents. Examples of non-PGE2-based EP3 agonists include, but are not limited to, AE5-599, MB28767, GR 63799X, ONO-NT012, and ONO-AE-248. Examples of non-PGE2-based EP4 agonists include, but are not limited to, ONO-4819, APS-999 Na, AH23848, and ONO-AE1-329. Further examples of non-PGE2-based EP4 agonists can be found in WO2000 / 038663; U.S. Patent No. 6,747,037; and U.S. Patent No. 6,610,719, each of which is incorporated by reference in the disclosure of such agonists.

[0156] In some embodiments, the activator of prostaglandin E receptor signaling is a Wnt agonist. Examples of Wnt agonists include, but are not limited to, Wnt polypeptides and glycogen synthase kinase 3 (GSK3) inhibitors. Examples of Wnt polypeptides suitable for use as compounds that stimulate the prostaglandin EP receptor signaling pathway include, but are not limited to, Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, or their biologically active fragments. GSK3 inhibitors suitable for use as agents that stimulate the prostaglandin EP receptor signaling pathway bind to GSK3a or GSK3 and reduce the activity of GSK3a or GSK3. Examples of GSK3 inhibitors include BIO(6-bromoindilbine-3), as exemplified in U.S. Patents 6,057,117 and 6,608,063, as well as U.S.2004 / 0092535 and U.S.2004 / 0209878. ’ Examples include, but are not limited to, GSK-3 inhibitors (oxime), LiCl, or other GSK-3 inhibitors, as well as the ATP-antagonistic and selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also known as CT-99021 and CT-98023, respectively) (Chiron Corporation (Emeryville, CA)).

[0157] In some embodiments, the activators of prostaglandin E receptor signaling are agents that increase signaling through the cAMP / P13K / AKT second messenger pathway, such as agents selected from the group consisting of dibutyryl cAMP (DBcAMP), phorbol ester, forskolin, sclarelin, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4-dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, iloprost, pituitary adenylyl cyclase-activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), as well as derivatives of these agents.

[0158] In some embodiments, the activator of prostaglandin E receptor signaling is an agent selected from the group consisting of bapta-AM, fendiline, nicardipine, and derivatives thereof, such as Ca 2+ It is an agent that increases signal transduction through the second messenger pathway.

[0159] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through NO / angiotensin signaling, such as an agent selected from the group consisting of L-Arg, sodium nitroprusside, sodium vanadate, bradykinin, and derivatives thereof.

[0160] In some embodiments, the cells further come into contact with the polycationic polymer. The cells may come into contact with the poloxamer and the polycationic polymer simultaneously. Alternatively, the cells may come into contact with the poloxamer before coming into contact with the polycationic polymer. In some embodiments, the cells come into contact with the polycationic polymer before coming into contact with the poloxamer.

[0161] In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or polyethylene glycol / poly-L-lysine block copolymer.

[0162] In some embodiments, the cells are further contacted with a swelling agent during the transduction process. The cells may be, for example, pluripotent hematopoietic stem cells, and the swelling agent may be a pluripotent hematopoietic stem cell swelling agent, such as a pluripotent hematopoietic stem cell swelling agent known in the art or described herein.

[0163] In some embodiments, during the transduction procedure, the cells are further exposed to an agent that inhibits mTor signaling. The agent that inhibits mTor signaling may be, for example, rapamycin, among other suppressors of mTor signaling.

[0164] In some embodiments, the cells are incubated with a viral vector (for example, in combination with one or more of the agents described above) for a period of about 6 to about 48 hours (e.g., about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours). In some embodiments, cells are incubated with a viral vector (for example, in combination with one or more of the agents described above) for a period of about 12 to about 24 hours (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In some embodiments, cells are incubated with a viral vector (for example, in combination with one or more of the agents described above) for a period of about 16 to about 22 hours (e.g., about 16, 17, 18, 19, 20, 21, or 22 hours). In some embodiments, cells are incubated with a viral vector (for example, in combination with one or more of the agents described above) for a period of about 17 to about 19 hours (e.g., about 17, 18, or 19 hours). In some embodiments, cells are incubated with a viral vector (for example, in combination with one or more of the agents described above) for a period of about 18 hours.

[0165] In some embodiments, cells are spun (i.e., subjected to centrifugation) (e.g., by centrifugation) while remaining in contact with the viral vector (e.g., in combination with one or more of the agents described above). This process, referred herein as "spinoculation," may occur with centripetal forces of, for example, about 200xg to about 2,000xg. In some embodiments, cells are spun with centripetal forces of about 300xg to about 1,200xg while remaining in contact with the viral vector (e.g., in combination with one or more of the agents described above). For example, cells may be spun with centripetal forces of about 300xg, 400xg, 500xg, 600xg, 700xg, 800xg, 900xg, 1,000xg, 1,100xg, or 1,200xg while remaining in contact with the viral vector (e.g., in combination with one or more of the agents described above). In some embodiments, cells are spun for about 10 minutes to about 3 hours (for example, about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes or longer). In some embodiments, cells are spun at room temperature, for example, at a temperature of about 25°C.

[0166] In a further embodiment, the present disclosure provides a method for expressing a transgene in a subject (e.g., a mammalian subject such as a human), characterized by administering to the subject a population of cells, or their progeny, that have been modified according to any of the above embodiments or embodiments of the present disclosure.

[0167] In a further embodiment, the present disclosure is a method for delivering a population of genetically modified cells to a subject (e.g., a mammalian subject such as a human), characterized by administering to the subject a population of cells, or their offspring, that have been modified according to any of the above embodiments or models of the present disclosure.

[0168] In yet another aspect, the Disclosure provides a method for providing cell therapy to a subject in need of cell therapy (e.g., a mammalian subject such as a human), characterized by administering to the subject a population of cells or their progeny modified in accordance with any of the above aspects or embodiments of the Disclosure.

[0169] In some embodiments of the three embodiments described herein, the cells are allogeneic to the subject. In some embodiments, the cells are HLA-matched to the subject. In some embodiments, the cells are autosomatic to the subject.

[0170] In some embodiments, a population of progenitor cells is isolated from a subject (e.g., an autologous cell population) or a donor (e.g., an allogeneic cell population) before contacting the cells with one or more agents described above or herein. The progenitor cells are then expanded ex vivo by incubation with one or more cell-expanding agents, for example, described herein or known in the art, thereby promoting cell proliferation and obtaining a population of cells to be administered to the subject. For example, the expanding agent may be StemRegenin-1, also known in the art as compound SR1, represented by the following formula (110).

[0171] [ka]

[0172] SR1 and other swelling agents are described, for example, in U.S. Patent Nos. 8,927,281 and 9,580,426, each of which is incorporated herein by reference in whole.

[0173] Further leavening agents that can be used in combination with the compositions and methods of this disclosure include compound UM-171, which is described in U.S. Patent No. 9,409,906, the disclosure of which is included in its entirety by reference in this invention. Further leavening agents that can be used herein include structural variants or stereoisomers of compound UM-171, such as the compound described in U.S. 2017 / 0037047, the disclosure of which is included in its entirety by reference in this invention. The structure of compound UM-171 is shown in formula (111) below.

[0174] [ka]

[0175] In some embodiments, the leavening agent is a bromide salt of compound (111), such as the compound represented by the following formula (112).

[0176] [ka]

[0177] Further swelling agents that can be used in combination with the compositions and methods of this disclosure include, for example, histone deacetylase (HDAC) inhibitors, as described in WO2000 / 023567, the disclosure of which is incorporated herein by reference. Exemplary agents that can be used to swell the populations of progenitor cells described herein include, among others, trichostatin A, trapoxin, trapoxin A, chlamydosin, sodium butyrate, dimethyl sulfoxide, suberanilohydroxamic acid, m-carboxycinnamate bishydroxamide, HC-toxin, Cyl-2, WF-3161, depdesin, and radisicol.

[0178] In some embodiments, the progenitor cells are CD34+ HSCs. Using HSC swelling agents described herein and known in the art, the progenitor cells can be swelled without losing their functional capabilities as HSCs.

[0179] In some embodiments, before isolating progenitor cells from a subject (e.g., an autologous cell population) or a donor (e.g., an allogeneic cell population), the subject or donor is administered one or more recruiters that stimulate the migration of pluripotent cells (e.g., CD34+ HSCs and HPCs) from the stem cell niche (e.g., bone marrow) to the peripheral circulation. Exemplary cell recruiters that can be used in combination with the compositions and methods of this disclosure are described herein and are known in the art. For example, the recruiter may be a CXC motif chemokine receptor (CXCR)2 (CXCR2) agonist. The CXCR2 agonist may be Gro-beta or its truncated variant. Gro-beta and its variants are described, for example, in U.S. Patents 6,080,398; 6,447,766; and 6,399,053, each of which is incorporated herein by reference in whole. Furthermore, or alternatively, the mobilizer may include a CXCR4 antagonist, such as prelixafor or a variant thereof. Prelixafor and structurally similar compounds are described, for example, in U.S. Patents 6,987,102; 7,935,692; and 7,897,590, each of which disclosures are incorporated by reference herein. Furthermore, or alternatively, the mobilizer may include granulocyte colony-stimulating factor (G-CSF). The use of G-CSF as an agent to induce the migration of pluripotent cells (e.g., CD34+ HSCs and / or HPCs) from the stem cell niche to the peripheral circulation is described, for example, in U.S. 2010 / 0178271, the entire disclosure of which is incorporated by reference herein.

[0180] In some embodiments, a population of endogenous pluripotent cells (e.g., a population of endogenous CD34+ HSCs or HPCs) is excised in the subject by administering one or more conditioning agents to the subject before administering a population of cells to the subject. In some embodiments, the method includes excising a population of endogenous pluripotent cells (e.g., a population of endogenous CD34+ HSCs or HPCs) in the subject by administering one or more conditioning agents to the subject before administering a population of cells to the subject. The one or more conditioning agents may be myeloablative conditioning agents that deplete various hematopoietic cells from the bone marrow of the subject. In some embodiments, the one or more conditioning agents are non-myeloablative conditioning agents that selectively target and excise a specific population of endogenous pluripotent cells, such as a population of endogenous CD34+ HSCs or HPCs.

[0181] In some embodiments, when administered to a population of cells, the administered cells, or their progeny, differentiate into one or more cell types selected from megakaryocytes, thrombi, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteolytic cells, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0182] In some embodiments, the subject is diagnosed with a deficiency in an endogenous protein encoded by the transgene. The subject may be diagnosed with, for example, one of the diseases listed in Table 2. In some embodiments, the subject is diagnosed with beta-thalassemia.

[0183] In some embodiments of any of the above aspects or embodiments of this disclosure, the transgene encodes a β-globin protein. The transgene may contain a nucleic acid having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the transgene contains a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the transgene contains a nucleic acid having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 (e.g., 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the transgene contains a nucleic acid having the nucleic acid sequence of SEQ ID NO: 1.

[0184] In some embodiments, the β-globin protein has an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the β-globin protein has an amino acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the β-globin protein has an amino acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the β-globin protein has the amino acid sequence of SEQ ID NO: 2.

[0185] In some embodiments, the β-globin protein has an amino acid sequence different from the amino acid sequence of SEQ ID NO: 2 due to one or more amino acid substitutions, insertions, and / or deletions. For example, the β-globin protein may have an amino acid sequence different from the amino acid sequence of SEQ ID NO: 2 due to one or more conserved or non-conserved amino acid substitutions. The β-globin protein may have an amino acid sequence different from the amino acid sequence of SEQ ID NO: 2 due to, for example, 1 to 50 conserved amino acid substitutions, 1 to 40 conserved amino acid substitutions, 1 to 30 conserved amino acid substitutions, 1 to 20 conserved amino acid substitutions, or 1 to 10 conserved amino acid substitutions, optionally combined with one or more non-conserved amino acid substitutions.

[0186] In another aspect, the Disclosure features a composition comprising a mixture formed by modifying eukaryotic cells in accordance with any of the methods described in the above aspects or embodiments of the Disclosure.

[0187] In a further embodiment, the present disclosure features a cell culture medium containing the composition of the above embodiment.

[0188] In yet another aspect, the Disclosure features a population of eukaryotic cells modified according to any of the methods described in the above aspects or embodiments of the Disclosure.

[0189] In another embodiment, the present disclosure features a pharmaceutical composition containing a population of cells as described above. The pharmaceutical composition may further contain one or more excipients, diluents, and / or carriers. In some embodiments, the pharmaceutical composition is formulated for administration by intravenous injection to a subject, for example, a mammalian subject (e.g., human).

[0190] In another aspect, the Disclosure features a kit containing a composition comprising a mixture formed by modifying eukaryotic cells in accordance with any of the methods described in the above aspects or embodiments of the Disclosure. Furthermore, or alternatively, the kit may contain a cell culture medium containing the composition. The kit may further contain accompanying documentation, including instructions for transducing target cells using the contents of the kit.

[0191] In another aspect, the Disclosure features a kit containing a population of eukaryotic cells modified according to any of the methods described in the above aspects or embodiments of the Disclosure. Furthermore, or alternatively, the kit may contain a pharmaceutical composition containing a population of eukaryotic cells modified according to any of the methods described in the above aspects or embodiments of the Disclosure. The kit may further contain a package insert instructing the user to administer the population of cells according to any of the cell administration methods described above or herein.

[0192] definition As used herein, the terms “excision,” “removal,” and “excision” mean the depletion of one or more cells within a cell population, either in vivo or ex vivo. In some embodiments of this disclosure, it may be desirable to excise endogenous cells within a patient (e.g., a patient being treated for a disease described herein) before administering a therapeutic composition, such as a therapeutic cell population, to a target. This may be beneficial, for example, to provide an environment in which newly administered cells can be transplanted. Excision of an endogenous cell population can be carried out, for example, by using an antibody-drug conjugate that binds to an antigen expressed on target cells, followed by the killing of the target cells, thereby selectively targeting a particular cell type. Furthermore, or alternatively, excision can be carried out in a nonspecific manner, using a cytotoxin that is not localized to a particular cell type, but instead can exert cytotoxic effects on a variety of different cells. Examples of excision include the depletion of at least 5% of the cells in a cell population (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more) in vivo or in vitro. Quantifying the number of cells in a cell sample can be done using various cell counting techniques, such as counting chambers, Coulter counters, flow cytometry, or other cell counting methods known in the art.

[0193] As used herein, the term “about” means a quantity that changes by up to about 30% (for example, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) relative to a reference quantity.

[0194] As used herein in the context of proteins of interest, the term “activity” means the biological functionality associated with the protein’s wild-type form. For example, in the context of enzymes, the term “activity” means the protein’s ability to perform substrate turnover in a manner that yields the product of the corresponding chemical reaction. The activity level of an enzyme can be detected and quantified, for example, using substrate turnover assays known in the art. As another example, in the context of membrane-bound receptors, the term “activity” may mean, for example, the signaling initiated by the receptor upon binding to its homologous ligand. The activity level of receptors involved in signal transduction pathways can be detected and quantified, for example, by observing an increase in the results of receptor signaling, such as an increase in the transcription of one or more genes (which can be detected, for example, using polymerase chain reaction techniques known in the art).

[0195] As used herein, terms such as “administer” and “dosage” mean directly giving a therapeutic agent (e.g., a population of cells, such as a population of pluripotent cells (e.g., embryonic stem cells, induced pluripotent stem cells, or CD34+ cells)) to a patient via any effective route. Exemplary routes of administration are described herein, including, in particular, systemic routes of administration such as intravenous injection.

[0196] As used herein, the term “allogeneic” means cells, tissues, nucleic acid molecules, or other substances obtained from or derived from different subjects of the same species. For example, in the context of a population of cells expressing one or more proteins as described herein (e.g., a population of pluripotent cells), allogeneic cells include cells that are (i) obtained from a non-therapeutic subject and then (ii) transduced or transmitted with a vector that directs the expression of one or more desired proteins. The phrase “directs expression” means including one or more polynucleotides that encode one or more proteins to be expressed. The polynucleotides may contain further sequence motifs that enhance the expression of the proteins of interest.

[0197] As used herein, the term “annealing” means the formation of a stable duplex of nucleic acids by hybridization mediated by interchain hydrogen bonding, for example, according to Watson-Crick base pairing. The duplex nucleic acids may be, for example, at least 50% complementary to each other (e.g., about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary to each other). A “stable duplex” formed during the annealing of one nucleic acid to another is a duplex structure that is not denatured by harsh washing. Exemplary harsh washing conditions, known in the art, include temperatures approximately 5°C lower than the melting temperature of the individual strands of the duplex, and low concentrations of monovalent salts (e.g., NaCl concentration) of less than 0.2 M (0.2M, 0.19M, 0.18M, ​​0.17M, 0.16M, 0.15M, 0.14M, 0.13M, 0.12M, 0.11M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, or less).

[0198] As used herein, the term “autologous” means cells, tissues, nucleic acid molecules, or other substances obtained from or derived from the cells, tissues, nucleic acid molecules, etc., of an individual. For example, in the context of a population of cells expressing one or more proteins as described herein (e.g., a population of pluripotent cells), autologous cells include cells obtained from a patient receiving treatment who has been transduced or transmitted with a vector that directs the expression of one or more target proteins.

[0199] As used herein, the term “cell type” refers to a group of cells that share a statistically separable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen-presenting properties. Cells of a common cell type may include cells isolated from common tissues in vivo (e.g., epithelial tissue, nerve tissue, connective tissue, or muscle tissue) and / or common organs, tissue systems, blood vessels, or other structures and / or regions.

[0200] As used herein, the terms “modify” and “conditioning” mean the process by which a subject is prepared to receive a transplant tissue containing a population of cells (e.g., a population of pluripotent cells such as CD34+ cells). Such an operation facilitates the transplantation of the cell transplant tissue by selectively depleting endogenous cells (e.g., in particular, endogenous CD34+ cells) to create a gap, which is then filled with exogenous cell transplantation. According to the methods described herein, a subject may be conditioned for cell transplant tissue operations by administering to the subject one or more agents, radiotherapy, or a combination thereof that can excrete endogenous cells (e.g., in particular, CD34+ cells). Conditioning regimens useful in combination with the compositions and methods of this disclosure may be myeloablative or non-myeloablative. Other cell excision agents and methods known in the art (e.g., antibody-drug conjugates) may also be used.

[0201] As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, static charge, and stereovolume. These properties are summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0202] [Table 1]

[0203] According to this table, the families of conserved amino acids include (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Therefore, a conserved mutation or substitution is a mutation or substitution that replaces an amino acid with a member of the same amino acid family (for example, substituting Thr with Ser, or Arg with Lys).

[0204] As used herein, the terms “embryonic stem cells” and “ES cells” mean embryonic totipotent or pluripotent stem cells derived from the inner cell mass of a blastocyst, which can be maintained in an in vitro culture medium under suitable conditions. ES cells can differentiate into cells of any of the three vertebrate germ layers: endoderm, ectoderm, or mesoderm. ES cells are also characterized by their ability to be cultured indefinitely under suitable in vitro culture conditions. ES cells are described, for example, in Thomson et al., Science 282:1145 (1998), and their disclosure relating to the structure and functionality of embryonic stem cells is incorporated herein by reference.

[0205] As used herein, the term “endogenous” means a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell).

[0206] As used herein, the term “blooming agent” means a substance capable of promoting the ex vivo proliferation of a given cell type. Therefore, “pluripotent hematopoietic stem cell blooming agent” or “HSC blooming agent” means a substance capable of promoting the ex vivo proliferation of a population of pluripotent hematopoietic stem cells. Examples of pluripotent hematopoietic stem cell blooming agents include those that promote the proliferation of a population of pluripotent hematopoietic stem cells in such a way that the cells retain their functional capacity as pluripotent hematopoietic stem cells. Exemplary pluripotent hematopoietic stem cell blooming agents that can be used in combination with the compositions and methods of this disclosure include, but are not limited to, aryl hydrocarbon receptor antagonists, such as those described in U.S. Patent Nos. 8,927,281 and 9,580,426, in which the entirety of each disclosure and, in particular, compound SR1 is incorporated herein by reference. Further pluripotent hematopoietic stem cell blooming agents that can be used in combination with the compositions and methods of this disclosure include compound UM-171, described in U.S. Patent No. 9,409,906, whose disclosure is incorporated in its entirety by reference herein, and other compounds. Further examples of pluripotent hematopoietic stem cell swelling agents include structural and / or stereoisomer variants of compound UM-171, such as the compounds described in US2017 / 0037047, the disclosure of which is incorporated herein by reference in its entirety. Further pluripotent hematopoietic stem cell swelling agents suitable for use in this disclosure include, for example, histone deacetylase (HDAC) inhibitors such as trichostatin A, trapoxin, trapoxin A, chlamydosine, sodium butyrate, dimethyl sulfoxide, suberanilohydroxamic acid, bishydroxamide m-carboxycinnamate, HC-toxin, Cyl-2, WF-3161, depdesine, and radicicol, the disclosure of which is incorporated herein by reference in WO2000 / 023567.

[0207] As used herein, the term “expression” refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. In the context of genes encoding protein products, terms such as “gene expression” are used interchangeably with terms such as “protein expression.” The expression of a target gene or protein in a subject can be revealed, for example, by detecting an increase in the activity of the corresponding protein in a sample obtained from the subject (e.g., evaluated using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the amount or concentration of the corresponding protein (e.g., evaluated using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), and / or (e.g., in the case of an enzyme, evaluated using an enzyme activity assay described herein or known in the art). As used herein, if one or more of the above events are detectable intracellularly or in the culture medium in which the cell is present, the cell is considered to "express" the target gene or protein.For example, the gene or protein in question is considered to be "expressed" by a cell or a population of cells if (i) the cell or population of cells produce a corresponding RNA transcript, such as an mRNA template (e.g., using the RNA detection procedure described herein), (ii) the RNA transcript is processed (e.g., splicing, editing, 5' cap formation, and / or 3' end processing, using the RNA detection procedure described herein), (iii) the RNA template is translated into a protein product (e.g., using the protein detection procedure described herein), and / or (iv) post-translational modifications of the protein product (e.g., using the protein detection procedure described herein) are detectable.

[0208] As used herein, the term “functional capacity” means, in the case of pluripotent cells such as pluripotent hematopoietic stem cells, the functional properties of a stem cell, including: 1) pluripotency (meaning the ability to differentiate into multiple different blood lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombus cells (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteolytic cells, and lymphocytes (e.g., NK cells, B cells, and T cells); 2) self-renewal (meaning the ability of a stem cell to produce daughter cells that have the same capacity as the parent cell and, furthermore, the ability to be repeatedly generated without exhaustion throughout the lifespan of the individual); and 3) the ability of the stem cell or its progeny to home to a stem cell niche and be reintroduced into a transplant recipient when proliferative and sustained cell proliferation and differentiation are re-established.

[0209] As used herein, the terms “pluripotent hematopoietic stem cell” and “HSC” mean immature blood cells that have the ability to self-replicate and differentiate into diverse lineages of mature blood cells, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombus cells (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteolytic cells, and lymphocytes (e.g., NK cells, B cells, and T cells). It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are thought to include a subpopulation of cells possessing the stem cell properties described above, while in mice, HSCs are CD34-. In addition, HSC refers to long-term regrowth HSCs (LT-HSCs) and short-term regrowth HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on their functional capacity and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CO2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL-7ra), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL-7ra). In addition, under homeostatic conditions, ST-HSCs are less resting (i.e., more active) and more proliferative than LT-HSCs.However, LT-HSCs have greater self-renewal capacity (i.e., they survive throughout adulthood and can be transplanted sequentially through successive recipients), while ST-HSCs have limited self-renewal (i.e., they survive for only a limited period and do not have the ability to be transplanted sequentially). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative and therefore can differentiate more quickly into progeny.

[0210] As used herein, the term "HLA-matched" means a donor-recipient pair in which there is no mismatch in any of the HLA antigens, such as a donor providing a hematopoietic stem cell graft to a recipient requiring pluripotent hematopoietic stem cell transplantation therapy. Endogenous T cells and NK cells are less likely to recognize the incoming graft as a foreign body, and therefore less likely to mount an immune response against the transplanted tissue. Thus, HLA-matched (i.e., all six alleles match) donor-recipient pairs have a reduced risk of graft rejection.

[0211] As used herein, the term “HLA mismatched” means a donor-recipient pair in which at least one HLA antigen is mismatched between the donor and recipient, particularly with respect to HLA-A, HLA-B, HLA-C, and HLA-DR, such as a donor providing a hematopoietic stem cell graft to a recipient requiring pluripotent hematopoietic stem cell transplantation therapy. In some embodiments, some haplotypes match while others are mismatched. Endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign in the case of an HLA mismatched donor-recipient pair, and such T cells and NK cells are therefore more likely to mount an immune response against the transplanted tissue. Therefore, an HLA mismatched donor-recipient pair may have an increased risk of graft rejection compared to an HLA-matched donor-recipient pair.

[0212] As used herein, the terms “induced pluripotent stem cells,” “iPS cells,” and “iPSCs” mean pluripotent stem cells that can be directly induced from differentiated somatic cells. Human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells that may include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and / or related transcription factors such as NANOG, LIN28, and / or Glis1. Human iPS cells can also be generated by using, for example, miRNAs, small molecules that mimic the action of transcription factors, or lineage-specific factors. Human iPS cells are characterized by their ability to differentiate into cells of any of the three vertebrate germ layers, such as the endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to be cultured indefinitely under suitable in vitro culture conditions. Human iPS cells are described, for example, in Takahashi and Yamanaka, Cell 126:663 (2006), and their disclosure relating to the structure and functionality of iPS cells is incorporated herein by reference.

[0213] As used herein, the terms “interfering ribonucleic acid” and “interfering RNA” mean RNA such as short interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA) that suppresses the expression of a target RNA transcript by (i) annealing to a target RNA transcript and forming a nucleic acid duplex, (ii) promoting the nuclease-mediated degradation of the RNA transcript, and / or (iii) slowing, inhibiting, or preventing the translation of the RNA transcript, for example by sterically eliminating the formation of a functional ribosome-RNA transcript complex or by otherwise weakening the formation of a functional protein product from the target RNA transcript. Interfering RNA as described herein may be provided to a patient, for example, in the form of a single-stranded or double-stranded oligonucleotide, or in the form of a vector (e.g., a viral vector) containing a transgene encoding the interfering RNA. Exemplary interfering RNA platforms are disclosed, for example, in Lam et al., Molecular Therapy - Nucleic Acids 4:e252 (2015); Rao et al., Advanced Drug Delivery Reviews 61:746-769 (2009); and Borel et al., Molecular Therapy 22:692-701 (2014), each of which is incorporated herein by reference in its entirety.

[0214] As used herein in the context of viral transduction protocols, the terms “multiplicity of infection” or “MOI” mean the ratio of (i) the number of virions added to a population of cells targeted for transduction to (ii) the number of cells in the population. For example, 1 × 10⁶ cells targeted for transduction. 6 A group of individual cells, 1 × 10 7 A transduction protocol involving contact with a virion (for example, a lentiviral virion such as the lentiviral virion described herein) is characterized by a multiplicity of 10 infections.

[0215] As used herein in the context of hematopoietic stem and / or progenitor cells, the term “mobilization” means the release of cells from a stem cell niche (e.g., bone marrow) into the peripheral circulation. A “mobilizer” is an agent capable of inducing the release of hematopoietic stem and / or progenitor cells from a stem cell niche into the peripheral circulation.

[0216] As used herein, the terms “myelolytic” or “myelolysis” typically mean a conditioning arrangement that substantially cripples or destroys the hematopoietic system due to exposure to cytotoxic agents or radiation. Myelolysis encompasses complete myelolysis resulting from high doses of cytotoxic agents or total body radiation that destroy the hematopoietic system.

[0217] As used herein, the terms “non-myeloablative” or “myelosuppressive” mean a conditioning arrangement that does not remove substantially all hematopoietic cells of host origin.

[0218] As used herein, the term "poloxamer" refers to a nonionic ternary block copolymer consisting of a central hydrophobic chain of polyoxypropylene with two hydrophilic chains of polyoxyethylene adjacent to each other. Poloxamers are also known by the trade names "Pluronics" or "Symperonics" (BASF). The block copolymer can be represented by the following formula: HO(C2H4O) x (C3H6O) y (C2H4O) zH. The length of the polymer block can be customized. As a result, many different poloxamers exist. Suitable poloxamers for use in combination with the compositions and methods of this disclosure include those having an average molecular weight of at least about 10,000 g / mol, at least about 11,400 g / mol, at least about 12,600 g / mol, at least about 13,000 g / mol, at least about 14,600 g / mol, or at least about 15,000 g / mol. Since the synthesis of block copolymers is related to the natural degree of change from one batch to another, the values ​​cited above (and those used herein to characterize a given poloxamer) may not be exactly attainable during synthesis, and the average values ​​will vary to a certain extent. Thus, as used herein, the term “poloxamer” can be used interchangeably with the term “poloxamer” (referring to several poloxamer components, also called a mixture of poloxamers) unless otherwise explicitly stated. As used herein, the term "average" relating to the monomer units or molecular weight of poloxamers(s) means that it is not technically possible to produce poloxamers of the same composition, and therefore all having the same molecular weight. Poloxamers produced according to this technique are presented as a mixture of poloxamers, each exhibiting variability in molecular weight, but collectively averaging the molecular weight as specified herein. BASF and Sigma Aldrich are preferred suppliers of poloxamers for use in combination with the compositions and methods of this disclosure.

[0219] As used herein, the term “pluripotent cell” means a cell that has the ability to develop into two or more differentiated cell types, such as cell types of the hematopoietic lineage (e.g., granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombus cells (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteolytic cells, and lymphocytes (e.g., NK cells, B cells, and T cells)). Examples of pluripotent cells are ESCs, iPSCs, and CD34+ cells.

[0220] As used herein, the term “promoter” refers to a recognition site on DNA to which RNA polymerase binds. Polymerase promotes the transcription of a transgene. Exemplary promoters suitable for use in combination with the compositions and methods described herein are, for example, described in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which relates to nucleic acid regulatory elements and is incorporated herein by reference. In addition, the term “promoter” may mean a synthetic promoter, which is a regulatory DNA sequence that does not occur naturally in a biological system. Synthetic promoters contain a portion of a naturally occurring promoter combined with a polynucleotide sequence that does not occur naturally, and can be optimized using various transgenes, vectors, and target cell types to express recombinant DNA.

[0221] The “sequence complementarity percentage (%)” with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acid in the reference polynucleotide sequence, if necessary, by aligning the sequence and introduction gap to achieve the maximum sequence complementarity percentage. A given nucleotide is considered “complementary” to the reference nucleotide described herein if two nucleotides form a standard Watson-Crick base pair. To avoid misunderstanding, in the context of this disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, a suitable Watson-Crick base pair is referred to as “matching,” while each nucleotide that does not form a pair, and nucleotides that do not form a pair properly, are referred to as “mismatching.” Alignment for the purpose of measuring nucleic acid sequence complementarity percentage can be achieved in various ways within the capabilities of those skilled in the art using commonly available computer software, such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum complementarity over the entire length of the sequences being compared. As an example, the proportion of sequence complementarity of a given nucleic acid sequence A to a given nucleic acid sequence B (which can also be called a given nucleic acid sequence A having a specific proportion of complementarity to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) [In the formula, X is the number of complementary base pairs in the alignment of A and B (performed by computer software such as BLAST), and Y is the total number of nucleic acids in B.]. It will be understood that if the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the sequence complementarity ratio of A to B will not be equal to the sequence complementarity ratio of B to A. As used herein, a query nucleic acid sequence is considered "perfectly complementary" to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0222] The “sequence identity percentage (%)” of a candidate sequence to a reference polynucleotide or reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or reference polypeptide sequence, after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum possible sequence identity percentage. Alignment for the purpose of measuring nucleic acid or amino acid sequence identity percentage can be achieved in various ways within the capabilities of a person skilled in the art using commonly available computer software, such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to obtain the maximum alignment over the full length of the sequences being compared. For example, a sequence identity percentage value can be generated using BLAST, a sequence comparison computer program. As an example, the sequence identity percentage of a given nucleic acid or amino acid sequence A to or with a given nucleic acid or amino acid sequence B (which can also be referred to as a given nucleic acid or amino acid sequence A having a specific sequence identity percentage to or with a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) [In the formula, X is the number of nucleotides or amino acids scored as identical by a sequence alignment program (e.g., BLAST) in the program alignment of A and B, and Y is the total number of nucleic acids in B.] If the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the sequence identity ratio of A to B is considered to be not equal to the sequence identity ratio of B to A.

[0223] As used herein, the term “pharmaceutically acceptable” means a compound, substance, composition, and / or dosage form that is free from excessive toxicity, irritation, allergic reactions, and other problematic ailments, has a reasonable benefit-to-risk ratio, and is suitable for contact with the tissues of a subject, such as those of a mammal (e.g., human).

[0224] As used herein, the term “regulatory sequence” includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of an antibody chain gene. Such regulatory sequences are described, for example, in Perdew et al., Regulation of Gene Expression (Humana Press, New York, NY, (2014)), which is incorporated herein by reference.

[0225] As used herein, the terms “stem cell” and “undifferentiated cell” mean a cell that is undifferentiated or partially differentiated, possessing the developmental capacity to differentiate into multiple cell types. Stem cells can proliferate and produce more such stem cells while maintaining their functional capacity. Stem cells can divide asymmetrically. This is known as inevitable asymmetric differentiation, in which one daughter cell retains the functional capacity of the parent stem cell, while another daughter cell expresses several other specific functions, phenotypes, and / or developmental capabilities that differ from the parent cell. A daughter cell can be induced to produce offspring that proliferate and differentiate into one or more mature cell types, while also retaining one or more cells that have the developmental potential of the parent. Differentiated cells can originate from pluripotent cells, and pluripotent cells themselves may originate from pluripotent cells, etc. Alternatively, some stem cells in a population can divide symmetrically into two stem cells. Thus, the term “stem cell” means any subset of cells that, under certain circumstances, possess the developmental capacity to differentiate into more specialized or differentiated phenotypes, and under certain circumstances, maintain the ability to proliferate substantially without differentiation. In some embodiments, the term “stem cell” generally refers to a naturally occurring parent cell whose offspring (progeny) specialize, often in different directions, by acquiring completely distinct characteristics through differentiation, such as in the progressive diversification of embryonic cells and tissues. Some differentiated cells also have the ability to produce cells with greater developmental potential. Such ability may be innate or may be artificially induced by treatment with various factors. Cells that begin as stem cells can progress to a differentiated phenotype, but can then be “reversed” and induced to re-express the stem cell phenotype. This term is often also referred to by those skilled in the art as “dedifferentiation,” “reprogramming,” or “retrodifferentiation.”

[0226] As used herein, the term “transgene” means a recombinant nucleic acid (e.g., DNA or cDNA) that codes for a gene product (e.g., a gene product as described herein). The gene product may be RNA, a peptide, or a protein. In addition to the coding region for the gene product, the transgene may include, or be operably conjugated to, one or more elements that facilitate or enhance expression, such as promoters, enhancers, destabilization domains, response elements, reporter elements, insulator elements, polyadenylation signals, and / or other functional elements. Embodiments of this disclosure may utilize any known, suitable promoters, enhancers, destabilization domains, response elements, reporter elements, insulator elements, polyadenylation signals, and / or other functional elements.

[0227] As used herein, the terms “subject” and “patient” are used interchangeably and mean a living organism (e.g., mammal, e.g., human) that has been diagnosed with and / or is receiving treatment for a disease characterized by a gene or protein deficiency as described herein.

[0228] As used herein, the terms “transduction” and “transduction” mean a method of introducing a viral vector construct or a portion thereof into a cell, and subsequently expressing the transgene encoded by the vector construct or a portion thereof within the cell.

[0229] As used herein, the term “transduction efficiency” means the proportion of cells in a given population that have been transduced with at least one copy of the vector (for example, a viral vector such as the lentiviral vector described herein). For example, 1 × 10⁻⁶ 6 Individual cells are exposed to a virus (e.g., lentivirus), and after transduction, 0.5 × 10⁻⁶ cells are produced. 6The transduction efficiency of the procedure is 50% when an individual cell is measured to have at least one copy within its genome. Exemplary methods for measuring transduction efficiency include polymerase chain reaction (PCR) and flow cytometry.

[0230] As used herein, “treatment” and “to treat” mean an approach to obtain a beneficial or desired outcome, such as a clinical outcome. Beneficial or desired outcomes may include, but are not limited to, reduction or remission of one or more signs or symptoms, whether detectable or undetectable; a reduction in the severity of the disease or symptoms; stabilization of the state of the disease, disability, or symptoms (i.e., no worsening); prevention of the progression of the disease or symptoms; delay or slowing of the progression of the disease or symptoms; improvement or relief of the disease or symptoms; and (partial or complete) remission. To “improve” or “relieve” a disease or symptom means that the severity of the disease, disability, or symptom and / or undesirable clinical signs are reduced and / or the time course of progression is slowed or prolonged compared to the degree or time course without treatment. “Treatment” may also mean extending survival compared to the predicted survival time without treatment. Those who require treatment include those who already have symptoms or the disease, those who are prone to developing symptoms or the disease, and those who need to prevent symptoms or the disease.

[0231] As used herein, the term “vector” includes nucleic acid vectors (e.g., DNA vectors such as plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in WO1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for the expression of the gene of interest. Expression vectors suitable for use in the compositions and methods described herein include polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of a mammalian cell. Vectors usable for the expression of one or more proteins described herein include plasmids containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. In addition, vectors useful for the expression of one or more proteins described herein may contain polynucleotide sequences that improve the translation rate of the corresponding one or more genes, or improve the stability or nuclear export of mRNA resulting from gene transcription. Examples of such sequence factors include 5' and 3' untranslated regions, IRESs, and polyadenylation signaling sites for directing the efficient transcription of one or more genes supported by the expression vector. Expression vectors suitable for use with the composition methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, noseoslysin, or zeosin.

[0232] As used herein, the term “vector copy number” or “VCN” means the copy number of a vector, or a portion thereof (e.g., the portion encoding the target transgene), within the genome of a cell. The mean VCN can be measured for a population of cells or for individual cell colonies. Exemplary methods for measuring VCN include PCR and flow cytometry.

[0233] As used herein, the term “β-globin,” along with the names of other genes or proteins cited herein, includes the wild-type form of the corresponding gene or protein, as well as its variants (e.g., splice variants, truncated, concatemers, and fusion constructs). In the context of β-globin, an example of such a variant is a protein that, if the β-globin variant retains the functionality of wild-type β-globin, has at least 70% sequence identity with any of the amino acid sequences of the wild-type β-globin protein (e.g., SEQ ID NO: 2) (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more).

[0234] In the present invention, the term "alkyl" refers to a monovalent, arbitrarily branched alkyl group having 1 to 6 or more carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and n-hexyl.

[0235] As used herein, the term "lower alkyl" means an alkyl group having 1 to 6 carbon atoms.

[0236] As used herein, the term "aryl" means an unsaturated aromatic carbocyclic group of 6 to 14 carbon atoms having one ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl). Preferred aryls include phenyl, naphthyl, and phenantrenyl.

[0237] As used herein, the terms "aralkyl" and "arylalkyl" are used interchangeably and refer to alkyl groups containing an aryl moiety. Similarly, terms such as "aryl lower alkyl" refer to lower alkyl groups containing an aryl moiety.

[0238] As used herein, the term "alkylaryl" means an alkyl group having an aryl substituent, including benzyl and phenethyl.

[0239] As used herein, the term "heteroaryl" means a monocyclic heterocyclic aromatic group, or a bicyclic or tricyclic fused heterocyclic aromatic group. Specific examples of heterocyclic aromatic groups include optionally substituted pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydrodibenzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzodienyl Examples include indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolidinil, quinazolinil, phthalazinil, quinoxalinil, cinnolinil, naphthilidinil, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinil, carbazolyl, xanthenyl, and benzoquinolyl.

[0240] As used herein, the term "alkyl heteroaryl" means an alkyl group having a heteroaryl substituent, including 2-furylmethyl, 2-thienylmethyl, and 2-(1H-indole-3-yl)ethyl.

[0241] As used herein, the term “lower alkenyl” means an alkenyl group having preferably 2 to 6 carbon atoms and at least 1 or 2 alkenyl unsaturated moieties. Exemplary alkenyl groups include ethenyl (-CH=CH2) and n-2-propenyl (allyl, -CH2CH=CH2).

[0242] As used herein, the term "alkenylaryl" means an alkenyl group having an aryl substituent, including 2-phenylvinyl.

[0243] As used herein, the term "alkenyl heteroaryl" means an alkenyl group having a heteroaryl substituent, such as 2-(3-pyridinyl)vinyl.

[0244] As used herein, the term "lower alkynyl" means an alkynyl group having preferably 2 to 6 carbon atoms and at least 1 to 2 alkynyl unsaturated moieties. Preferred alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0245] As used herein, the term "alkynylaryl" means an alkynyl group having an aryl substituent, including phenylethynyl.

[0246] As used herein, the term "alkynyl heteroaryl" means an alkynyl group having a heteroaryl substituent, including 2-thienylethynyl.

[0247] As used herein, the term "cycloalkyl" refers to monocyclic cycloalkyl groups having 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0248] As used herein, the term “lower cycloalkyl” means a saturated carbocyclic group of 3 to 8 carbon atoms having a monocycle (e.g., cyclohexyl) or multiple fused rings (e.g., norvonyl). Preferred cycloalkyls include cyclopentyl, cyclohexyl, and norvonyl.

[0249] As used herein, the term "heterocycloalkyl" means a cycloalkyl group in which one or more ring carbon atoms are replaced by heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl groups include pyrrolidinyl, piperidinyl, oxopiperidinyl, morpholinyl, piperazinyl, oxopiperazinyl, thiomorpholinyl, azepanyl, diazepanyl, oxazepanyl, thiazepanyl, dioxothiazepinyl, azokanil, tetrahydrofuranil, and tetrahydropyranil.

[0250] As used herein, the term "alkylcycloalkyl" means an alkyl group having a cycloalkyl substituent, including cyclohexylmethyl and cyclopentylpropyl.

[0251] As used herein, the term "alkyl heterocycloalkyl" means a C1-C6 alkyl group having a heterocycloalkyl substituent, including 2-(1-pyrrolidinyl)ethyl, 4-morpholinylmethyl, and (1-methyl-4-piperidinyl)methyl.

[0252] As used herein, the term "carboxyl" means the group -C(O)OH.

[0253] As used herein, the term "alkylcarboxy" means a C1-C5 alkyl group having a carboxyl substituent, including 2-carboxyethyl.

[0254] As used herein, the term "acyl" means the group -C(O)R [wherein R may be, for example, a C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents].

[0255] As used herein, the term "acyloxy" means the group -OC(O)R [wherein R may be, for example, a C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents].

[0256] As used herein, the term "alkoxy" means the group -OR [wherein R is an optionally substituted alkyl group, such as an optionally substituted C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents]. Examples of alkoxy groups include methoxy, ethoxy, and phenoxy.

[0257] As used herein, the term "alkoxycarbonyl" means the group -C(O)OR [wherein R is, for example, hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other possible substituents].

[0258] As used herein, the term "alkylalkoxycarbonyl" means an alkyl group having an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl.

[0259] As used herein, the term "aminocarbonyl" means the group -C(O)NRR' [wherein R and R' can be independently hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents].

[0260] As used herein, the term "alkylaminocarbonyl" means an alkyl group having an aminocarbonyl substituent, including 2-(dimethylaminocarbonyl)ethyl.

[0261] As used herein, the term "acylamino" means the group -NRC(O)R' [wherein R and R' can be independently hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents].

[0262] As used herein, the term "alkylacylamino" means an alkyl group having an acylamino substituent, including 2-(propionylamino)ethyl.

[0263] As used herein, the term “ureid” means the group -NRC(O)NR'R” [wherein R, R', and R'' may independently be hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, or heterocycloalkyl, among other substituents]. Exemplary ureid groups include moieties in which R' and R'', together with the nitrogen atom to which they are bonded, form a 3- to 8-membered heterocycloalkyl ring.

[0264] As used herein, the term "alkylureido" means an alkyl group having a ureido substituent, including 2-(N'-methylureido)ethyl.

[0265] As used herein, the term “amino” means the group -NRR' [wherein R and R' may independently be hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, or heterocycloalkyl, among other substituents]. Exemplary amino groups include moieties in which R and R', together with the nitrogen atom to which they are bonded, can form a 3- to 8-membered heterocycloalkyl ring.

[0266] As used herein, the term "alkylamino" means an alkyl group having an amino substituent, including 2-(1-pyrrolidinyl)ethyl.

[0267] As used herein, the term "ammonium" means a positively charged base -N + RR'R'' [wherein R, R', and R'' can independently be, for example, a C1-C6 alkyl, a C1-C6 alkylaryl, a C1-C6 alkylheteroaryl, a cycloalkyl, or a heterocycloalkyl, among other substituents]. Exemplary ammonium groups include moieties in which R' and R'', together with the nitrogen atom to which they are bonded, form a 3- to 8-membered heterocycloalkyl ring.

[0268] As used herein, the term "halogen" means fluorine, chlorine, bromine, and iodine atoms.

[0269] As used herein, the term "sulfonyloxy" means the group -OSO2-R [wherein R is hydrogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl (e.g., -OSO2-CF3 group), aryl, heteroaryl, C1-C6 alkylaryl, and C1-C6 alkylheteroaryl].

[0270] As used herein, the term "alkylsulfonyloxy" means an alkyl group having a sulfonyloxy substituent, including 2-(methylsulfonyl)ethyl.

[0271] As used herein, the term "sulfonyl" means the group "-SO2-R" [wherein R is hydrogen, aryl, heteroaryl, C1-C6 alkyl, halogen-substituted C1-C6 alkyl (e.g., -SO2-CF3 group), C1-C6 alkylaryl, or C1-C6 alkylheteroaryl].

[0272] As used herein, the term "alkylsulfonyl" means an alkyl group having a sulfonyl substituent, including 2-(methylsulfonyl)ethyl.

[0273] As used herein, the term "sulfinyl" means the group "-S(O)-R" [wherein R is hydrogen, a C1-C6 alkyl group, a halogen-substituted C1-C6 alkyl group (e.g., a -SO-CF3 group), an aryl group, a heteroaryl group, a C1-C6 alkylaryl group, or a C1-C6 alkylheteroaryl group].

[0274] As used herein, the term "alkylsulfinyl" means a C1-C5 alkyl group having a sulfinyl substituent, including 2-(methylsulfinyl)ethyl.

[0275] As used herein, the term "sulfanyl" means the group -SR [wherein R is, for example, alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents]. Exemplary sulfanyl groups include methylsulfanyl and ethylsulfanyl.

[0276] As used herein, the term "alkylsulfanyl" means an alkyl group having a sulfanyl substituent, including 2-(methylsulfanyl)ethyl.

[0277] As used herein, the term "sulfonylamino" means the group -NRSO2-R' [wherein R and R' may independently be hydrogen, a C1-C6 alkylaryl, a heteroaryl, a C1-C6 alkylaryl, or a C1-C6 alkylheteroaryl, among other substituents].

[0278] As used herein, the term "alkylsulfonylamino" means an alkyl group having a sulfonylamino substituent, including 2-(ethylsulfonylamino)ethyl.

[0279] The groups described above, such as "alkyl," "alkenyl," "alkynyl," "aryl," and "heteroaryl," can be optionally substituted with one or more substituents selected from, for example, alkyl (e.g., C1-C6 alkyl), alkenyl (e.g., C2-C6 alkenyl), alkynyl (e.g., C2-C6 alkynyl), cycloalkyl, heterocycloalkyl, alkylaryl (e.g., C1-C6 alkylaryl), alkylheteroaryl (e.g., C1-C6 alkylheteroaryl), alkylcycloalkyl (e.g., C1-C6 alkylcycloalkyl), alkylheterocycloalkyl (e.g., C1-C6 alkylheterocycloalkyl), amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, etc., provided that the valency is possible. In some embodiments, substitutions involve vicinal functional substituents, thereby resulting in ring closure of adjacent substituents, such as in situations where lactams, lactones, cyclic anhydrides, acetals, thioacetals, and aminals are formed, among other things.

[0280] As used herein, the term “optionally condensed” means a cyclic chemical group that can be condensed in a cyclic system, such as a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group. Examples of ring systems that can be condensed to any condensed chemical group include, for example, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, indazolyl, benzimimidazolyl, quinolinyl, isoquinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl, sinnolinyl, indolidinyl, naphthylidinyl, pteridinyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, indolinyl, isoindolinyl, 2,3,4,5-tetrahydrobenzo[b]oxepinyl, 6,7,8,9-tetrahydro-5H-benzocycloheptenyl, and chromanil.

[0281] As used herein, the term “pharmaceutically acceptable salt” means a salt of a compound described herein that retains the desired biological activity of the non-ionized parent compound, to which the salt is formed. Examples of such salts include, but are not limited to, acid addition salts formed by inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), as well as salts formed by organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. Compounds also include those of the formulas -NR, R', R” + Z -[In the formula, each of R, R', and R'' can independently be, for example, hydrogen, alkyl, benzyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, heterocycloalkyl, etc., and Z is a counterion such as chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, carboxylate (e.g., benzoate, succinate, acetate, glycolate, maleate, fumarate, citrate, tartarate, ascorbate, cinnamoate, mandeloate, and diphenylacetate).] It can be administered as a pharmaceutically acceptable quaternary salt, such as a quaternary ammonium salt of [the formula, where R, R', and R'' can independently be, for example, hydrogen, alkyl, benzyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, heterocycloalkyl, etc., and Z is a counterion such as chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, carboxylate (e.g., benzoate, succinate, acetate, glycolate, maleate, maleate, fumarate, citrate, tartarate, ascorbate, cinnamoate, mandeloate, and diphenylacetate)].

[0282] For example, in the context of protein kinase C (PKC) inhibitors such as staurosporines, as used herein, the term “variant” means an agent that contains one or more modifications compared to a reference agent, and (i) retains the functional properties of the reference agent (e.g., the ability to inhibit PKC activity), and / or (ii) is converted to the reference agent in cells (e.g., CD34+ cells, or the types of cells described herein). In the context of small molecule PKC inhibitors such as staurosporines, structural variants of a reference compound include variants that differ from the reference compound by the inclusion and / or arrangement of one or more substituents, variants that are isomers of the reference compound, such as structural isomers (e.g., positional isomers) or stereoisomers (e.g., enantiomers or diastereomers), and prodrugs of the reference compound. In the context of interfering RNA molecules, a variant may contain one or more nucleic acid substitutions compared to the parent interfering RNA molecule.

[0283] Structural compositions described herein include tautomers, geometric isomers (e.g., E / Z isomers and cis / trans isomers), enantiomers, diastereomers, and racemates, as well as pharmaceutically acceptable salts thereof. Such salts include, for example, acid addition salts formed with pharmaceutically acceptable acids such as hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulfonate, benzenesulfonic acid, and p-toluenesulfonate.

[0284] As used herein, a chemical structural formula of a compound having one or more stereocenters that does not show the stereochemical composition shall be interpreted as encompassing one or more stereoisomers of the compound shown, or a mixture of one or more such stereoisomers (e.g., one enantiomer or diastereomer of the compound shown, or a mixture of enantiomers (e.g., a racemic mixture) or a mixture of diastereomers). As used herein, a chemical structural formula of a compound having one or more stereocenters that does not specifically show the stereochemical composition shall be interpreted as referring to a substantially pure form of the particular stereoisomer shown. "Substantially pure" means a compound having a purity greater than 85%, e.g., 85% to 99%, 85% to 99.9%, 85% to 99.99%, or 85% to 100%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 100% when evaluated using chromatography and nuclear magnetic resonance techniques known in the art. [Brief explanation of the drawing]

[0285] [Figure 1A]The graph below, shown in Example 1, illustrates the effect of poloxamer P407 on lentiviral transduction efficiency when incubated with CD34+ pluripotent hematopoietic stem cells at various concentrations. In short, CD34+ cells were transduced with a lentiviral vector containing a transgene encoding green fluorescent protein (GFP). As shown along the x-axis, cells were either left untreated ("untreated"), incubated with lentivirus alone ("vector only"), lentivirus combined with a vehicle ("vector + vehicle"), or lentivirus combined with a specific concentration of P407. The values ​​along the y-axis represent the multiplier of the amount of transduced cells 12 days after exposure to each specified condition, compared to the amount of transduced cells 12 days after treatment with lentiviral vector alone ("vector only"). The amount of transduced cells was measured by measuring the expression of the encoded transgene, and this was evaluated using flow cytometry to detect GFP-mediated fluorescence. The values ​​along the y-axis represent the mean ± standard error (SEM) of the mean for an independent experiment with n=4. [Figure 1B] The graph below, shown in Example 1, illustrates the effect of poloxamer P407 on lentiviral vector copy numbers when incubated with CD34+ pluripotent hematopoietic stem cells at various concentrations. CD34+ cells were transduced with a lentiviral vector containing a transgene encoding GFP. As shown along the x-axis, cells were incubated either untreated ("untreated"), with lentivirus alone ("vector only"), with lentivirus combined with a vehicle ("vector + vehicle"), or with lentivirus combined with a specific concentration of P407. The values ​​along the y-axis represent the multiplier of increase in the vector copy number of cells 12 days after exposure to each of the specified conditions, compared to the vector copy number of cells 12 days after exposure to lentiviral vector alone ("vector only"). The values ​​along the y-axis represent the mean ± standard error (SEM) of the mean for n=3 independent experiments. [Figure 2]A-C are graphs showing the effects of poloxamer P407 and P338 on lentiviral transduction of human pluripotent hematopoietic stem cells, as shown in Example 2 below. CD34+ pluripotent hematopoietic stem cells derived from peripherally mobilized blood were either left untreated ("untreated") or incubated with a lentiviral vector containing a transgene encoding GFP. In these cells incubated with lentiviral vectors, the cells were transduced either with the vector alone ("vector only", with infection multiplicity of 5-20) or in the presence of various concentrations of poloxamer P407 (final concentration of 1 μg / mL to 10 mg / mL) or poloxamer P338 (final concentration of 10 μg / mL to 4 mg / mL). A is a plot summarizing the percentage of viable cells 18-24 hours after transduction, as measured by flow cytometry detection of 7AAD-annexin V- cells. Plot B summarizes the multiplicative change in the proportion of transdextrin cells induced by the addition of P407 or P338 compared to cells treated with the vector alone. The proportion of transdextrin cells was assessed by flow cytometry detection of transgene expression 12 days after transduction. Plot C summarizes the multiplicative change in the mean transgene copy number (VCN) compared to cells treated with the vector alone. VCN was measured by droplet digital PCR detection of integrated transgene sequences in genomic DNA recovered from cell culture medium 12 days after transduction. The data shown are mean ± SEM for independent experiments using healthy human donor cells, n=3-9 (Figures 2A and 2B) and n=3-6 (Figure 2C). Statistical analysis was performed using paired t-tests for the test conditions for vector and poloxamer vehicle-treated cells. Here, "ns" indicates that no statistical significance was detected (p<0.05). [Figure 3]Figures A and B are graphs showing the effects of poloxamer P407 or poloxamer P338 in combination with protamine sulfate in lentiviral transduction of human pluripotent hematopoietic stem cells, as shown in Example 3 below. CD34+ pluripotent hematopoietic stem cells derived from peripherally mobilized blood were transduced with a lentiviral vector encoding GFP in the presence of poloxamer P407 (7 μg / mL) or poloxamer P338 (1 mg / mL) in combination with protamine sulfate ("PS", 3.5 μg / mL). The plots summarize the multiplicative changes in the percentage of transduced cells, evaluated by flow cytometry (Figure 3A) and mean transgene copy number (VCN, Figure 3B), induced by the addition of P407 or P338 in combination with protamine sulfate, compared to cells treated with the vector alone. Twelve days after transduction, the proportion of transgenes was assessed by flow cytometry detection of transgene expression, and the VCN was measured by droplet digital PCR detection of the integrated transgene sequence in genomic DNA. The data represent the mean ± range of n=5-8 independent experiments using healthy human donor cells. Statistical analysis was performed using a t-test (p<0.05). [Figure 4]Figures A and B are graphs showing the effects of poloxamer P407 and protamine sulfate in combination with either cyclosporine H or lithium carbonate in lentiviral transduction of human pluripotent hematopoietic stem cells, as shown in Example 3 below. CD34+ stem cells newly isolated from peripherally mobilized blood were transduced with a clinical therapeutic lentiviral vector in the presence of poloxamer P407 (7 μg / mL), which is a combination of protamine sulfate ("PS", 3.5 μg / mL) and either cyclosporine H ("CSH", 8 μM, dark gray) or lithium carbonate (Li2CO3, shown as "LiCO" in the figure, 2 mM, light gray). The plots summarize the integrated transgene sequence in genomic DNA isolated from colony-forming units (CFUs) (Figure 4A), and the percentage of transgene colonies measured by droplet digital PCR detection, with a mean transgene copy number (VCN) per cell detected 12 days after transduction, achieved using a certain range of vector doses (moi, infection multiplicity, Figure 4B). White bars indicate transduction efficiency and VCN achieved with vector addition alone. The data represent the mean ± range of n=4 independent experiments involving the isolation of healthy human donor CD34+ cells at a clinical scale. [Modes for carrying out the invention]

[0286] The compositions and methods described herein can be used to modify eukaryotic cells, such as pluripotent cells including pluripotent hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). The compositions and methods of this disclosure can be used to genetically recombinate such cells to express a target gene and / or to proliferate ex vivo. In some embodiments of this disclosure, a population of pluripotent cells, such as a population of HSCs and / or HPCs, is contacted with a viral vector encoding a transgene. The transgene may encode a protein product or a regulatory ribonucleic acid (RNA) molecule that modulates the expression of a different gene. In some embodiments, the transgene encodes a protein that is deficient or non-functional in a patient (e.g., a mammalian patient such as a human) suffering from a genetic disease, such as a loss-of-function mutation. The cells can be contacted with the virus in a manner that promotes transduction of the cells to express the desired transgene. In some embodiments, the cells are then administered to a patient suffering from the disease to restore gene expression in the individual.

[0287] Various viral vectors can be used in combination with the compositions and methods of this disclosure. For example, the viral vector may be a retrovirus such as a lentivirus. Other viral vectors that can be used to achieve transduction of target cells are described herein.

[0288] To complement the degree of transduction and / or the rate at which target cells are transduced, cells may be brought into contact with a poloxamer such as a poloxamer having an excess molar mass of 10,000 g / mol, a molar mass of more than 2,000 g / mol of polyoxypropylene subunits, and / or an ethylene oxide content of more than 40% by mass.

[0289] The following chapters describe the use of various viral vectors and agents that can be used to complement viral transduction of target cells, as well as a range of therapeutic uses of transduction cells.

[0290] Poloxamer Poloxamers that can be used in combination with the compositions and methods disclosed herein include those with an average molar mass of polyoxypropylene subunits greater than 2,050 g / mol (e.g., approximately 2,055 g / mol, 2,060 g / mol, 2,075 g / mol, 2,080 g / mol, 2,085 g / mol, 2,090 g / mol, 2,095 g / mol, 2,100 g / mol, 2,200 g / mol, 2,300 g / mol, 2,400 g / mol, 2,500 g / mol, 2,600 g / mol, 2,700 g / mol, 2,800 g / mol, 2,900 g / mol, and 3,000 g / mol). Examples include those having an average molar mass of polyoxypropylene subunits of 3,100 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol.

[0291] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,250 g / mol (e.g., about 2,300 g / mol, 2,400 g / mol, 2,500 g / mol, 2,600 g / mol, 2,700 g / mol, 2,800 g / mol, 2,900 g / mol, 3,000 g / mol, 3,100 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol). It has an average molar mass of polyoxypropylene subunits of 1 mol, 3,600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol.

[0292] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,750 g / mol (e.g., about 2,800 g / mol, 2,900 g / mol, 3,000 g / mol, 3,100 g / mol, 3,200 g / mol, 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3, It has an average molar mass of polyoxypropylene subunits of 800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol.

[0293] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,250 g / mol (for example, an average molar mass of polyoxypropylene subunits of about 3,300 g / mol, 3,400 g / mol, 3,500 g / mol, 3,600 g / mol, 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol).

[0294] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,625 g / mol (for example, an average molar mass of polyoxypropylene subunits of about 3,700 g / mol, 3,800 g / mol, 3,900 g / mol, 4,000 g / mol, 4,100 g / mol, 4,200 g / mol, 4,300 g / mol, 4,400 g / mol, 4,500 g / mol, 4,600 g / mol, 4,700 g / mol, 4,800 g / mol, 4,900 g / mol, or 5,000 g / mol).

[0295] In some embodiments, the poloxamer has a molecular weight of from about 2,050 g / mol to about 4,000 g / mol (e.g., about 2,050 g / mol, 2,055 g / mol, 2,060 g / mol, 2,065 g / mol, 2,070 g / mol, 2,075 g / mol, 2,080 g / mol, 2,085 g / mol, 2,090 g / mol, 2,095 g / mol, 2,100 g / mol, 2,105 g / mol, 2,110 g / mol, 2,115 g / mol, 2,120 g / mol, 2,125 g / mol, 2,130 g / mol, 2,135 g / mol, 2,140 g / mol, 2,145 g / mol, 2,150 g / mol, 2,155 g / mol, 2,160 g / mol, 2,165 g / mol, 2,170 g / mol, 2,175 g / mol, 2,180 g / mol, 2,185 g / mol, 2,190 g / mol, 2,195 g / mol, 2,200 g / mol, 2,205 g / mol, 2,210 g / mol, 2,215 g / mol, 2,220 g / mol, 2,225 g / mol, 2,230 g / mol, 2,235 g / mol, 2,240 g / mol, 2,245 g / mol, 2,250 g / mol, 2,255 g / mol, 2,260 g / mol, 2,265 g / mol, 2,270 g / mol, 2,275 g / mol, 2,280 g / mol, 2,285 g / mol, 2,290 g / mol, 2,295 g / mol, 2,300 g / mol, 2,305 g / mol, 2,310 g / mol, 2,315 g / mol, 2,320 g / mol, 2,325 g / mol, 2,330 g / mol, 2,335 g / mol, 2,340 g / mol, 2,345 g / mol, 2,350 g / mol, 2,355 g / mol, 2,360 g / mol, 2,365 g / mol, 2,370 g / mol, 2,375 g / mol, 2,380 g / mol, 2,385 g / mol, 2,390 g / mol, 2,395 g / mol, 2,400 g / mol, 2,405 g / mol, 2,410 g / mol, 2,415 g / mol, 2,420 g / mol, 2,425 g / mol, 2,430 g / mol, 2,435 g / mol, 2,440 g / mol, 2,445 g / mol, 2,450 g / mol, 2,455 g / mol, 2,460 g / mol, 2,465 g / mol, 2,470 g / mol, 2,475 g / mol, 2,[480g / mol、2,485g / mol、2,490g / mol、2,495g / mol、2,500g / mol、2,505g / mol、2,510g / mol、2,515g / mol、2,520g / mol、2,525g / mol、2,530g / mol、2,535g / mol、2,540g / mol、2,545g / mol、2,550g / mol、2,555g / mol、2,560g / mol、2,565g / mol、2,570g / mol、2,575g / mol、2,580g / mol、2,585g / mol、2,590g / mol、2,595g / mol、2,600g / mol、2,605g / mol、2,610g / mol、2,615g / mol、2,620g / mol、2,625g / mol、2,630g / mol、2,635g / mol、2,640g / mol、2,645g / mol、2,650g / mol、2,655g / mol、2,660g / mol、2,665g / mol、2,670g / mol、2,675g / mol、2,680g / mol、2,685g / mol、2,690g / mol、2,695g / mol、2,700g / mol、2,705g / mol、2,710g / mol、2,715g / mol、2,720g / mol、2,725g / mol、2,730g / mol、2,735g / mol、2,740g / mol、2,745g / mol、2,750g / mol、2,755g / mol、2,760g / mol、2,765g / mol、2,770g / mol、2,775g / mol、2,780g / mol、2,785g / mol、2,790g / mol、2,795g / mol、2,800g / mol、2,805g / mol、2,810g / mol、2,815g / mol、2,820g / mol、2,825g / mol、2,830g / mol、2,835g / mol、2,840g / mol、2,845g / mol、2,850g / mol、2,855g / mol、2,860g / mol、2,865g / mol、2,870g / mol、2,875g / mol、2,880g / mol、2,885g / mol、2,890g / mol、2,895g / mol、2,900g / mol、2,905g / mol、2,910g / mol、2,915g / mol、2,920g / mol、2,925g / mol、2,930g / mol、2,935g / mol、2,940g / mol、2,945g / mol、2,950g / mol、2,955g / mol、2,960g / mol、2,965g / mol、2,970g / mol、2,975g / mol、2,980g / mol、2,985g / mol、2,990g / mol、2,995g / mol、3,000g / mol、3,005g / mol、3,010g / mol、3,015g / mol、3,020g / mol、3,025g / mol、3,030g / mol、3,035g / mol、3,040g / mol、3,045g / mol、3,050g / mol、3,055g / mol、3,060g / mol、3,065g / mol、3,070g / mol、3,075g / mol、3,080g / mol、3,085g / mol、3,090g / mol、3,095g / mol、3,100g / mol、3,105g / mol、3,110g / mol、3,115g / mol、3,120g / mol、3,125g / mol、3,130g / mol、3,135g / mol、3,140g / mol、3,145g / mol、3,150g / mol、3,155g / mol、3,160g / mol、3,165g / mol、3,170g / mol、3,175g / mol、3,180g / mol、3,185g / mol、3,190g / mol、3,195g / mol、3,200g / mol、3,205g / mol、3,210g / mol、3,215g / mol、3,220g / mol、3,225g / mol、3,230g / mol、3,235g / mol、3,240g / mol、3,245g / mol、3,250g / mol、3,255g / mol、3,260g / mol、3,265g / mol、3,270g / mol、3,275g / mol、3,280g / mol、3,285g / mol、3,290g / mol、3,295g / mol、3,300g / mol、3,305g / mol、3,310g / mol、3,315g / mol、3,320g / mol、3,325g / mol、3,330g / mol、3,335g / mol、3,340g / mol、3,345g / mol、3,350g / mol、3,355g / mol、3,360g / mol、3,365g / mol、3,370g / mol、3,375g / mol、3,380g / mol、3,385g / mol、3,390g / mol、3,395g / mol、3,400g / mol、3,405g / mol、3,410g / mol、3,415g / mol、3,420g / mol、3,425g / mol、3,430g / mol、3,435g / mol、3,440g / mol、3,445g / mol、3,450g / mol、3,455g / mol、3,460g / mol、3,465g / mol、3,470g / mol、3,475g / mol、3,480g / mol、3,485g / mol、3,490g / mol、3,495g / mol、3,500g / mol、3,505g / mol、3,510g / mol、3,515g / mol、3,520g / mol、3,525g / mol、3,530g / mol、3,535g / mol、3,540g / mol、3,545g / mol、3,550g / mol、3,555g / mol、3,560g / mol、3,565g / mol、3,570g / mol、3,575g / mol、3,580g / mol、3,585g / mol、3,590g / mol、3,595g / mol、3,600g / mol、3,605g / mol、3,610g / mol、3,615g / mol、3,620g / mol、3,625g / mol、3,630g / mol、3,635g / mol、3,640g / mol、3,645g / mol、3,650g / mol、3,655g / mol、3,660g / mol、3,665g / mol、3,670g / mol、3,675g / mol、3,680g / mol、3,685g / mol、3,690g / mol、3,695g / mol、3,700g / mol、3,705g / mol、3,710g / mol、3,715g / mol、3,720g / mol、3,725g / mol、3,730g / mol、3,735g / mol、3,740g / mol、3,745g / mol、3,750g / mol、3,755g / mol、3,760g / mol、3,765g / mol、3,770g / mol、3,775g / mol、3,780g / mol、3,785g / mol、3,790g / mol、3,795g / mol、3,800g / mol、3,805g / mol、3,810g / mol、3,815g / mol、3,820g / mol、3,825g / mol、3,830g / mol、3,835g / mol、3,840g / mol、3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol l, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / mol, 3,925g / mol, 3,930g It has an average molar mass of polyoxypropylene subunits of 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0296] In some embodiments, the poloxamer has a molecular weight of from about 2,750 g / mol to about 4,000 g / mol (e.g., about 2,750 g / mol, 2,755 g / mol, 2,760 g / mol, 2,765 g / mol, 2,770 g / mol, 2,775 g / mol, 2,780 g / mol, 2,785 g / mol, 2,790 g / mol, 2,795 g / mol, 2,800 g / mol, 2,805 g / mol, 2,810 g / mol, 2,815 g / mol, 2,820 g / mol, 2,825 g / mol, 2,830 g / mol, 2,835 g / mol, 2,840 g / mol, 2,845 g / mol, 2,850 g / mol, 2,855 g / mol, 2,860 g / mol, 2,865 g / mol, 2,870 g / mol, 2,875 g / mol, 2,880 g / mol, 2,885 g / mol, 2,890 g / mol, 2,895 g / mol, 2,900 g / mol, 2,905 g / mol, 2,910 g / mol, 2,915 g / mol, 2,920 g / mol, 2,925 g / mol, 2,930 g / mol, 2,935 g / mol, 2,940 g / mol, 2,945 g / mol, 2,950 g / mol, 2,955 g / mol, 2,960 g / mol, 2,965 g / mol, 2,970 g / mol, 2,975 g / mol, 2,980 g / mol, 2,985 g / mol, 2,990 g / mol, 2,995 g / mol, 3,000 g / mol, 3,005 g / mol, 3,010 g / mol, 3,015 g / mol, 3,020 g / mol, 3,025 g / mol, 3,030 g / mol, 3,035 g / mol, 3,040 g / mol, 3,045 g / mol, 3,050 g / mol, 3,055 g / mol, 3,060 g / mol, 3,065 g / mol, 3,070 g / mol, 3,075 g / mol, 3,080 g / mol, 3,085 g / mol, 3,090 g / mol, 3,095 g / mol, 3,100 g / mol, 3,105 g / mol, 3,110 g / mol, 3,115 g / mol, 3,120 g / mol, 3,125 g / mol, 3,130 g / mol, 3,135 g / mol, 3,140 g / mol, 3,145 g / mol, 3,150 g / mol, 3,155 g / mol, 3,160 g / mol, 3,165 g / mol, 3,170 g / mol, 3,175 g / mol, 3,180g / mol、3,185g / mol、3,190g / mol、3,195g / mol、3,200g / mol、3,205g / mol、3,210g / mol、3,215g / mol、3,220g / mol、3,225g / mol、3,230g / mol、3,235g / mol、3,240g / mol、3,245g / mol、3,250g / mol、3,255g / mol、3,260g / mol、3,265g / mol、3,270g / mol、3,275g / mol、3,280g / mol、3,285g / mol、3,290g / mol、3,295g / mol、3,300g / mol、3,305g / mol、3,310g / mol、3,315g / mol、3,320g / mol、3,325g / mol、3,330g / mol、3,335g / mol、3,340g / mol、3,345g / mol、3,350g / mol、3,355g / mol、3,360g / mol、3,365g / mol、3,370g / mol、3,375g / mol、3,380g / mol、3,385g / mol、3,390g / mol、3,395g / mol、3,400g / mol、3,405g / mol、3,410g / mol、3,415g / mol、3,420g / mol、3,425g / mol、3,430g / mol、3,435g / mol、3,440g / mol、3,445g / mol、3,450g / mol、3,455g / mol、3,460g / mol、3,465g / mol、3,470g / mol、3,475g / mol、3,480g / mol、3,485g / mol、3,490g / mol、3,495g / mol、3,500g / mol、3,505g / mol、3,510g / mol、3,515g / mol、3,520g / mol、3,525g / mol、3,530g / mol、3,535g / mol、3,540g / mol、3,545g / mol、3,550g / mol、3,555g / mol、3,560g / mol、3,565g / mol、3,570g / mol、3,575g / mol、3,580g / mol、3,585g / mol、3,590g / mol、3,595g / mol、3,600g / mol、3,605g / mol、3,610g / mol、3,615g / mol、3,620g / mol、3,625g / mol、3,630g / mol、3,635g / mol, 3,640g / mol, 3,645g / mol, 3,650g / mol, 3,655g / mol, 3,660g / mol, 3,665g / mol, 3,670g / mol, 3,675g / mol, 3,680g / m ol, 3,685g / mol, 3,690g / mol, 3,695g / mol, 3,700g / mol, 3,705g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,73 0g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol, 3,760g / mol, 3,765g / mol, 3,770g / mol, 3,775g / mol , 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,810g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3 ,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / m It has an average molar mass of polyoxypropylene subunits of ol, 3,925 g / mol, 3,930 g / mol, 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0297] In some embodiments, the poloxamer has a molecular weight of from about 3,250 g / mol to about 4,000 g / mol (e.g., about 3,250 g / mol, 3,255 g / mol, 3,260 g / mol, 3,265 g / mol, 3,270 g / mol, 3,275 g / mol, 3,280 g / mol, 3,285 g / mol, 3,290 g / mol, 3,295 g / mol, 3,300 g / mol, 3,305 g / mol, 3,310 g / mol, 3,315 g / mol, 3,320 g / mol, 3,325 g / mol, 3,330 g / mol, 3,335 g / mol, 3,340 g / mol, 3,345 g / mol, 3,350 g / mol, 3,355 g / mol, 3,360 g / mol, 3,365 g / mol, 3,370 g / mol, 3,375 g / mol, 3,380 g / mol, 3,385 g / mol, 3,390 g / mol, 3,395 g / mol, 3,400 g / mol, 3,405 g / mol, 3,410 g / mol, 3,415 g / mol, 3,420 g / mol, 3,425 g / mol, 3,430 g / mol, 3,435 g / mol, 3,440 g / mol, 3,445 g / mol, 3,450 g / mol, 3,455 g / mol, 3,460 g / mol, 3,465 g / mol, 3,470 g / mol, 3,475 g / mol, 3,480 g / mol,  680g / mol, 3,685g / mol, 3,690g / mol, 3,695g / mol, 3,700g / mol, 3,705g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,730g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol, 3,760g / mol, 3 ,765g / mol, 3,770g / mol, 3,775g / mol, 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,810g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3 ,850g / mol, 3,855g / mol, 3,860g / mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3,915g / mol, 3,920g / mol, 3,925g / mol, 3,930g / mol, It has an average molar mass of polyoxypropylene subunits of 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol.

[0298] In some embodiments, poloxamer is present in concentrations of approximately 3,625 g / mol to approximately 4,000 g / mol (for example, approximately 3,625 g / mol, 3,630 g / mol, 3,635 g / mol, 3,640 g / mol, 3,645 g / mol, 3,650 g / mol, 3,655 g / mol, 3,660 g / mol, 3,665 g / mol, 3,670 g / mol, 3,675 g / mol, 3,680 g / mol, 3,685 g / mol, 3,690 g / mol, 3,695 g / mol, 3,70700 g / mol, 3,680 g / mol, 3,685 g / mol, 3,690 g / mol, 3,695 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 3,600 g / mol, 5g / mol, 3,710g / mol, 3,715g / mol, 3,720g / mol, 3,725g / mol, 3,730g / mol, 3,735g / mol, 3,740g / mol, 3,745g / mol, 3,750g / mol, 3,755g / mol l, 3,760g / mol, 3,765g / mol, 3,770g / mol, 3,775g / mol, 3,780g / mol, 3,785g / mol, 3,790g / mol, 3,795g / mol, 3,800g / mol, 3,805g / mol, 3,8 10g / mol, 3,815g / mol, 3,820g / mol, 3,825g / mol, 3,830g / mol, 3,835g / mol, 3,840g / mol, 3,845g / mol, 3,850g / mol, 3,855g / mol, 3,860g / mol mol, 3,865g / mol, 3,870g / mol, 3,875g / mol, 3,880g / mol, 3,885g / mol, 3,890g / mol, 3,895g / mol, 3,900g / mol, 3,905g / mol, 3,910g / mol, 3 It has an average molar mass of polyoxypropylene subunits of 915 g / mol, 3,920 g / mol, 3,925 g / mol, 3,930 g / mol, 3,935 g / mol, 3,940 g / mol, 3,945 g / mol, 3,950 g / mol, 3,955 g / mol, 3,960 g / mol, 3,965 g / mol, 3,970 g / mol, 3,975 g / mol, 3,980 g / mol, 3,985 g / mol, 3,990 g / mol, 3,995 g / mol, or 4,000 g / mol).

[0299] In some embodiments, poloxamer is present in amounts exceeding 40% by mass (for example, about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%). It has an average ethylene oxide content of 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more.

[0300] In some embodiments, the poloxamer has an average ethylene oxide content of more than 50% by mass (for example, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0301] In some embodiments, the poloxamer has an average ethylene oxide content of more than 60% by mass (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0302] In some embodiments, the poloxamer has an average ethylene oxide content of more than 70% by mass (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0303] In some embodiments, the poloxamer has an average ethylene oxide content of about 40% to about 90% (for example, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%).

[0304] In some embodiments, the poloxamer has an average ethylene oxide content of about 50% to about 85% (for example, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%).

[0305] In some embodiments, the poloxamer has an average ethylene oxide content of about 60% to about 80% (for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%).

[0306] In some embodiments, poloxamer is present in concentrations greater than 10,000 g / mol (e.g., approximately 10,100 g / mol, 10,200 g / mol, 10,300 g / mol, 10,400 g / mol, 10,500 g / mol, 10,600 g / mol, 10,700 g / mol, 10,800 g / mol, 10,900 g / mol, 11,000 g / mol, 11,100 g / mol). 0g / mol, 11,200g / mol, 11,300g / mol, 11,400g / mol, 11,500g / mol, 11,600g / mol, 11,700g / mol, 11, 800g / mol, 11,900g / mol, 12,000g / mol, 12,100g / mol, 12,200g / mol, 12,300g / mol, 12,400g / mol, 1 2,500g / mol, 12,600g / mol, 12,700g / mol, 12,800g / mol, 12,900g / mol, 13,000g / mol, 13,100g / mol l, 13,200g / mol, 13,300g / mol, 13,400g / mol, 13,500g / mol, 13,600g / mol, 13,700g / mol, 13,800g / It has an average molar mass of 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0307] In some embodiments, poloxamer is present in concentrations greater than 11,000 g / mol (for example, about 11,100 g / mol, 11,200 g / mol, 11,300 g / mol, 11,400 g / mol, 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 1 It has an average molar mass of 3,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0308] In some embodiments, poloxamer is present in concentrations greater than 12,000 g / mol (e.g., about 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 1 It has an average molar mass of 3,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0309] In some embodiments, poloxamer is present in concentrations greater than 12,500 g / mol (for example, about 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g It has an average molar mass of 1 / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0310] In some embodiments, poloxamer is present in concentrations of approximately 10,000 g / mol to approximately 15,000 g / mol (for example, approximately 10,000 g / mol, 10,100 g / mol, 10,200 g / mol, 10,300 g / mol, 10,400 g / mol, 10,500 g / mol, 10,600 g / mol, 10,700 g / mol, 10,800 g / mol, 10,900 g / mol). l, 11,000g / mol, 11,100g / mol, 11,200g / mol, 11,300g / mol, 11,400g / mol, 11,500g / mol, 11,600g / mol , 11,700g / mol, 11,800g / mol, 11,900g / mol, 12,000g / mol, 12,100g / mol, 12,200g / mol, 12,300g / mol, 12,400g / mol, 12,500g / mol, 12,600g / mol, 12,700g / mol, 12,800g / mol, 12,900g / mol, 13,000g / mol, 1 3,100g / mol, 13,200g / mol, 13,300g / mol, 13,400g / mol, 13,500g / mol, 13,600g / mol, 13,700g / mol, 13 It has an average molar mass of 800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0311] In some embodiments, poloxamer is present in concentrations of approximately 11,000 g / mol to approximately 15,000 g / mol (for example, approximately 11,000 g / mol, 11,100 g / mol, 11,200 g / mol, 11,300 g / mol, 11,400 g / mol, 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol). It has an average molar mass of 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0312] In some embodiments, poloxamer is present in concentrations of approximately 11,500 g / mol to approximately 15,000 g / mol (for example, approximately 11,500 g / mol, 11,600 g / mol, 11,700 g / mol, 11,800 g / mol, 11,900 g / mol, 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol). It has an average molar mass of g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0313] In some embodiments, poloxamer is present in concentrations of approximately 12,000 g / mol to approximately 15,000 g / mol (for example, approximately 12,000 g / mol, 12,100 g / mol, 12,200 g / mol, 12,300 g / mol, 12,400 g / mol, 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, It has an average molar mass of 13,400 g / mol, 13,500 g / mol, 13,600 g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0314] In some embodiments, poloxamer is present in concentrations of approximately 12,500 g / mol to approximately 15,000 g / mol (for example, approximately 12,500 g / mol, 12,600 g / mol, 12,700 g / mol, 12,800 g / mol, 12,900 g / mol, 13,000 g / mol, 13,100 g / mol, 13,200 g / mol, 13,300 g / mol, 13,400 g / mol, 13,500 g / mol, 13,600 g / mol). It has an average molar mass of g / mol, 13,700 g / mol, 13,800 g / mol, 13,900 g / mol, 14,000 g / mol, 14,100 g / mol, 14,200 g / mol, 14,300 g / mol, 14,400 g / mol, 14,500 g / mol, 14,600 g / mol, 14,700 g / mol, 14,800 g / mol, 14,900 g / mol, or 15,000 g / mol.

[0315] Poloxamer P288, P335, P338, and P407 Suitable poloxamers that can be used in combination with the compositions and methods disclosed herein include those with the chemical formula HO(C2H4O) x (C3H6O) y (C2H4O) z One example is "poloxamer 288" (also known in the art as "P288" and poloxamer "F98"), which has H [wherein the formula, the sum of x and y is approximately 236.36, and z is approximately 44.83]. The average molecular weight of P288 is approximately 13,000 g / mol.

[0316] In some embodiments, poloxamer is of the formula HO(C2H4O) x (C3H6O) y (C2H4O) z Variants of P288, such as a variant of H[wherein the formula, the sum of x and y is approximately 220 to approximately 250, and z is approximately 40 to approximately 50]. In some embodiments, the average molecular weight of poloxamer is approximately 12,000 g / mol to approximately 14,000 g / mol.

[0317] Suitable poloxamers that can be used in combination with the compositions and methods disclosed herein include those with the chemical formula HO(C2H4O) x (C3H6O) y (C2H4O) z Further examples include "poloxamer 335" (also known in the art as "P335" and poloxamer "P105") having H [wherein the formula, the sum of x and y is approximately 73.86, and z is approximately 56.03]. The average molecular weight of P335 is approximately 6,500 g / mol.

[0318] In some embodiments, poloxamer is of the formula HO(C2H4O) x (C3H6O) y (C2H4O) z Variants of P335, such as a variant of H[wherein the formula, the sum of x and y is approximately 60 to approximately 80, and z is approximately 50 to approximately 60]. In some embodiments, the average molecular weight of poloxamer is approximately 6,000 g / mol to approximately 7,000 g / mol.

[0319] Suitable poloxamers that can be used in combination with the compositions and methods disclosed herein include those with the chemical formula HO(C2H4O) x (C3H6O) y (C2H4O) z Further examples include "poloxamer 338" (also known in the art as "P338" and poloxamer "F108") having H [wherein the formula, the sum of x and y is approximately 265.45, and z is approximately 50.34]. The average molecular weight of P335 is approximately 14,600 g / mol.

[0320] In some embodiments, poloxamer is of the formula HO(C2H4O) x (C3H6O) y (C2H4O) z Variants of P338, such as a variant of H[wherein the formula, the sum of x and y is approximately 260 to approximately 270, and z is approximately 45 to approximately 55]. In some embodiments, the average molecular weight of poloxamer is approximately 14,000 g / mol to approximately 15,000 g / mol.

[0321] Suitable poloxamers that can be used in combination with the compositions and methods disclosed herein include those with the chemical formula HO(C2H4O) x (C3H6O) y (C2H4O) z Further examples include "Poloxamer 407" (also known in the art as "P407" and "Poloxamer F127") having H [wherein the formula, the sum of x and y is approximately 200.45, and z is approximately 65.17]. The average molecular weight of P335 is approximately 12,600 g / mol.

[0322] In some embodiments, poloxamer is of the formula HO(C2H4O) x (C3H6O) y (C2H4O) z Variants of P407, such as a variant of H[wherein the formula, the sum of x and y is approximately 190 to approximately 210, and z is approximately 60 to approximately 70]. In some embodiments, the average molecular weight of poloxamer is approximately 12,000 g / mol to approximately 13,000 g / mol.

[0323] For clarity, the terms “average molar mass” and “average molecular weight” are used herein to mean the same thing and refer to the same quantity. The average molar mass, ethylene oxide content, and propylene oxide content of poloxamers described herein can be measured using the methods disclosed in Alexandridis and Hatton, Colloids and Surfaces A: Physicochemical and Engineering Aspects 96:1-46 (1995), the entire disclosure of which is incorporated by reference herein.

[0324] PKC control agent Various agents can be used to reduce PKC activity and / or expression. While not limited by mechanism, such agents can complement viral transduction by stimulating Akt signaling and / or maintaining cofilin, thereby promoting actin depolymerization. This actin depolymerization event may play a role in removing the physical barrier that inhibits the viral vector from entering the nucleus of target cells.

[0325] Staurosporine and its variants In some embodiments, the substance that reduces the activity and / or expression of PKC is a PKC inhibitor. The PKC inhibitor may be a staurosporine or a variant thereof. For example, the PKC inhibitor may be a compound represented by formula (I).

[0326] [ka] [In the formula, R1 is H, OH, optionally substituted alkoxide, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amide, halogen, optionally substituted C] 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkinyl, or optionally substituted acyl, R a and R b These are H and C, which are substituted independently of each other. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, or optionally substituted C 2-6Alkynyl, optionally substituted and optionally condensed aryl, optionally substituted and optionally condensed heteroaryl, optionally substituted and optionally condensed cycloalkyl, or optionally substituted and optionally condensed heterocycloalkyl, or R a and R b These atoms bond with the atoms they bind to, forming optionally substituted and optionally condensed heterocycloalkyl rings. R c O, NR d , or S, R d H, and C as arbitrarily substituted. 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 It is alkinyl, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl.

[0327] [ka] This represents an arbitrary combination, n is an integer from 0 to 4. m is an integer between 0 and 4. It may be a compound represented by, or a salt thereof.

[0328] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO1991 / 009034, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (II)

[0329] [ka] [In the formula, R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amide, halogen, oxo, or thiocarbonyl, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkinyl, or optionally substituted acyl, R a and R b These atoms bond with the atoms they bind to, forming optionally substituted and optionally condensed heterocycloalkyl rings. R c is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. It is represented by, or a salt thereof.

[0330] Further examples of such staurosporin variants are given by equation (III)

[0331] [ka] [In the formula, R1 is H, OH, oxo, or thiocarbonyl, R2 is H, and C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkinyl, or optionally substituted acyl, Ring A is an optionally substituted and optionally condensed heterocycloalkyl ring, R c is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. It is represented by, or a salt thereof.

[0332] Further examples of such staurosporin variants are given by equation (IV)

[0333] [ka] [In the formula, R1 is H, OH, or oxo, Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring. R c is either O or S, W is O, NH, or S. Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. It is represented by, or a salt thereof.

[0334] Further examples of such staurosporin variants are given by equation (V)

[0335] [ka] [In the formula, R1 is H, OH, or oxo, R c is either O or S, W is O, NH, or S. Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. p is either 0 or 1. It is represented by, or a salt thereof.

[0336] Further examples of such staurosporin variants are given by equation (VI).

[0337] [ka] [In the formula, R1 is H, OH, or oxo, Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. s is an integer between 0 and 8. It is represented by, or a salt thereof.

[0338] Further examples of such staurosporin variants are given by equation (VII)

[0339] [ka] [In the formula, R1 is H, OH, or oxo, R2 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R3 is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. It is represented by, or a salt thereof.

[0340] Further examples of such staurosporin variants are given by equation (VIII)

[0341] [ka] [In the formula, R1 is H, OH, or oxo, R2 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R3 is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. It is represented by, or a salt thereof.

[0342] Further examples of such staurosporin variants are given by equation (IX)

[0343] [ka] [In the formula, each X independently represents a halogen, an optionally substituted haloalkyl, cyano, an optionally substituted amino, hydroxyl, thiol, an optionally substituted alkoxy, an optionally substituted alkylthio, an optionally substituted acyloxy, an optionally substituted alkoxycarbonyl, an optionally substituted carboxy, ureido, an optionally substituted alkylsulfonyl, an optionally substituted arylsulfonyl, an optionally substituted heteroarylsulfonyl, an optionally substituted cycloalkylsulfonyl, an optionally substituted heterocycloalkylsulfonyl, an optionally substituted alkylsulfanyl, an optionally substituted arylsulfanyl, or an optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. It is represented by, or a salt thereof.

[0344] Further examples of such staurosporin variants are given by equation (1)

[0345] [ka] It is represented by, or a salt thereof.

[0346] In some embodiments, the PKC inhibitor is staurosporine, i.e., (2S,3R,4R,6R)-3-methoxy-2-methyl-4-(methylamino)-29-oxa-1,7,17-triazaoctactacyclo[12.12.2.12,6.07,28.08,13.015,19.020,27.021,26]nonacosa-8,10,12,14,19,21,23,25,27-nonaen-16-one, represented by formula (2).

[0347] [ka] or its salt.

[0348] Further examples of such staurosporin variants are given by equation (X)

[0349] [ka] [In the formula, R1 is H, OH, or oxo, Each Z independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. t is an integer between 0 and 6. It is represented by, or a salt thereof.

[0350] Further examples of such staurosporin variants are given by equation (XI)

[0351] [ka] [In the formula, R1 is H, OH, or oxo, R4 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. It is represented by, or a salt thereof.

[0352] An example of such a staurosporin variant is given by equation (XII)

[0353] [ka] [In the formula, R1 is H, OH, or oxo, R4 is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. It is represented by, or a salt thereof.

[0354] Further examples of such staurosporin variants are given by equation (XIII)

[0355] [ka] [In the formula, each X independently represents a halogen, an optionally substituted haloalkyl, cyano, an optionally substituted amino, hydroxyl, thiol, an optionally substituted alkoxy, an optionally substituted alkylthio, an optionally substituted acyloxy, an optionally substituted alkoxycarbonyl, an optionally substituted carboxy, ureido, an optionally substituted alkylsulfonyl, an optionally substituted arylsulfonyl, an optionally substituted heteroarylsulfonyl, an optionally substituted cycloalkylsulfonyl, an optionally substituted heterocycloalkylsulfonyl, an optionally substituted alkylsulfanyl, an optionally substituted arylsulfanyl, or an optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently consists of a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4. m is an integer between 0 and 4. It is represented by, or a salt thereof.

[0356] A further example of such a staurosporin variant is given by equation (3).

[0357] [ka] It is represented by, or a salt thereof.

[0358] Further examples of such staurosporin variants are given by equation (4).

[0359] [ka] It is represented by, or a salt thereof.

[0360] Further examples of such staurosporin variants are:

[0361] [ka] or its salt.

[0362] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO1993 / 007153, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XIV)

[0363] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. It is represented by, or a salt thereof.

[0364] In some embodiments, the PKC inhibitor is a compound represented by formula (XV).

[0365] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0366] In some embodiments, the PKC inhibitor is

[0367] [ka] It is a compound selected from either or its salts.

[0368] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Patent No. 5,093,330, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XVI)

[0369] [ka] [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] It is represented by, or its salt, or a quaternized variant.

[0370] In some embodiments, the PKC inhibitor is a compound represented by formula (XVII).

[0371] [ka] [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] Alternatively, it is its salt, or a quaternized variant.

[0372] In some embodiments, the PKC inhibitor is

[0373] [ka] A compound selected from TIFF2026108645000077.tif187170TIFF2026108645000078.tif224170TIFF2026108645000079.tif227170TIFF2026108645000080.tif226170TIFF2026108645000081.tif114170, or a salt thereof.

[0374] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Patent No. 5,264,431, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XVIII)

[0375] [ka] [In the formula, R is H, OH, C 1-6 It is an alkoxy or oxo, R2 is

[0376] [ka] And optionally, the sugar moiety in the formula is derived from D-glucose, D-galactose, or D-mannose. R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R4 is OH, C 1-6 Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 Alkoxycarbonylamino, C 2-20These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 It is substituted with an alkoxy, R5 is H or C 1-6 It is alkyl, R6 is either free or aliphatic C 2-22 It is a hydroxyl esterified with a carboxylic acid, or C 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R7 is either free or aliphatic C 2-22 OH, C esterified with a carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, azide, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 These are alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 It is substituted with an alkoxycarbonyl group. It is represented by, or a salt thereof.

[0377] In some embodiments, the PKC inhibitor is a compound represented by formula (XIX).

[0378] [ka] [In the formula, R is H, OH, C 1-6 It is an alkoxy or oxo, R2

[0379] [ka] and; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R4 is OH, C 1-6 Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 Alkoxycarbonylamino, C 2-20 These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 It is substituted with an alkoxy, R5 is H or C 1-6 It is alkyl, R6 is either free or aliphatic C 2-22 It is a hydroxyl esterified with a carboxylic acid, or C 1-6 Alkoxycarbonyloxy, C 1-6Alkyl sulfonyl oxy, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, or C 1-6 It is substituted with an alkoxy, R7 is either free or aliphatic C 2-22 OH, C esterified with a carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyl oxy, azide, free or aliphatic C 2-22 amino acids acylated with carboxylic acids, C 1-6 Alkylamino, di-C 1-6 Alkylamino, C 1-6 These are alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 It is substituted with an alkoxycarbonyl group. or its salt.

[0380] In some embodiments, the PKC inhibitor is N-(1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(2-N-acetyl-muramyl)staurosporine, N-(6-O-mesyl-1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(6-azido-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-6-deoxy-2-N-acetylmyl)staurosporine N-(6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(2-N-acetyl-demethylmuramyl)staurosporine, N-(1-α-O-benzyl-2-N-acetylhomomuramyl)staurosporine, N-(1-α-O-benzyl-2-N-acetyl-L-homomuramyl)staurosporine, 1-α-anomer of N-(2-N-acetyl-L-homomuramyl)staurosporine, N-(1-α-O-benzyl-4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α-O -benzyl-4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-2-N-acetylmuramyl)staurosporine, 1-α-anomer of N-(4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, 1-α-anomer of N-(4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4,6-O-triacetyl The compound is selected from N-(1-deoxy-6-O-acetyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-truolsulfonyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-azido-2-N-acetylmuramyl)staurosporine, and N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, or a salt thereof.

[0381] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Patent No. 5,461,146, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XX)

[0382] [ka] [In the formula, Z1 is H or OH, Z2 is either H or OH. R1 is H, a halogen, or an optionally substituted alkyl group. R2 is H or halogen, R is an OH group or an optionally substituted alkoxy. X is an optionally substituted alkyl or optionally substituted acyl, optionally, in the formula, X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents the phenyl moiety. It is represented by [ ] or a salt thereof.

[0383] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Patent No. 5,756,494, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XXI)

[0384] [ka] [In the formula, Z1 is H or OH, Z2 is either H or OH. R1 is H, a halogen, or an optionally substituted alkyl group. R2 is H or halogen, R is an OH group or an optionally substituted alkoxy. X is an optionally substituted alkyl or optionally substituted acyl, optionally, in the formula, X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents the phenyl moiety. It is represented by [ ] or a salt thereof.

[0385] In some embodiments, the PKC inhibitor is a staurosporine variant described in US2005 / 0020570, the entire disclosure of which is incorporated by reference in this invention. Examples of such staurosporine variants are formulas (XXII), (XXIII), (XXIV), or (XXV)

[0386] [ka] [In the formula, each R1 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl, Each R2 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms, or 30 or fewer carbon atoms in each case. Each X is independently O, OH, and H, or a pair of hydrogen atoms. Each Q is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each n is an independent integer between 0 and 4. Each m is an independent integer between 0 and 4. It is represented by, or a salt thereof.

[0387] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII).

[0388] [ka] [In the formula, each R1 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl, Each R2 is independently an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-- or N,N-disubstituted aminosulfonyl. Each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, or 30 or fewer carbon atoms. Each R8 is independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms in each case, and 9 or fewer heteroatoms in each case. Each R9 is independently an optionally substituted acyl, optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, carbonyl, carbonyldioxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R 10 These are independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms in each case, and a heteroatom having 9 or fewer heteroatoms in each case. Each X is independently O, OH, and H, or a pair of hydrogen atoms. Each n is an independent integer between 0 and 4. Each m is an independent integer between 0 and 4. Each n' is an independent integer between 0 and 4. Each m' is an independent integer between 0 and 4. It is represented by, or a salt thereof.

[0389] In some embodiments, the PKC inhibitor is a staurosporine variant described in US Patent No. 5,624,949, the entire disclosure of which is incorporated by reference in this invention. An example of such a staurosporine variant is formula (XXVIII)

[0390] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. It is represented by, or a salt thereof.

[0391] In some embodiments, the PKC inhibitor is a compound represented by formula (XXIX).

[0392] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0393] In some embodiments, the PKC inhibitor is a compound represented by formula (XXX).

[0394] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6It is alkyl. or its salt.

[0395] In some embodiments, the PKC inhibitor is a compound represented by formula (XXXI).

[0396] [ka] [In the formula, R1 is H, or optionally substituted with C] 1-6 It is alkyl, R2 is an arbitrarily substituted C 1-6 It is alkyl. or its salt.

[0397] In some embodiments, the PKC inhibitor is

[0398] [ka] The compound is selected from TIFF2026108645000095.tif201170, TIFF2026108645000096.tif212170, TIFF2026108645000097.tif222170, and TIFF2026108645000098.tif161170.

[0399] Interfering RNA Exemplary PKC modifiers that can be used in combination with the compositions and methods of this disclosure include interfering RNA molecules, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA), which reduce PKC gene expression. Methods for preparing interfering RNA molecules are known in the art and are described in detail, for example, in WO2004 / 044136 and U.S. Patent No. 9,150,605, each of which is incorporated herein by reference in whole.

[0400] Spinoculation In some embodiments of this disclosure, cells targeted for transduction can be spun, for example by centrifugation, while being cultured with a viral vector (for example, in combination with one or more additional agents described herein). This “spinoculation” process may occur with a centripetal force of, for example, about 200xg to about 2,000xg. The centripetal force may be, for example, about 300xg to about 1,200xg (e.g., about 300xg, 400xg, 500xg, 600xg, 700xg, 800xg, 900xg, 1,000xg, 1,100xg, or 1,200xg or more). In some embodiments, cells are spun for about 10 minutes to about 3 hours (for example, about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes or longer). In some embodiments, cells are spun at room temperature, for example, at a temperature of about 25°C.

[0401] Exemplary transduction procedures involving a spinoculation step are described, for example, in Millington et al., PLoS One 4:e6461 (2009); Guo et al., Journal of Virology 85:9824-9833 (2011); O'Doherty et al., Journal of Virology 74:10074-10080 (2000); and Federico et al., Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools, Methods in Molecular Biology 1448, Chapter 4 (2016), each of which is incorporated herein by reference.

[0402] target cell Cells that can be used in combination with the compositions and methods described herein include cells that can undergo further differentiation. For example, one type of cell that can be used in combination with the compositions and methods described herein is a pluripotent cell. A pluripotent cell is a cell that has the ability to develop into two or more differentiated cells. Examples of pluripotent cells are ESCs, iPSCs, and CD34+ cells. ESCs and iPSCs have the ability to differentiate into ectoderm cells that give rise to the skin and nervous system, endoderm cells that form the gastrointestinal tract and airways, endocrine glands, liver, and pancreas, and mesoderm cells that form bone, cartilage, muscle, connective tissue, and most of the circulatory system.

[0403] Cells that can be used in combination with the compositions and methods described herein include pluripotent hematopoietic stem cells and hematopoietic progenitor cells. Hematopoietic stem cells (HSCs) are immature blood cells that self-replicate and have the ability to differentiate into mature blood cells, including but not limited to a diverse lineage of granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombus cells (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteolytic cells, and lymphocytes (e.g., NK cells, B cells, and T cells). Human HSCs are CD34+. In addition, HSCs refer to long-term regrowing HSCs (LT-HSCs) and short-term regrowing HSCs (ST-HSCs). Any of these HSCs can be used in combination with the compositions and methods described herein.

[0404] HSCs and other pluripotent progenitor cells can be obtained from blood products. Blood products are products obtained from the body or organs of the body containing hematopoietic cells. Such sources include unfractionated bone marrow, umbilical cord, placenta, peripheral blood, or mobilized peripheral blood. All of the aforementioned crude or unfractionated blood products can be enriched in various ways for cells that have the characteristics of HSCs or bone marrow progenitor cells. For example, differentiated cells can be selected based on the cell surface molecules they express, as they mature. Blood products can be fractionated by positive selection for CD34+ cells, containing a subpopulation of self-renewing, pluripotent pluripotent hematopoietic stem cells that can home to a pluripotent hematopoietic stem cell niche and be reintroduced into transplant recipients to re-establish proliferative and maintained hematopoiesis. Such selection can be achieved, for example, using commercially available magnetic anti-CD34 beads (Dynal, Lake Success, NY). Bone marrow progenitor cells can also be isolated based on the markers they express. Non-fractionated blood products can be obtained directly from donors or recovered from cryogenic storage. HSCs and bone marrow progenitor cells can also be obtained through differentiation of ES cells, iPS cells, or other reprogrammed mature cell types.

[0405] Cells that can be used in combination with the compositions and methods described herein include allogeneic cells and autologous cells. When using allogeneic cells, the cells may optionally be HLA-matched with those of the subject receiving cell treatment.

[0406] Viral vectors for transgene expression Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of mammalian cells by universal or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., retroviridae virus vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses, e.g., influenza virus), rhabdoviruses (e.g., rabies virus and varicella stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, variant vaccinia ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis viruses. Examples of retroviruses include avian leukemia sarcoma, avian type C virus, mammalian type C, B, and D viruses, onchoretrovirus, HTLV-BLV group, lentivirus, alpha-retrovirus, gamma-retrovirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include mouse leukemia virus, mouse sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in McVey et al., (US 5,801,030), the teachings of which are incorporated herein by reference.

[0407] Retrovirus vectors The delivery vectors used in the methods and compositions described herein may be retroviral vectors. One type of retroviral vector usable in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, efficiently transduce a wide range of mitotic and undist cell types and confer stable, long-term transgene expression. An outline of optimized methods for packaging and transducing LVs is described in Delenda, The Journal of Gene Medicine 6: S125 (2004), and its disclosure is incorporated herein by reference.

[0408] The use of lentivirus-based gene transfer technology relies on the in vitro generation of recombinant lentivirus particles containing a highly depleted viral genome that encapsulates the target transgene. Specifically, recombinant lentiviruses are recovered by trans co-expression in a cell line that tolerates a transcription vector, consisting of (1) a packaging construct, i.e., a vector expressing (or trans-expressing) a Gag-Pol precursor with Rev; (2) a vector generally expressing a heterogeneous envelope receptor; and (3) viral cDNA from which all open reading frames have been removed, but which maintains the sequences necessary for replication, encapsulation, and expression in which the sequences are expressed and inserted.

[0409] Examples of lentiviral vectors used in the methods and compositions described herein include one or more 5'-long-terminal repeats (LTRs), HIV signal sequences, HIV Psi signal 5'-splice sites (SDs), delta-GAG elements, Rev response elements (RREs), 3'-splice sites (SAs), elongation elements (EFs), 1-alpha promoters, and 3'-autoinactivating LTRs (SIN-LTRs). Lentiviral vectors optionally include a central polypurine band (cPPT) and a WPRE, as described in US6,136,597, whose disclosure is incorporated herein by reference for woodchuck virus post-transcriptional regulatory elements (WPREs). Lentiviral vectors may further include a pHR' backbone, for example, those shown below.

[0410] Lentigen LVs, such as those described in Lu et al., Journal of Gene Medicine 6:963 (2004), can be used to express DNA molecules and / or transduce cells. Examples of LVs used in the methods and compositions described herein include 5'-long-terminal repeats (LTRs), HIV signaling sequences, HIV Psi signaling 5'-splice sites (SDs), delta-GAG elements, Rev response elements (RREs), 3'-splice sites (SAs), elongation elements (EFs), 1-alpha promoters, and 3'-self-inactivating LTRs (SIN-LTRs). It will be readily apparent to those skilled in the art that one or more of these regions may be optionally replaced with other regions that perform similar functions.

[0411] Enhancer elements can be used to increase the expression of modified DNA molecules or to increase the efficiency of lentiviral integration. Examples of LVs used in the methods and compositions described herein include nef sequences. Examples of LVs used in the methods and compositions described herein include cPPT sequences, which improve vector integration. cPPT acts as a secondary starting point for (+)-strand DNA synthesis, introducing partial strand overlap at the center of its endogenous HIV genome. The introduction of cPPT sequences into the transcription vector backbone significantly increased nuclear transport and the total amount of genome integrated into the target cell's DNA. Examples of LVs used in the methods and compositions described herein include woodchuck post-transcriptional regulatory elements (WPREs). WPREs act at the transcriptional level, increasing the total amount of intracellular mRNA by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts. Addition of WPREs to LVs results in substantial improvements in the level of transgene expression from several different promoters, both in vitro and in vivo. Examples of LVs used in the methods and compositions described herein include both cPPT sequences and WPRE sequences. Examples of vectors include IRES sequences, which enable the expression of multiple polypeptides from a single promoter.

[0412] In addition to IRES sequences, other elements that enable the expression of multiple polypeptides are useful. Examples of vectors used in the methods and compositions described herein include multiple promoters that enable the expression of two or more polypeptides. Examples of vectors used in the methods and compositions described herein include protein cleavage sites that enable the expression of two or more polypeptides. Examples of protein cleavage sites that enable the expression of two or more polypeptides are described in Klump et al., Gene Ther.; 8:811 (2001), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004), and these disclosures are incorporated herein by reference because they relate to protein cleavage sites that enable the expression of two or more polypeptides. It will be readily apparent to those skilled in the art that other elements that enable the expression of multiple polypeptides, to be identified in the future, are useful and may be available in vectors suitable for use with the compositions and methods described herein.

[0413] The vectors used in the methods and compositions described herein may be clinical-grade vectors.

[0414] Treatment methods Exemplary diseases treatable using compositions and methods disclosed herein Examples of transgenes that can be introduced into target cells using the compositions and methods of this disclosure and ultimately delivered to a patient (e.g., by administering target cells to a patient) include those encoding therapeutic proteins. Recipients of transgenes (e.g., recipients of cells transduced to express the transgene) may suffer from diseases characterized by deficiencies in the encoded protein. For example, a transgene that can be expressed in target cells and delivered to a patient according to the compositions and methods of this disclosure is a transgene encoding β-globin, which is particularly useful for treating patients with β-therasemia. Exemplary nucleic acid and amino acid sequences of human β-globin cDNA and protein are shown below. Exemplary wild-type human β-globin cDNA sequences: ATGGTGCATCTGACCCCGGAAGAAAAAAGCGCGGTGACCGCGCTGGGGCAAAGTGAACGTGGATGAAGTGGGCGGCGAAGCGCTGGGCCGCCTGCTGGTGGTGTATCCGTGGACCCAGCCTTTTTGAAAGCTTTGGCGATCTGAGCACCCCGGATGCGGTGATGGGCAACCCGAAAGTGAAAGCGCATGGCAAAAAAGTGCTGGGCGCGTTTAGCG ATGGCCTGGCGCATCTGGATAACCTGAAAGGCACCTTTGCGACCCTGAGCGAACTGCATTGCGATAAACTGCATGTGGATCCGGAAAACTTTCGCCTGCTGGGCAACGTGCTGGTGTGCGTGCTGGCGCATCATTTTGGCAAAGAATTTACCCCGCCGGTGCAGGCGGCGTATCAGAAAGTGGTGGCGGGCGTGGCGAACGCGCTGGCGCATAAATATCAT (Sequence ID 1) Exemplary wild-type human β-globin amino acid sequence: MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH(Sequence ID 2)

[0415] Further examples of transgenes that can be used in combination with the compositions and methods of this disclosure include insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), calcitonin, growth hormone-releasing factor (GRF), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), prolactin, melatonin, vasopressin, β-endorphin, met-enkephalin, leu-enkephalin, prolactin-releasing factor, prolactin inhibitor, adrenocorticotropin-releasing hormone, thyroid-stimulating hormone-releasing hormone (TRH), follicle-stimulating hormone (FSH), and luteinizing hormone. (LH), chorionic gonadotropin (CG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, endostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), bFGF2, acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, or bone morphogenetic protein (BMF) BMP1 Examples of hormones and growth and differentiation factors include, but are not limited to, any of the 15 transforming growth factor β (TGFβ), any of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BNDF), neurotrophin NT-3, NT-4 / 5, and NT-6, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), any of the neuruturin, percefin, agrin, semaphorin / colapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0416] Further examples of transgenes that can be used in combination with the compositions and methods of this disclosure include thrombopoietin (TPO), interleukin (IL) IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, and IL-17, mononuclear cytochemoattractant protein (MCP-1), leukemia suppressor (LIF), and granulocyte macul Examples include, but are not limited to, cytokines and lymphokines that encode proteins that modulate the immune system, such as clophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), mononuclear cell colony-stimulating factor (M-CSF), Fas ligand, tumor necrosis factor α and β (TNFα and TNFβ), interferon (IFN) IFN-α, IFN-β, and IFN-γ, stem cell factors, and flk-2 / flt3 ligands. Transgenes encoding protein products produced by the immune system are also included in this disclosure. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and recombinant MHC molecules including single-chain MHC molecules. Other useful gene products include membrane cofactor proteins (MCPs), complement regulatory proteins such as CR1, CR2, and CD59.

[0417] Further examples of suitable transgenes include those encoding receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Examples of such receptors include, among others, the trk family of receptors such as flt-1, flk-1, TIE-2; TrkA, MuSK, Eph, PDGF receptor, EGF receptor, HER2, insulin receptor, and IGF-1 receptor; the FGF family of receptors; the TGFβ receptor, interleukin receptor, interferon receptor, serotonin receptor, α-adrenergic receptor, β-adrenergic receptor, GDNF receptor, and p75 neurotrophin receptor. Further examples include transgenes encoding extracellular matrix proteins such as integrins; counter-receptors for transmembrane junction proteins such as intercellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3, and ICAM-4); vascular adhesion molecules (VCAM); and selectins, namely E-selectin, P-selectin, and L-selectin. The present invention encompasses receptors for cholesterol regulation, including LDL receptors, HDL receptors, VLDL receptors, and scavenger receptors. Further examples include transgenes encoding apolipoprotein ligands for these receptors, including ApoAI, ApoAIV, and ApoE.Further transgenes include antimicrobial peptides such as defensins and madinin; transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, and NF-κB; the leucine zipper family; C2H4 zinc finger proteins including Zif268, EGR1, and EGR2; C6 zinc finger proteins including glucocorticoid and estrogen receptors; POU domain proteins exemplified by Pit1; homeodomain proteins including HOX-1; and m Examples include basic helix-loop-helix proteins including yc, MyoD, and myogenin; ETS box-containing proteins; TFE3; E2F; ATF1; ATF2; ATF3; ATF4; ZF5; NFAT; CREB; HNF-4; C / EBP; SP1; CCAAT box-binding proteins; interferon regulator 1 (IRF-1); Wilms tumor protein; ETS-binding proteins; STAT; GATA box-binding proteins, e.g., GATA-3; and those encoding the forkhead family of winged helix proteins.

[0418] Other useful transgenes include carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate triase, arginase, fumarylacetate hydrolase, phenylalanine hydroxylase, α-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VII, factor VIII, factor IX, factor II, factor V, factor X, factor XII, factor XI, von Willebrand factor, superoxide dismutase, and glutathione. Onperoxidase and reductase, heme oxygenase, angiotensin-converting enzyme, endothelin-1, atrial natriuretic peptide, prourokinase, urokinase, plasminogen activator, heparin subfactor II, activated protein C (factor V Leiden), protein C, antithrombin, cystathionine β-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, Surin, β-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase (also called P-protein), H-protein, T-protein, Menkes disease protein, tumor suppressor (e.g., p53), cystic fibrosis transmembrane regulator (CFTR), Wilson's disease gene PWD product, Cu / Zn superoxide dismutase, aromatic amino acid decarboxylase, tyrosine hydroxylase, acetylcholine synthetase, prohormone converterase, protease inhibitors, lactors Examples include gastrointestinal enzymes including ze, lipase, trypsin, chymotrypsin, and pepsin; adenosine deaminase, α1 antitrypsin; tissue metalloproteinase inhibitors (TIMP); GLUT-1, GLUT-2, trehalose phosphate synthase, hexokinase I, II, and III; glucokinase; collagen, elastin, fibronectin, thrombospongin, vitronectin, and one or more individual chains or species of tenascin; and those encoding suicide genes such as thymidine kinase and cytosine deaminase.Other useful proteins include acid β-glucosidase, α-galactosidase a, α-1-idulonidase, iduloate sulfatase, lysosomal acid α-glucosidase, sphingomyelinase, hexosaminidase A, hexomimidase A and B, aryl sulfatase A, acid lipase, acid ceramidase, galactosylceramidase, α-fucosidase, α-, β-manosidosis, aspartylglycosamidase, neuramidase, and galac Examples of enzymes involved in lysosomal storage disorders include tosylceramidase, heparan-N-sulfatase, N-acetyl-α-glucosaminidase, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B, β-glucuronidase, and hexosaminidases A and B.

[0419] Other useful transgenes include those encoding polypeptides that do not exist naturally, such as chimeric or hybrid polypeptides containing non-natural amino acid sequences with insertions, deletions, or amino acid substitutions. For example, single-stranded recombinant immunoglobulins may be useful in certain immunocompromised patients. Other useful proteins include transected receptors lacking transmembrane and cytoplasmic domains. These transected receptors can be used to antagonize the function of corresponding ligands by binding without the associated signaling by the receptor. Other types of non-natural gene sequences include sense and antisense molecules, such as ribozymes, which can be used to regulate gene expression, as well as catalytic nucleic acids.

[0420] Examples of transgenes that can be expressed in target cells and subsequently administered to patients to treat diseases characterized by deficiencies or dysfunctions of the encoded product include those encoding the protein products listed in Table 2 below.

[0421] [Table 2] TIFF2026108645000100.tif224170TIFF2026108645000101.tif190170TIFF2026108645000102.tif195170TIFF2026108645000103.tif208170

[0422] Selection of donor cells In some embodiments, the subject to be treated is a donor that provides cells (e.g., pluripotent cells such as CD34+HSCs or HPCs) that have been subsequently modified to express one or more therapeutic proteins of this disclosure before being re-administered to a patient. In such cases, the extracted cells (e.g., CD34+HSCs or HPCs) may be re-injected into the subject after, for example, integration of a transgene encoding one or more therapeutic proteins of this disclosure and / or disruption of allelic variants having harmful mutations, thereby allowing the cells to subsequently home to hematopoietic tissue and establish proliferative hematopoiesis, thereby restoring the expression of the transgene in the patient. When the cells to be treated also serve as a cell donor, the transplanted cells (e.g., HSCs or HPCs) are less likely to be rejected. This is due to the fact that the injected cells originate from the patient and express the same HLA class I and class II antigens as those expressed by the patient. Alternatively, the subject and donor may be different. In some embodiments, the subject and donor may be related and, for example, HLA-matched. As described herein, HLA-matched donor-recipient pairs reduce the risk of graft rejection because endogenous T cells and NK cells in the transplant recipient are less likely to recognize the incoming hematopoietic cell or progenitor cell graft as a foreign body, and therefore less likely to mount an immune response against the transplanted tissue. Exemplary HLA-matched donor-recipient pairs are genetically related donors and recipients, such as familial donor-recipient pairs (e.g., sibling donor-recipient pairs). In some embodiments, the subject and donor are HLA mismatched, which occurs when at least one HLA antigen is mismatched between the donor and recipient, particularly for HLA-A, HLA-B, and HLA-DR. To reduce the likelihood of graft rejection, for example, some haplotypes may be matched between the donor and recipient, while others are mismatched.

[0423] Pharmaceutical compositions and medications When a population of cells co-expressing one or more therapeutic proteins of this disclosure is administered to a target, the number of cells administered depends, for example, on the expression level of the desired protein(s), the patient, the formulation method, the method of administration (e.g., administration time and route), the patient's age, weight, sex, the severity of the disease being treated, and whether the patient is being treated with an agent that excises endogenous pluripotent cells (e.g., in particular endogenous CD34+ cells, hematopoietic stem cells or progenitor cells, or microglia). The number of cells administered may, for example, be 1 × 10⁶. 4 cells / kg ~ 1 × 10⁻¹⁶ 14 The number of cells / kg may be or more. Cells can be administered in an undifferentiated state or after partial or complete differentiation into the target cell type. The number of pluripotent cells may be administered in any suitable dosage form. [Examples]

[0424] The following examples are provided to those skilled in the art to illustrate how the compositions and methods described herein may be used, manufactured, and evaluated, and are intended purely to illustrate the present disclosure and not to limit the scope of what the inventors consider to be their disclosure.

[0425] Example 1. When used at low concentrations, poloxamer plays a role in increasing the efficiency of viral transduction. This example describes a series of experiments designed to evaluate the effect of poloxamers, particularly poloxamer P407, on lentiviral transduction efficiency. Human CD34+ pluripotent hematopoietic stem cells were recruited into peripheral circulation and isolated from donor subjects. The cells were cultured at 10 infection multiplicities in the presence of lentiviral vectors. The lentiviral vectors used in these experiments contained genes encoding green fluorescent protein (GFP), and detection of cells expressing the transgene was possible by monitoring fluorescence at approximately 510 nm.

[0426] During the transduction procedure, cells were exposed to a lentiviral vector encoding GFP, either alone or in combination with poloxamer P407 at concentrations of 7 μg / mL, 10 μg / mL, or 100 μg / mL. For cells transduced in the presence of poloxamer, the cells were simultaneously exposed to lentivirus and P407. After 18 hours of incubation, the cells were washed to remove the lentivirus (and, in the case of co-cultured cells, poloxamer). The ratio of cells expressing the transgene to the vector copy number in each cell population was then evaluated 12 days after the transduction procedure. The results of these studies are reported in Figures 1A and 1B, respectively.

[0427] To assess the amount of cells expressing the transgene, each cell population was monitored using flow cytometry to detect GFP+ cells. Surprisingly, as shown in Figure 1A, low concentrations of P407 appeared to promote a general increase in cells expressing the transgene, proportional to the number of cells expressing the transgene. Vector copy number was assessed using polymerase chain reaction technology designed to measure the number of lentiviral genomes present in each cell. As shown in Figure 1B, cells transduced with 7 μg / mL of P407 appeared to show an increase in vector copy number compared to cells transduced with 10 μg / mL.

[0428] Example 2. Effects of poloxamer P407 and P338 on lentiviral transduction of pluripotent hematopoietic stem cells. This example describes a series of experiments designed to compare the effects of poloxamer P407 and P338 on lentiviral transduction efficiency. Human CD34+ pluripotent hematopoietic stem cells were recruited into peripheral circulation and isolated from donor subjects. The cells were cultured in the presence of lentiviral vectors at infection multiplicity levels of 5–20. The lentiviral vectors used in these experiments contained transgenes encoding GFP, and cells expressing the transgene were detected by monitoring fluorescence at approximately 510 nm.

[0429] During the transduction procedure, cells were exposed to lentiviral vectors encoding GFP, either alone or in combination with poloxamer P407 or poloxamer 338 at varying concentrations. The ratio of cells expressing the transgene to the vector copy number in each cell population was then evaluated 12 days after the transduction procedure. The results of these studies are reported in Figures 2A–2C.

[0430] As shown in Figure 2A, both CD34+ stem cells were resistant to both poloxamer P407 and poloxamer P338. This is because neither poloxamer had a detrimental effect on cell viability at the concentrations tested. Surprisingly, as shown in Figures 2B and 2C, low concentrations of P407, such as less than 10 μg / mL, were found to enhance lentiviral transduction to a greater extent than poloxamer P338 at the same or higher concentrations.

[0431] Example 3. Poloxamer can be used in combination with protamine sulfate, cyclosporine H, and / or lithium carbonate to improve lentiviral transduction. This example describes a series of experiments designed to evaluate the effect of poloxamers, such as poloxamer P407, in combination with protamine sulfate on lentiviral transduction efficiency. Briefly, human CD34+ pluripotent hematopoietic stem cells were recruited into peripheral circulation and isolated from donor subjects according to the methods described in Examples 1 and 2 above. The cells were then transduced with a GFP-encoding vector in the presence of either poloxamer P407 (7 μg / mL) or poloxamer P338 (1 mg / mL), combined with protamine sulfate (3.5 μg / mL), respectively. Twelve days after transduction, the proportion of transgenes was assessed by flow cytometry detection of transgene expression, and the VCN was measured by droplet digital PCR detection of the integrated transgene sequence in genomic DNA. The results of these experiments are shown in Figures 3A and 3B.

[0432] As shown in Figures 3A and 3B, poloxamer P407 showed a remarkable improvement in lentiviral transduction efficiency in the presence of protamine sulfate compared to poloxamer P338 in the presence of protamine sulfate.

[0433] In addition to analyzing the effects of poloxamer in combination with protamine sulfate, a series of experiments were conducted using poloxamer P407 in combination with either protamine sulfate and either cyclosporine H or lithium carbonate (Li2CO3) to evaluate the effects of combinations of the three constituent iodines on lentiviral transduction efficiency. In these experiments, a clinical therapeutic lentiviral vector encoding a β-globin transgene was transduced into newly isolated CD34+ stem cells from peripherally mobilized blood in the presence of poloxamer P407 (7 μg / mL), which was a combination of protamine sulfate (3.5 μg / mL) and either cyclosporine H (8 μM) or Li2CO3 (2 mM). The results of these experiments are shown in Figures 4A and 4B, and in Table 3 below.

[0434] [Table 3]

[0435] As shown in Figures 4A, 4B, and Table 3, lentiviral transduction efficiency can be improved using poloxamers such as poloxamer P407 in the presence of additional agents such as protamine sulfate, cyclosporine H, and Li2CO3.

[0436] Other Embodiments Various modifications and variations of the Disclosure described herein will be apparent to those skilled in the art without departing from the scope and spirit of the Disclosure. Although the Disclosure is described in relation to specific embodiments, it should be understood that the claimed disclosure should not be excessively limited to such specific embodiments. In fact, various modifications of the form described for carrying out the Disclosure, which will be apparent to those skilled in the art, are intended to be within the scope of the Disclosure.

[0437] Other embodiments are found in the claims.

[0438] SEQUENCE LISTING <110> Orchard Therapeutics (Europe) Limited <120> COMPOSITIONS AND METHODS FOR MODIFYING EUKARYOTIC CELLS <130> PA26-091 <150> US 62 / 857,587 <151> 2019-06-05 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 441 <212> DNA <213> Homo sapiens <400> 1 atggtgcatc tgaccccgga agaaaaaagc gcggtgaccg cgctgtgggg caaagtgaac 60 gtggatgaag tgggcggcga agcgctggggc cgcctgctgg tggtgtatcc gtggacccag 120 cgcttttttg aaagctttgg cgatctgagc accccggatg cggtgatggg caacccgaaa 180 gtgaaagcgc atggcaaaaa agtgctggggc gcgtttagcg atggcctggc gcatctggat 240 aacctgaaag gcacctttgc gaccctgagc gaactgcatt gcgataact gcatgtgt 300 ccggaaaact tcgcctgct gggcaacgtg ctggtgtgcg tgctggcgca tcattttggc 360 aaagaattta ccccgccggt gcaggcggcg tatcagaag tggtggcggg cgtggcgac 420 gcgctggcgc aataatca t 441 <210> 2 <211> 147 <212> PRT <213> Homo sapiens <400> 2 Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 20 25 30 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp 35 40 45 Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His 50 55 60 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp 65 70 75 80 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys 85 90 95 Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val 100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln 115 120 125 Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His 130 135 140 Lys Tyr His 145

Claims

1. A method for transducing eukaryotic cells to express a transgene, wherein the method comprises contacting the cells with (i) a viral vector encoding the transgene and (ii) a poloxamer, wherein the concentration of the poloxamer is less than 10 μg / mL when it comes into contact with the cells.

2. A method for expressing a transgene in eukaryotic cells, the method comprising contacting the cells with (i) a viral vector encoding the transgene and (ii) a poloxamer, wherein the concentration of the poloxamer is less than 10 μg / mL when it comes into contact with the cells.

3. A method for promoting the translocation of a viral vector encoding a transgene into the nucleus of a eukaryotic cell, the method comprising contacting the cell with (i) the viral vector and (ii) poloxamer, wherein the concentration of the poloxamer is less than 10 μg / mL when in contact with the cell.

4. The method according to any one of claims 1 to 3, wherein the cells are mammalian cells.

5. The method according to claim 4, wherein the cells are human cells.

6. The method according to any one of claims 1 to 5, wherein the cells are pluripotent cells.

7. The method according to any one of claims 1 to 6, wherein the cells are CD34+ cells.

8. The method according to any one of claims 1 to 6, wherein the cells are embryonic stem cells.

9. The method according to any one of claims 1 to 6, wherein the cells are induced pluripotent stem cells.

10. The method according to any one of claims 1 to 6, wherein the cells are pluripotent hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs).

11. The method according to any one of claims 1 to 10, further comprising contacting the cells with a substance that reduces the activity and / or expression of protein kinase C (PKC).

12. The method according to claim 11, wherein the substance that reduces the activity and / or expression of PKC activates Akt signal transduction.

13. The method according to claim 11 or 12, wherein the substance that reduces the activity and / or expression of PKC is a PKC inhibitor or an agent that reduces the translation of a ribonucleic acid (RNA) transcript encoding PKC.

14. The method according to claim 13, wherein the substance that reduces the activity and / or expression of PKC is a PKC inhibitor.

15. The method according to claim 13, wherein the substance that reduces the activity and / or expression of PKC is an agent that reduces the translation of an RNA transcript encoding PKC.

16. The method according to claim 15, wherein the agent comprises nucleic acid.

17. The method according to claim 16, wherein the nucleic acid includes interfering RNA.

18. The method according to claim 17, wherein the interfering RNA is short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

19. The method according to claim 16, wherein the nucleic acid comprises an antisense oligonucleotide.

20. The method according to any one of claims 16 to 19, wherein the nucleic acid anneals to an endogenous RNA transcript encoding PKC.

21. The method according to claim 20, wherein the nucleic acid is at least 85% complementary to an endogenous RNA transcript encoding PKC.

22. The method according to claim 21, wherein the nucleic acid is at least 90%, 95%, 96%, 97%, 98%, 99% complementary, or 100% complementary, to an endogenous RNA transcript encoding PKC.

23. The PKC inhibitor is a compound represented by formula (I). 【Chemistry 1】 [In the formula, R １ This includes H, OH, optionally substituted alkoxides, optionally substituted acyloxys, optionally substituted aminos, optionally substituted alkylaminos, optionally substituted amides, halogens, and optionally substituted C. １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido, R ２ H, and C as arbitrarily substituted. １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkinyl, or optionally substituted acyl, R ａ and R ｂ are each independently H, optionally substituted C １－６ alkyl, optionally substituted C ２－６ alkenyl, or optionally substituted C ２－６ alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl, or R ａ and R ｂ combine with the atoms to which they are attached to form an optionally substituted and optionally fused heterocycloalkyl ring, R ｃ O, NR ｄ , or S, R ｄ H, and C as arbitrarily substituted. １－６ Alkyl, optionally substituted C ２－６ Alkenyl, or optionally substituted C ２－６ It is alkinyl, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. 【Chemistry 2】 This represents an arbitrary combination, n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 14, or a salt thereof.

24. The compound is a compound represented by formula (II). 【Transformation 3】 [In the formula, R １ is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amide, halogen, oxo, or thiocarbonyl. R ２ H, and C as arbitrarily substituted. １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkinyl, or optionally substituted acyl, R ａ and R ｂ These atoms bond with the atoms they bind to, forming optionally substituted and optionally condensed heterocycloalkyl rings. R ｃ is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 23, or a salt thereof.

25. The compound is a compound represented by formula (III). 【Chemistry 4】 [In the formula, R １ is H, OH, oxo, or thiocarbonyl, R ２ H, and C as arbitrarily substituted. １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkinyl, or optionally substituted acyl, Ring A is an arbitrarily substituted and arbitrarily condensed heterocycloalkyl ring, R ｃ is either O or S, Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 24, or a salt thereof.

26. The compound is a compound represented by formula (IV). 【Transformation 5】 [In the formula, R １ is H, OH, or oxo, Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring. R ｃ is either O or S, W is O, NH, or S. Each X is independently a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 25, or a salt thereof.

27. The compound is a compound represented by formula (V). 【Transformation 6】 [In the formula, R １ is H, OH, or oxo, R ｃ is either O or S, W is O, NH, or S. Each Z can independently be a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. p is either 0 or 1. The method according to claim 26, wherein the salt thereof.

28. The compound is a compound represented by formula (VI). 【Transformation 7】 [In the formula, R １ is H, OH, or oxo, Each Z can independently be a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. s is an integer between 0 and 8. The method according to claim 27, or a salt thereof.

29. The compound is a compound represented by formula (VII). 【Transformation 8】 [In the formula, R １ is H, OH, or oxo, R ２ is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R ３ This is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. The method according to claim 28, or a salt thereof.

30. The compound is a compound represented by formula (VIII). 【Chemistry 9】 [In the formula, R １ is H, OH, or oxo, R ２ is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy. R ３ This is H, OH, an optionally substituted alkoxy, an optionally substituted acyloxy, an optionally substituted amino, or an optionally substituted amide. The method according to claim 29, or a salt thereof.

31. The aforementioned compound is a compound represented by formula (IX). 【Chemistry 10】 [In the formula, each X independently represents a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, or optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 23, or a salt thereof.

32. The compound is a compound represented by formula (1). 【Chemistry 11】 The method according to claim 23, or a salt thereof.

33. The aforementioned compound is staurosporine, i.e., (2S,3R,4R,6R)-3-methoxy-2-methyl-4-(methylamino)-29-oxa-1,7,17-triazaoctactacyclo[12.12.2.12,6.07,28.08,13.015,19.020,27.021,26]nonacosa-8,10,12,14,19,21,23,25,27-nonaen-16-one, represented by formula (2). 【Chemistry 12】 The method according to claim 32, or a salt thereof.

34. The aforementioned compound is a compound represented by formula (X). 【Chemistry 13】 [In the formula, R １ is H, OH, or oxo, Each Z can independently be a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. t is an integer between 0 and 6. The method according to claim 23, or a salt thereof.

35. The compound is a compound represented by formula (XI). 【Chemistry 14】 [In the formula, R １ is H, OH, or oxo, R ４ [This is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy.] The method according to claim 34, or a salt thereof.

36. The aforementioned compound is a compound represented by formula (XII). 【Chemistry 15】 [In the formula, R １ is H, OH, or oxo, R ４ [This is H, OH, an optionally substituted alkoxy, or an optionally substituted acyloxy.] The method according to claim 35, or a salt thereof.

37. The aforementioned compound is a compound represented by formula (XIII). 【Chemistry 16】 [In the formula, each X independently represents a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, or optionally substituted heteroaryl Sulfanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. Each Y independently comprises a halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkylsulfonyl, optionally substituted arylsulfonyl, optionally substituted heteroarylsulfonyl, optionally substituted cycloalkylsulfonyl, optionally substituted heterocycloalkylsulfonyl, optionally substituted alkylsulfanyl, optionally substituted arylsulfanyl, optionally substituted heteroarylsulfonyl Phanyl, optionally substituted cycloalkylsulfanyl, optionally substituted heterocycloalkylsulfanyl, optionally substituted alkylsulfinyl, optionally substituted arylsulfinyl, optionally substituted heteroarylsulfinyl, optionally substituted cycloalkylsulfinyl, optionally substituted heterocycloalkylsulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and condensed aryl, optionally substituted and condensed heteroaryl, optionally substituted and condensed cycloalkyl, or optionally substituted and condensed heterocycloalkyl. n is an integer from 0 to 4, m is an integer between 0 and 4. The method according to claim 23, or a salt thereof.

38. The compound is a compound represented by formula (3). 【Chemistry 17】 The method according to claim 23, or a salt thereof.

39. The compound is a compound represented by formula (4). [Chemistry 18] The method according to claim 38, or a salt thereof.

40. The aforementioned compound, 【Chemistry 19】 The method according to claim 23, wherein a salt thereof is selected.

41. The compound is a compound represented by formula (XIV). 【Chemistry 20】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 23, or a salt thereof.

42. The aforementioned compound is a compound represented by formula (XV). 【Chemistry 21】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 41, or a salt thereof.

43. The aforementioned compound, 【Chemistry 22】 The method according to claim 42, wherein the compound is selected from a salt thereof.

44. The compound is a compound represented by formula (XVI). 【Chemistry 23】 [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] The method according to claim 23, or the salt thereof, or a quaternized variant thereof.

45. The compound is a compound represented by formula (XVII). 【Chemistry 24】 [In the formula, R is H, an optionally substituted alkyl, an optionally substituted acyl, an optionally substituted sulfonyl, an optionally substituted sulfinyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted cycloalkyl, and an optionally substituted heterocycloalkyl.] The method according to claim 44, or the salt thereof, or a quaternized variant thereof.

46. The aforementioned compound, 【Chemistry 25】 【change】 【change】 【change】 【change】 【change】 The method according to claim 45, wherein the compound is selected from a salt thereof.

47. The aforementioned compound is a compound represented by formula (XVIII). 【Chemistry 26】 [In the formula, R is H, OH, C] １－６ It is an alkoxy or oxo, R ２ teeth, 【Chemistry 27】 And optionally, the sugar moiety in the formula is derived from D-glucose, D-galactose, or D-mannose. R ３ H, OH, C １－６ Alkanoyloxy, C １－６ These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, or C １－６ It is substituted with an alkoxy, R ４ is OH, C １－６ Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C １－６ Alkylamino, D-C １－６ Alkylamino, C １－６ Alkoxycarbonylamino, C ２－２０ These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, C １－６ It is substituted with an alkoxy, R ５ is H or C １－６ It is alkyl, R ６ These are either free or aliphatic C ２－２２ It is a hydroxyl esterified with a carboxylic acid, or C １－６ Alkoxycarbonyloxy, C １－６ Alkyl sulfonyl oxy, free or aliphatic C ２－２２ amino acids acylated with carboxylic acids, C １－６ These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, or C １－６ It is substituted with an alkoxy, R ７ is either free or OH esterified with an aliphatic C ２－２２ carboxylic acid, C １－６ alkoxycarbonyloxy, C １－６ alkylsulfonyloxy, azide, either free or amino acylated with an aliphatic C ２－２２ carboxylic acid, C １－６ alkylamino, di-C １－６ alkylamino, C １－６ alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which is substituted within the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C １－６ alkyl, C １－６ alkoxy, or C １－６ alkoxycarbonyl. ] The method according to claim 23, or a salt thereof.

48. The aforementioned compound is a compound represented by formula (XIX). 【Chemistry 28】 [In the formula, R is H, OH, C] １－６ It is an alkoxy or oxo, R ２ is 【Chemistry 29】 And; R ３ H, OH, C １－６ Alkanoyloxy, C １－６ These are alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each containing a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, or C １－６ It is substituted with an alkoxy, R ４ is OH, C １－６ Alkanoyloxy, Benzoyloxy, Benzyloxy, Amino, C １－６ Alkylamino, D-C １－６ Alkylamino, C １－６ Alkoxycarbonylamino, C ２－２０ These are alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, C １－６ It is substituted with an alkoxy, R ５ is H or C １－６ It is alkyl, R ６ These are either free or aliphatic C ２－２２ It is a hydroxyl esterified with a carboxylic acid, or C １－６ Alkoxycarbonyloxy, C １－６ Alkyl sulfonyl oxy, free or aliphatic C ２－２２ amino acids acylated with carboxylic acids, C １－６ These are alkoxycarbonylamino, azide, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, or C １－６ It is substituted with an alkoxy, R ７ is either free or aliphatic C ２－２２ OH, C esterified with a carboxylic acid １－６ Alkoxycarbonyloxy, C １－６ Alkyl sulfonyl oxy, azide, free or aliphatic C ２－２２ amino acids acylated with carboxylic acids, C １－６ Alkylamino, D-C １－６ Alkylamino, C １－６ These are alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino, or benzyloxycarbonylamino, each of which contains a halogen, hydroxyl, trifluoromethyl, or C within the phenyl portion. １－６ Alkyl, C １－６ Alkoxy, or C １－６ It is substituted with an alkoxycarbonyl group. The method according to claim 47, or a salt thereof.

49. The aforementioned compounds are N-(1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(2-N-acetylmuramyl)staurosporine, N-(6-O-mesyl-1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(6-azido-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-amino-6-deoxy-2-N-acetylmuramyl)staurosporine N-(6-O-mesyl-2-N-acetylmuramyl) staurosporine, N-(2-N-acetyl-demethylmuramyl) staurosporine, N-(1-α-O-benzyl-2-N-acetylhomomuramyl) staurosporine, N-(1-α-O-benzyl-2-N-acetyl-L-homomuramyl) staurosporine, 1-α-anomer of N-(2-N-acetyl-L-homomuramyl) staurosporine, N-(1-α-O-benzyl-4,6-O-diacetyl-2-N-acetylmuramyl) staurosporine, N-(1-α-O-benzyl-4-O N-(1-deoxy-2-N-acetylmuramyl) staurosporine, N-(1-deoxy-2-N-acetylmuramyl) staurosporine, 1-α-anomer of N-(4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl) staurosporine, 1-α-anomer of N-(4,6-O-diacetyl-2-N-acetylmuramyl) staurosporine, N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl) staurosporine, N-(1-α,4,6-O-triacetyl-2-N-acetylmuramyl) staurosporine The method according to claim 48, selected from N-(1-deoxy-6-O-acetyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-truolsulfonyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-azido-2-N-acetylmuramyl)staurosporine, and N-(1-deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, or a salt thereof.

50. The aforementioned compound is a compound represented by formula (XX). 【Transformation 30】 [In the formula, Z １ is H or OH, Z ２ is H or OH, R １ is H, halogen, or optionally substituted alkyl, R ２ is H or halogen, R is an OH group or an optionally substituted alkoxy group. X is an optionally substituted alkyl or optionally substituted acyl, optionally in the formula, where X is CH ２ -NH-Serine, CO ２ CH ３ ,CH ２ NHCO ２ C ６ H ５ CONHC ６ H ５ , or CH ２ NHCO ２ CH3, in the formula, C ６ H ５ The method according to claim 23, wherein represents the phenyl portion, or a salt thereof.

51. The aforementioned compound is a compound represented by formula (XXI). 【Chemistry 31】 [In the formula, Z １ is H or OH, Z ２ is H or OH, R １ is H, halogen, or optionally substituted alkyl, R ２ is H or halogen, R is an OH group or an optionally substituted alkoxy group. X is an optionally substituted alkyl or optionally substituted acyl, optionally in the formula, where X is CH ２ -NH-Serine, CO ２ CH ３ ,CH ２ NHCO ２ C ６ H ５ CONHC ６ H ５ , or CH ２ NHCO ２ CH3, in the formula, C ６ H ５ The method according to claim 50, wherein represents the phenyl portion, or a salt thereof.

52. The compound is a compound represented by formula (XXII), (XXIII), (XXIV), or (XXV). 【Chemistry 32】 [In the formula, each R １ These are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R ２ These are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R ５ Each is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms, or 30 or fewer carbon atoms in each case. Each X is independently O, OH, and H, or a pair of hydrogen atoms. Each Q is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each n is an independent integer between 0 and 4. Each m is an independent integer between 0 and 4. The method according to claim 23, or a salt thereof.

53. The compound is a compound represented by formula (XXVI) or (XXVII). 【Transformation 33】 [In the formula, each R １ These are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R ２ These are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, monosubstituted or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R ５ Each is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms and 9 or fewer heteroatoms in each case, or 30 or fewer carbon atoms. Each R ８ Each is independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms in each case, and a heteroatom having 9 or fewer heteroatoms in each case. Each R ９ These are independently an optionally substituted acyl, an optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, carbonyl, carbonyldioxy, esterified carboxy, carbamoyl, N-1 or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-1 or N,N-disubstituted aminosulfonyl. Each R １０ Each is independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical having 29 or fewer carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical having 20 or fewer carbon atoms in each case, and a heteroatom having 9 or fewer heteroatoms in each case. Each X is independently O, OH, and H, or a pair of hydrogen atoms. Each n is an independent integer between 0 and 4. Each m is an independent integer between 0 and 4. Each n' is an independent integer between 0 and 4. Each m' is an independent integer between 0 and 4. The method according to claim 47, or a salt thereof.

54. The aforementioned compound is a compound represented by formula (XXVIII). 【Transformation 34】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 23, or a salt thereof.

55. The aforementioned compound is a compound represented by formula (XXIX). 【Chemistry 35】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 54, or a salt thereof.

56. The aforementioned compound is a compound represented by formula (XXX). 【Transformation 36】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 55, or a salt thereof.

57. The aforementioned compound is a compound represented by formula (XXXI). 【Chemistry 37】 [In the formula, R １ is H, or optionally substituted C １－６ It is alkyl, R ２ is an arbitrarily substituted C １－６ It is alkyl. The method according to claim 56, or a salt thereof.

58. The aforementioned compound, 【Transformation 38】 【change】 【change】 【change】 【change】 The method according to claim 23, selected from the following.

59. The method according to any one of claims 1 to 58, wherein the concentration of the poloxamer is about 10 ng / mL to about 9 μg / mL when in contact with the cells.

60. The method according to claim 59, wherein the concentration of the poloxamer is about 50 ng / mL to about 8 μg / mL when in contact with the cells.

61. The method according to claim 60, wherein the concentration of the poloxamer is about 100 ng / mL to about 7 μg / mL when in contact with the cells.

62. The method according to claim 61, wherein the concentration of the poloxamer is about 250 ng / mL to about 6 μg / mL when in contact with the cells.

63. The method according to claim 62, wherein the concentration of the poloxamer is about 500 ng / mL to about 5 μg / mL when in contact with the cells.

64. The method according to claim 63, wherein the concentration of the poloxamer is approximately 1 μg / mL when it comes into contact with the cells.

65. The method according to any one of claims 1 to 64, wherein the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,050 g / mol.

66. The method according to claim 65, wherein the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,250 g / mol.

67. The method according to claim 66, wherein the poloxamer has an average molar mass of polyoxypropylene subunits greater than 2,750 g / mol.

68. The method according to claim 67, wherein the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,250 g / mol.

69. The method according to claim 68, wherein the poloxamer has an average molar mass of polyoxypropylene subunits greater than 3,625 g / mol.

70. The method according to any one of claims 1 to 69, wherein the poloxamer has an average molar mass of polyoxypropylene subunits of about 2,050 g / mol to about 4,000 g / mol.

71. The method according to claim 70, wherein the poloxamer has an average molar mass of polyoxypropylene subunits ranging from about 2,250 g / mol to about 4,000 g / mol.

72. The method according to claim 71, wherein the poloxamer has an average molar mass of polyoxypropylene subunits of about 2,750 g / mol to about 4,000 g / mol.

73. The method according to claim 72, wherein the poloxamer has an average molar mass of polyoxypropylene subunits of about 3,250 g / mol to about 4,000 g / mol.

74. The method according to claim 73, wherein the poloxamer has an average molar mass of polyoxypropylene subunits ranging from about 3,625 g / mol to about 4,000 g / mol.

75. The method according to any one of claims 1 to 74, wherein the poloxamer has an average ethylene oxide content of more than 40% by mass.

76. The method according to claim 75, wherein the poloxamer has an average ethylene oxide content of more than 50% by mass.

77. The method according to claim 76, wherein the poloxamer has an average ethylene oxide content of more than 60% by mass.

78. The method according to claim 77, wherein the poloxamer has an average ethylene oxide content of more than 70% by mass.

79. The method according to any one of claims 1 to 78, wherein the poloxamer has an average molar mass of more than 10,000 g / mol.

80. The method according to claim 79, wherein the poloxamer has an average molar mass of more than 11,000 g / mol.

81. The method according to claim 80, wherein the poloxamer has an average molar mass of more than 12,000 g / mol.

82. The method according to claim 81, wherein the poloxamer has an average molar mass of more than 12,500 g / mol.

83. The method according to any one of claims 1 to 82, wherein the poloxamer has an average molar mass of about 10,000 g / mol to about 15,000 g / mol.

84. The method according to claim 83, wherein the poloxamer has an average molar mass of about 11,000 g / mol to about 15,000 g / mol.

85. The method according to claim 84, wherein the poloxamer has an average molar mass of about 11,500 g / mol to about 15,000 g / mol.

86. The method according to claim 85, wherein the poloxamer has an average molar mass of about 12,000 g / mol to about 15,000 g / mol.

87. The method according to claim 86, wherein the poloxamer has an average molar mass of about 12,500 g / mol to about 15,000 g / mol.

88. The method according to any one of claims 1 to 64, wherein the poloxamer is P407, P338, P288, or P188.

89. The method according to any one of claims 1 to 88, wherein the viral vector is selected from the group consisting of retroviridae family viruses, adeno-associated viruses, adenoviruses, parvoviruses, coronaviruses, rhabdoviruses, paramyxoviruses, picornaviruses, alphaviruses, herpesviruses, and poxviruses.

90. The method according to claim 89, wherein the viral vector is a retroviridae family viral vector.

91. The method according to claim 90, wherein the retroviridae family viral vector is a lentiviral vector.

92. The method according to claim 91, wherein the retroviridae family virus vector is an alpha retrovirus vector or a gamma retrovirus vector.

93. The method according to any one of claims 89 to 92, wherein the retroviridae family virus vector comprises a central polypurine band, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-autoinactivating LTR.

94. The method according to any one of claims 1 to 93, wherein the viral vector is a pseudotype viral vector.

95. The aforementioned pseudotyped viral vectors include vesicular stomatitis virus (VSV), RD114 virus, murine leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan encephalitis virus (VEE), human formy virus (HFV), walleye dermatosarcoma virus (WDSV), Semryki forest virus (SFV), rabies virus, avian leukemia virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), canine arthritis encephalitis virus (CAEV), and Synnomble. The method according to claim 94, comprising one or more envelope proteins derived from a virus selected from virus (SNV), cherry twisted leaf virus (ChTLV), monkey T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), bird carcinoma virus (MH2), avian encephalomyelitis virus (AEV), alpha mosaic virus (AMV), bird sarcoma virus CT10, and equine infectious anemia virus (EIAV).

96. The method according to claim 95, wherein the pseudotyped viral vector comprises a VSV-G envelope protein.

97. The method according to any one of claims 1 to 96, wherein the contact occurs ex vivo.

98. The method according to claim 97, wherein the cells are newly cultured or cryopreserved prior to contact.

99. The method according to any one of claims 1 to 98, wherein the cells further come into contact with cyclosporine.

100. The method according to claim 99, wherein the cyclosporine is cyclosporine A or cyclosporine H.

101. The method according to any one of claims 1 to 100, wherein the cells further come into contact with an activator of prostaglandin E receptor signaling.

102. The method according to claim 101, wherein the activator of the prostaglandin E receptor signaling pathway is prostaglandin E2.

103. The method according to any one of claims 1 to 102, wherein the cells further come into contact with a polycationic polymer.

104. The method according to claim 103, wherein the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or polyethylene glycol / poly-L-lysine block copolymer.

105. The method according to any one of claims 1 to 104, wherein the cells are spun by centrifugation while in contact with the viral vector.

106. The method according to claim 105, wherein the cells are spun with a centripetal force of about 300x g to about 1,200x g.

107. The method according to claim 105 or 106, wherein the cells are spun at a temperature of about 25°C.

108. A method for expressing an introduced gene in a subject, the method comprising administering to the subject a population of cells modified according to any one of claims 1 to 107, or their progeny.

109. A method for delivering genetically modified cells to a population of cells, the method comprising administering to the population of cells modified according to any one of claims 1 to 107, or their progeny.

110. A method for providing cell therapy to a subject in need of cell therapy, the method comprising administering to the subject a population of cells modified according to any one of claims 1 to 107, or their progeny.

111. The method according to any one of claims 108 to 110, wherein the cells are allogeneic with respect to the subject.

112. The method according to claim 111, wherein the cells are HLA-matched to the subject.

113. The method according to any one of claims 108 to 110, wherein the cells are of autologous origin to the subject.

114. The method according to any one of claims 108 to 113, wherein, prior to the contact, a population of progenitor cells is isolated from the subject or donor, the progenitor cells are expressed ex vivo, and the population of cells to be administered to the subject is obtained.

115. The method according to claim 114, wherein the progenitor cells are CD34+ HSCs, and the progenitor cells swell without losing the functional capacity of the HSCs.

116. The method according to claim 114 or 115, wherein one or more pluripotent cell recruiters are administered to the subject or donor before isolation of the progenitor cells derived from the subject or donor.

117. The method according to any one of claims 108 to 116, wherein, before administering the population of cells to the subject, one or more conditioning agents are administered to the subject, thereby excising a population of endogenous pluripotent cells within the subject.

118. The method according to any one of claims 108 to 116, wherein the method includes administering one or more conditioning agents to the subject before administering the population of cells to the subject, thereby excising a population of endogenous pluripotent cells from the subject.

119. The method according to claim 117 or 118, wherein the one or more conditioning agents are non-myeloablative conditioning agents.

120. The method according to any one of claims 108 to 119, wherein when the population of cells is administered to the subject, the administered cells or their progeny differentiate into one or more cell types selected from megakaryocytes, thrombus cells, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteolytic cells, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

121. The method according to any one of claims 108 to 120, wherein the subject is a mammal.

122. The method according to claim 121, wherein the subject is a human.

123. The method according to any one of claims 108 to 122, wherein the subject has been diagnosed with a deficiency of the endogenous protein encoded by the transgene.

124. The method according to claim 123, wherein the subject is diagnosed with one of the diseases listed in Table 2.

125. The method according to claim 123, wherein the subject has been diagnosed with beta-thalassemia.

126. The method according to any one of claims 1 to 124, wherein the introduced gene encodes a beta-globin protein.

127. The method according to claim 126, wherein the introduced gene comprises a nucleic acid having at least 85% sequence identity with the nucleic acid sequence of Sequence ID No.

1.

128. The method according to claim 127, wherein the introduced gene comprises a nucleic acid having at least 90% sequence identity with the nucleic acid sequence of Sequence ID No.

1.

129. The method according to claim 128, wherein the introduced gene comprises a nucleic acid having at least 95% sequence identity with the nucleic acid sequence of Sequence ID No.

1.

130. The method according to claim 129, wherein the introduced gene comprises a nucleic acid having the nucleic acid sequence of Sequence ID No.

1.

131. The method according to any one of claims 126 to 130, wherein the β-globin protein has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

2.

132. The method according to claim 131, wherein the β-globin protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

2.

133. The method according to claim 132, wherein the β-globin protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

2.

134. The method according to claim 133, wherein the β-globin protein has the amino acid sequence of SEQ ID NO:

2.

135. A composition comprising a mixture formed by the method described in any one of claims 1 to 107.

136. A cell culture medium comprising the composition described in claim 135.

137. A population of eukaryotic cells modified according to the method described in any one of claims 1 to 107.

138. A pharmaceutical composition comprising a population of eukaryotic cells as described in claim 137, wherein the pharmaceutical composition further comprises one or more excipients, diluents, or carriers.

139. The pharmaceutical composition according to claim 138, wherein the pharmaceutical composition is formulated for administration to a target.

140. The pharmaceutical composition according to claim 139, wherein the subject is a mammal.

141. The pharmaceutical composition according to claim 140, wherein the subject is a human.

142. The pharmaceutical composition according to any one of claims 139 to 141, wherein the pharmaceutical composition is formulated for intravenous injection into the subject.

143. A kit comprising the composition according to claim 135, or the cell culture medium according to claim 136.

144. The kit according to claim 143, further comprising an accompanying document containing instructions for transducing the cells.

145. A kit comprising a population of cells according to claim 137, or a pharmaceutical composition according to any one of claims 138 to 142.

146. The kit according to claim 145, further comprising a package insert instructing a user to administer the kit to a population of cells in accordance with the method described in any one of claims 108 to 134.

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