Compositions and methods for modifying eukaryotic cells

Abstract

Compositions and methods are provided for modifying eukaryotic cells, e.g., to express a transgene of interest and / or to generate a population of expanded cells ex vivo.SOLUTION: A method of transducing a population of eukaryotic cells, such as a population of pluripotent cells, to express a gene of interest by contacting the cells with a viral vector, e.g., a lentiviral vector, and a diblock copolymer, e.g., a diblock copolymer comprised of a hydrophilic region and a hydrophobic region. For example, the diblock copolymer can be composed of polyoxyethylene (PEO) subunits and polyoxypropylene (PRO) subunits. In addition, the compositions and methods of the invention can be used to promote the ex vivo expansion or survival of a population of pluripotent cells (e.g., CD34 + hematopoietic stem or progenitor cells), for example, by contacting the cells with a diblock copolymer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compositions and methods for modifying eukaryotic cells, for example, genetically modifying eukaryotic cells to express a transgene of interest, as well as promoting cell growth and survival.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on October 16, 2020, is named 51139-023WO2_Sequence_Listing_10_16_20_ST25 and is 2,292 bytes in size. [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 therapy represents a promising future pathway, allowing genes of interest to be functionally expressed in patients in a stable manner. Preparing cells for this form of therapy often requires that the cells be genetically modified to express the desired gene. Improved methods for enhancing the genetic modification of eukaryotic cells are needed. Summary of the Invention

[0004] The present 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, etc., can be genetically modified, for example, to promote expression of a transgene of interest in the cells. For example, the compositions and methods of the present disclosure can be used to contact a population of pluripotent cells, such as a population of HSCs and / or HPCs, with a viral vector encoding a transgene of interest to transduce the cells and express the desired gene. The viral vector can be a retrovirus, such as a lentivirus. To stimulate viral transduction of target cells, the cells can be contacted with the viral vector and a diblock copolymer comprising a hydrophilic component and a hydrophobic component. For example, the diblock copolymer can comprise a polyoxyethylene (PEO) subunit and a polyoxypropylene (PPO) subunit. The compositions and methods of the present disclosure offer a range of important medical benefits, as cells prepared according to the procedures described herein can be provided to a subject (e.g., a mammalian subject, such as a human patient) with a pathology associated with an endogenous defect in the gene of interest. By administering the modified cells to the subject, the subject may experience restoration of expression of the defective gene. While not limited by mechanism, this therapeutic approach represents a manner in which subjects with genetic diseases may be treated, as well as a methodology for alleviating the symptoms of the disease.

[0005] The compositions and methods of the present disclosure are based, in part, on the discovery that diblock copolymers comprising a hydrophilic component (e.g., a PEO subunit) and a hydrophobic component (e.g., a PPO subunit) can promote viral transduction upon contact with target cells. These diblock copolymers can be used to transduce target cells while still maintaining robust genetic modification.

[0006] In a first aspect, the disclosure features a method of transforming a eukaryotic cell to express a transgene by contacting the cell with (i) a vector encoding the transgene and (ii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0007] In a further aspect, the disclosure features a method of expressing a transgene in a eukaryotic cell by contacting the cell with (i) a vector encoding the transgene and (ii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0008] In a further aspect, the disclosure features a method of promoting the transfer of a viral vector encoding a transgene to the nucleus of a eukaryotic cell by contacting the cell with (i) a viral vector and (ii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0009] In some embodiments of any of the aforementioned three aspects of the disclosure, the method further comprises contacting the cell with an agent that decreases the activity and / or expression of protein kinase C (PKC).

[0010] In a further aspect, the disclosure features a method of transforming a eukaryotic cell to express a transgene by contacting the cell with (i) a vector encoding the transgene, (ii) a substance that reduces PKC activity and / or expression, and (iii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0011] In a further aspect, the disclosure features a method of expressing a transgene in a eukaryotic cell by contacting the cell with (i) a vector encoding the transgene, (ii) a substance that reduces PKC activity and / or expression, and (iii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0012] In a further aspect, the disclosure features a method of promoting the transfer of a viral vector encoding a transgene to the nucleus of a eukaryotic cell by contacting the cell with (i) a viral vector, (ii) a substance that reduces PKC activity and / or expression, and (iii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0013] In yet another aspect, the disclosure features a method of promoting actin depolymerization in a eukaryotic cell by contacting the cell with (i) a substance that reduces PKC activity and / or expression, and (ii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0014] In a further aspect, the disclosure features a method of inhibiting phosphorylation of cofilin in a eukaryotic cell by contacting the cell with (i) a substance that reduces the activity and / or expression of PKC and (ii) a diblock copolymer, e.g., a diblock copolymer comprising PEO and PPO subunits.

[0015] In a further aspect, the disclosure features a method for increasing the concentration of dephosphorylated cofilin in a eukaryotic cell, the method including contacting the cell with (i) a substance that reduces the activity and / or expression of PKC and (ii) a diblock copolymer, e.g., a diblock copolymer including PEO and PPO subunits.

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

[0017] In a further aspect, the disclosure features a method of promoting survival and / or proliferation of a eukaryotic cell, the method including contacting the cell with (i) a substance that reduces the activity and / or expression of PKC and (ii) a diblock copolymer, e.g., a diblock copolymer including PEO and PPO subunits.

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

[0019] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: X1-[PEO] m -L-[PPO] n -X2 wherein m and n are integers; L is absent or a chemical linker, and X1 and X2 each independently represent an optionally present chemical substituent.

[0020] In some embodiments, the diblock copolymer has the following structure: X1-[PEO] m -[PPO] n -X2 wherein m and n are integers, and X1 and X2 each independently represent an optionally present chemical substituent.

[0021] In some embodiments, X and X are each independently selected from the group consisting of: H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6In some embodiments, X and X are each independently selected from the group consisting of alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, and ureido. 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or optionally substituted C 1-6 For example, in some embodiments, X and X are each independently absent, H, OH, HN, HCO, ethyl-O, n-butyl-O, tert-butyl-O, n-butyl, or tert-butyl.

[0022] In some embodiments of any of the above aspects, the PEO subunits of the diblock copolymer have a number average molecular weight (Mn) of about 5,000 g / mol to about 25,000 g / mol (e.g., the PEO subunits of the diblock copolymer have a number average molecular weight (Mn) of about 5,500 g / mol, 6,000 g / mol, 6,500 g / mol, 7,000 g / mol, 7,500 g / mol, 8,000 g / mol, 8,5000 g / mol, 9,000 g / mol, 9,500 g / mol, 10,000 g / mol, 10,500 g / mol, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500g / mol, 14,000g / mol, 14,500g / mol, 15,000g / mol, 15,500g / mol, 16,000g / mol, 16 ,500g / mol, 17,000g / mol, 17,500g / mol, 18,000g / mol, 18,500g / mol, 19,000g / mol, 19,50 (The PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol to about 19,000 g / mol. In some specific embodiments, the PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol, 9,500 g / mol, 13,800 g / mol, 15,500 g / mol, 18,000 g / mol, or 19,000 g / mol.)

[0023] In some embodiments of any of the above aspects, the PPO subunits of the diblock copolymer have an Mn of about 2,000 g / mol to about 10,000 g / mol (e.g., the PPO subunits of the diblock copolymer can have an Mn of about 2,000 g / mol, 2,500 g / mol, 3,000 g / mol, 3,500 g / mol, 4,000 g / mol, 4,500 g / mol, 5,000 g / mol, 5,500 g / mol, 6,000 g / mol, 6,500 g / mol, 7,000 g / mol, 7,500 g / mol, 8,000 g / mol, 8,500 g / mol, 9,000 g / mol, 9,500 g / mol, or 10,000 g / mol). For example, in some embodiments, the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol to about 5,500 g / mol, and in some particular embodiments, the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol or 5,500 g / mol.

[0024] In some embodiments, the diblock copolymer comprises more than 40% by weight (e.g., 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%, 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%, 96%, 97%, 98 %, 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).

[0025] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 50% by weight (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%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0026] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 60% by weight (e.g., 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).

[0027] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 70% by weight (e.g., about 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).

[0028] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 40% to about 90% (e.g., 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%).

[0029] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 50% to about 85% (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%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%).

[0030] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 60% to about 80% (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%).

[0031] In some embodiments of any of the above aspects, the diblock copolymer has an Mn greater than about 8,000 g / mol. For example, the diblock copolymer has an Mn of greater than about 10,000 g / mol (e.g., the diblock copolymer has an Mn of 10,500, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500 g / mol, 14,000 g / mol, 14,500 g / mol, 15,000 g / mol, 15,500 g / mol, 16,000 g / mol, 16,500 g / mol, 17,000 g / mol, 17,500 g / mol, 18,000 g / mol, 18,500 g / mol, 19,000 g / mol, 19,500 g / mol, 20,000 g / mol, 21,000 g / mol, 22,000 g / mol, 23,000 g / mol, 24,000 g / mol, 25,000 g / mol, 26,000 g / mol, 27,000 g / mol, 28,000 g / mol, 29,000 g / mol, 30,000 g / mol, 31,000 g / mol, 32,000 g / mol, 33,000 g / mol, 34,000 g / mol, 35,000 g / mol, 36,000 g / mol, 37,000 g / mol, 38,000 g / mol, 39,000 g / mol, 39,500 g / mol, 40,000 g / mol, 41,000 g / mol, 42,000 g / mol, (having a Mn of greater than 0,000 g / mol, 20,500 g / mol, 21,000 g / mol, 21,500 g / mol, 22,000 g / mol, 22,500 g / mol, 23,000 g / mol, 23,500 g / mol, 24,000 g / mol, 24,500 g / mol, 25,000 g / mol, 25,000 g / mol, 26,000 g / mol, 26,500 g / mol, 27,000 g / mol, 27,500 g / mol, 28,000 g / mol, 28,500 g / mol, 29,000 g / mol, 29,500 g / mol, 30,000 g / mol, or more).

[0032] In some embodiments, the diblock copolymer has a Mn of about 10,000 g / mol to about 30,000 g / mol (e.g., the diblock copolymer has a Mn of about 10,500 g / mol, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500 g / mol, 14,000 g / mol, 14,500 g / mol, 15,000 g / mol, 15,500 g / mol, 16,000 g / mol, 16,500 g / mol, 17,000 g / mol, 17,500 g / mol, 18,000 g / mol, 18,500 g / mol, 19,000 g / mol, 20,000 g / mol, 21,000 g / mol, 22,000 g / mol, 23,000 g / mol, 24,000 g / mol, 25,000 g / mol, 26,000 g / mol, 27,000 g / mol, 28,000 g / mol, 29,000 g / mol, 30,000 g / mol, 31,000 g / mol, 32,000 g / mol, 33,000 g / mol, 34,000 g / mol, 35,000 g / mol, 36,000 g / mol, 37,000 g / mol, 38,000 g / mol, 39,000 g / mol, 40,000 g / mol, 41,000 g / mol, 42,000 g / mol, 43,000 g 20,000g / mol, 20,500g / mol, 21,000g / mol, 21,500g / mol, 22,000g / mol, 22,500g / mol, 23,000g / mol, 23,500g / mol, 24,000g / mol, 24,500g / mol, 25,000g / mol, 25,000g / mol, 26,000g / mol, 26,500g / mol, 27,000g / mol, 27,500g / mol, 28,000g / mol, 28,500g / mol, 29,000g / mol, 29,500g / mol, or 30,000g / mol). For example, in some embodiments, the diblock copolymer has an Mn of about 12,000 g / mol to about 25,000 g / mol (e.g., about 12,500 g / mol to about 23,500 g / mol). In some specific embodiments, the diblock copolymer has an Mn of about 12,500 g / mol, 13,000 g / mol, 17,300 g / mol, 19,000 g / mol, 22,500 g / mol, or 23,500 g / mol.

[0033] In some embodiments of any of the above aspects, the diblock copolymer has a polydispersity index (Mw / Mn) of about 1 to about 1.2 (e.g., the diblock copolymer has a polydispersity index of about 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, or 1.20). For example, in some embodiments, the diblock copolymer has a polydispersity index of about 1.06 to about 1.17. In some particular embodiments, the diblock copolymer has a polydispersity index of from about 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, or 1.17.

[0034] In some embodiments of the diblock copolymer, m is from about 100 to about 500. For example, in some embodiments, m is from about 200 to about 450, e.g., from about 205 to about 432. In some embodiments, m is from 162 to 486 (e.g., 323). In some embodiments, m is from 159 to 477 (e.g., 318). In some embodiments, m is from 108 to 324 (e.g., 216). In some embodiments, m is from 103 to 309 (e.g., 205). In some embodiments, m is from 148 to 444 (e.g., 295). In some embodiments, m is from 171 to 513 (e.g., 341). In some embodiments, m is from 142 to 426 (e.g., 284). In some embodiments, m is from 100 to 300 (e.g., 200). In some embodiments, m is from 113 to 339 (e.g., 225). In some embodiments, m is 109 to 327 (e.g., 217). In some embodiments, m is 115 to 345 (e.g., 230). In some embodiments, m is 120 to 360 (e.g., 240).

[0035] In some particular embodiments, m is 200, 205, 216, 217, 225, 230, 240, 284, 314, 318, 323, 352, 409, or 432.

[0036] In some embodiments of the diblock copolymer, n is from about 10 to about 200. For example, in some embodiments, n is from about 40 to about 100, e.g., from about 50 to about 95. In some embodiments, n is from 43 to 129 (e.g., 86). In some embodiments, n is from 27 to 81 (e.g., 53). In some embodiments, n is from 29 to 87 (e.g., 57). In some embodiments, n is from 28 to 84 (e.g., 55). In some embodiments, n is from 30 to 90 (e.g., 60). In some embodiments, n is from 33 to 99 (e.g., 65). In some embodiments, n is from 28 to 84 (e.g., 55).

[0037] In some specific embodiments, n is 50, 53, 55, 57, 60, 65, 70, 86, or 95.

[0038] In some embodiments of the diblock copolymer, m is from about 100 to about 500, and n is from about 10 to about 200, e.g., from about 40 to 100, or from 50 to about 95. For example, in some embodiments, m is from about 200 to about 450, e.g., from about 205 to about 432, and n is from about 10 to about 200, e.g., from about 40 to 100, or from 50 to about 95.

[0039] In some embodiments, m is 162 to 486 (e.g., 323) and n is 43 to 129 (e.g., 86). In some embodiments, m is 162 to 486 (e.g., 323) and n is 27 to 81 (e.g., 53). In some embodiments, m is 162 to 486 (e.g., 323) and n is 29 to 87 (e.g., 57). In some embodiments, m is 162 to 486 (e.g., 323) and n is 28 to 84 (e.g., 55). In some embodiments, m is 162 to 486 (e.g., 323) and n is 30 to 90 (e.g., 60). In some embodiments, m is 162 to 486 (e.g., 323) and n is 33 to 99 (e.g., 65). In some embodiments, m is 162 to 486 (eg, 323) and n is 28 to 84 (eg, 55).

[0040] In some embodiments, m is 159 to 477 (e.g., 318) and n is 43 to 129 (e.g., 86). In some embodiments, m is 159 to 477 (e.g., 318) and n is 27 to 81 (e.g., 53). In some embodiments, m is 159 to 477 (e.g., 318) and n is 29 to 87 (e.g., 57). In some embodiments, m is 159 to 477 (e.g., 318) and n is 28 to 84 (e.g., 55). In some embodiments, m is 159 to 477 (e.g., 318) and n is 30 to 90 (e.g., 60). In some embodiments, m is 159 to 477 (e.g., 318) and n is 33 to 99 (e.g., 65). In some embodiments, m is 159 to 477 (eg, 318) and n is 28 to 84 (eg, 55).

[0041] In some embodiments, m is 108 to 324 (e.g., 216) and n is 43 to 129 (e.g., 86). In some embodiments, m is 108 to 324 (e.g., 216) and n is 27 to 81 (e.g., 53). In some embodiments, m is 108 to 324 (e.g., 216) and n is 29 to 87 (e.g., 57). In some embodiments, m is 108 to 324 (e.g., 216) and n is 28 to 84 (e.g., 55). In some embodiments, m is 108 to 324 (e.g., 216) and n is 30 to 90 (e.g., 60). In some embodiments, m is 108 to 324 (e.g., 216) and n is 33 to 99 (e.g., 65). In some embodiments, m is between 108 and 324 (eg, 216) and n is between 28 and 84 (eg, 55).

[0042] In some embodiments, m is 103 to 309 (e.g., 205) and n is 43 to 129 (e.g., 86). In some embodiments, m is 103 to 309 (e.g., 205) and n is 27 to 81 (e.g., 53). In some embodiments, m is 103 to 309 (e.g., 205) and n is 29 to 87 (e.g., 57). In some embodiments, m is 103 to 309 (e.g., 205) and n is 28 to 84 (e.g., 55). In some embodiments, m is 103 to 309 (e.g., 205) and n is 30 to 90 (e.g., 60). In some embodiments, m is 103 to 309 (e.g., 205) and n is 33 to 99 (e.g., 65). In some embodiments, m is between 103 and 309 (eg, 205) and n is between 28 and 84 (eg, 55).

[0043] In some embodiments, m is 148 to 444 (e.g., 295) and n is 43 to 129 (e.g., 86). In some embodiments, m is 148 to 444 (e.g., 295) and n is 27 to 81 (e.g., 53). In some embodiments, m is 148 to 444 (e.g., 295) and n is 29 to 87 (e.g., 57). In some embodiments, m is 148 to 444 (e.g., 295) and n is 28 to 84 (e.g., 55). In some embodiments, m is 148 to 444 (e.g., 295) and n is 30 to 90 (e.g., 60). In some embodiments, m is 148 to 444 (e.g., 295) and n is 33 to 99 (e.g., 65). In some embodiments, m is 148 to 444 (eg, 295) and n is 28 to 84 (eg, 55).

[0044] In some embodiments, m is 171 to 513 (e.g., 341) and n is 43 to 129 (e.g., 86). In some embodiments, m is 171 to 513 (e.g., 341) and n is 27 to 81 (e.g., 53). In some embodiments, m is 171 to 513 (e.g., 341) and n is 29 to 87 (e.g., 57). In some embodiments, m is 171 to 513 (e.g., 341) and n is 28 to 84 (e.g., 55). In some embodiments, m is 171 to 513 (e.g., 341) and n is 30 to 90 (e.g., 60). In some embodiments, m is 171 to 513 (e.g., 341) and n is 33 to 99 (e.g., 65). In some embodiments, m is 171 to 513 (eg, 341) and n is 28 to 84 (eg, 55).

[0045] In some embodiments, m is 142 to 426 (e.g., 284) and n is 43 to 129 (e.g., 86). In some embodiments, m is 142 to 426 (e.g., 284) and n is 27 to 81 (e.g., 53). In some embodiments, m is 142 to 426 (e.g., 284) and n is 29 to 87 (e.g., 57). In some embodiments, m is 142 to 426 (e.g., 284) and n is 28 to 84 (e.g., 55). In some embodiments, m is 142 to 426 (e.g., 284) and n is 30 to 90 (e.g., 60). In some embodiments, m is 142 to 426 (e.g., 284) and n is 33 to 99 (e.g., 65). In some embodiments, m is 142 to 426 (eg, 284) and n is 28 to 84 (eg, 55).

[0046] In some embodiments, m is 100 to 300 (e.g., 200) and n is 43 to 129 (e.g., 86). In some embodiments, m is 100 to 300 (e.g., 200) and n is 27 to 81 (e.g., 53). In some embodiments, m is 100 to 300 (e.g., 200) and n is 29 to 87 (e.g., 57). In some embodiments, m is 100 to 300 (e.g., 200) and n is 28 to 84 (e.g., 55). In some embodiments, m is 100 to 300 (e.g., 200) and n is 30 to 90 (e.g., 60). In some embodiments, m is 100 to 300 (e.g., 200) and n is 33 to 99 (e.g., 65). In some embodiments, m is 100 to 300 (eg, 200) and n is 28 to 84 (eg, 55).

[0047] In some embodiments, m is 113 to 339 (e.g., 225) and n is 43 to 129 (e.g., 86). In some embodiments, m is 113 to 339 (e.g., 225) and n is 27 to 81 (e.g., 53). In some embodiments, m is 113 to 339 (e.g., 225) and n is 29 to 87 (e.g., 57). In some embodiments, m is 113 to 339 (e.g., 225) and n is 28 to 84 (e.g., 55). In some embodiments, m is 113 to 339 (e.g., 225) and n is 30 to 90 (e.g., 60). In some embodiments, m is 113 to 339 (e.g., 225) and n is 33 to 99 (e.g., 65). In some embodiments, m is 113 to 339 (eg, 225) and n is 28 to 84 (eg, 55).

[0048] In some embodiments, m is 109 to 327 (e.g., 217) and n is 43 to 129 (e.g., 86). In some embodiments, m is 109 to 327 (e.g., 217) and n is 27 to 81 (e.g., 53). In some embodiments, m is 109 to 327 (e.g., 217) and n is 29 to 87 (e.g., 57). In some embodiments, m is 109 to 327 (e.g., 217) and n is 28 to 84 (e.g., 55). In some embodiments, m is 109 to 327 (e.g., 217) and n is 30 to 90 (e.g., 60). In some embodiments, m is 109 to 327 (e.g., 217) and n is 33 to 99 (e.g., 65). In some embodiments, m is 109 to 327 (eg, 217) and n is 28 to 84 (eg, 55).

[0049] In some embodiments, m is 115 to 345 (e.g., 230) and n is 43 to 129 (e.g., 86). In some embodiments, m is 115 to 345 (e.g., 230) and n is 27 to 81 (e.g., 53). In some embodiments, m is 115 to 345 (e.g., 230) and n is 29 to 87 (e.g., 57). In some embodiments, m is 115 to 345 (e.g., 230) and n is 28 to 84 (e.g., 55). In some embodiments, m is 115 to 345 (e.g., 230) and n is 30 to 90 (e.g., 60). In some embodiments, m is 115 to 345 (e.g., 230) and n is 33 to 99 (e.g., 65). In some embodiments, m is 115 to 345 (eg, 230) and n is 28 to 84 (eg, 55).

[0050] In some embodiments, m is 120 to 360 (e.g., 240) and n is 43 to 129 (e.g., 86). In some embodiments, m is 120 to 360 (e.g., 240) and n is 27 to 81 (e.g., 53). In some embodiments, m is 120 to 360 (e.g., 240) and n is 29 to 87 (e.g., 57). In some embodiments, m is 120 to 360 (e.g., 240) and n is 28 to 84 (e.g., 55). In some embodiments, m is 120 to 360 (e.g., 240) and n is 30 to 90 (e.g., 60). In some embodiments, m is 120 to 360 (e.g., 240) and n is 33 to 99 (e.g., 65). In some embodiments, m is 120 to 360 (eg, 240) and n is 28 to 84 (eg, 55).

[0051] In some embodiments of the diblock polymer, m is 205, 216, 314, 352, 409, or 432, and n is 50, 60, 70, or 95. In some embodiments, m is 205 and n is 60. In some embodiments, m is 216 and n is 60. In some embodiments, m is 216 and n is 50. In some embodiments, m is 216 and n is 70. In some embodiments, m is 314 and n is 60. In some embodiments, m is 352 and n is 60. In some embodiments, m is 409 and n is 95. In some embodiments, m is 432 and n is 60.

[0052] Due to variations that occur during the synthesis of diblock copolymers containing PPO and PEO subunits, one skilled in the art will understand that the values ​​of m and n can vary, for example, by up to two-fold above and below the recited values. Thus, a value of n=50 represents a heterogeneous mixture of diblock copolymers where n can be from 25 to 100, e.g., 25 to 75, 26 to 74, 27 to 73, 28 to 72, 29 to 71, 30 to 70, 31 to 69, 32 to 68, 33 to 67, 34 to 66, 35 to 65, 36 to 64, 37 to 63, 38 to 62, 39 to 61, 40 to 60, 41 to 59, 42 to 58, 43 to 57, 44 to 56, 45 to 55, etc. Similarly, a value of n=60 represents a heterogeneous mixture of diblock copolymers where n can be from 30 to 120, e.g., 30 to 90. Similarly, a value of n=70 represents a heterogeneous mixture of diblock copolymers where n can be from 35 to 140, such as from 35 to 105.

[0053] In some embodiments of the diblock copolymer, the ratio of m:n is from about 1 to about 12. For example, in some embodiments, the ratio of m:n is from about 2 to about 8, e.g., from about 3.4 to about 7.2. In some embodiments, the ratio of m:n is from about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 ...8, 5.9, 5.1, 5.2, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.1, 5.2, 5.3, 5.4, 5.5 .4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 9.5, 9.6, 8.7, 8.8, 8.9, 9 or higher. In some particular embodiments, the ratio of m:n is about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, or more.

[0054] In some embodiments, the diblock copolymer has the following structure:

[0055] [ka]

[0056] In some embodiments, the diblock copolymer has a structure selected from the following species: In each structure, the recited values ​​of n and m should be understood to represent a heterogeneous mixture of diblock copolymers in which n and m can vary from up to two-fold below the recited value to two-fold above the recited value: [PEO] 323 -[PPO]86 -OH, HOCH2CH2-[PEO] 323 -[PPO] 86 -On-butyl, [PEO] 318 -[PPO] 53 -OH, HOCH2CH2-[PEO] 318 -[PPO] 53- On-butyl, [PEO] 216 -[PPO] 53 -OH, HOCH2CH2-[PEO] 216 -[PPO] 53 -On-butyl, [PEO] 205 -[PPO] 53 -OH, HOCH2CH2-[PEO] 205 -[PPO] 53 -On-butyl, [PEO] 295 -[PPO] 57 -OH, HOCH2CH2-[PEO] 295 -[PPO] 57 -On-butyl, [PEO] 341 -[PPO] 57 -OH, HOCH2CH2-[PEO] 341 -[PPO] 57 -On-butyl, [PEO] 284 -[PPO] 57 -OH, HOCH2CH2-[PEO] 284 -[PPO] 57 -On-butyl, [PEO] 200 -[PPO] 55 -OH, HOCH2CH2-[PEO] 200 -[PPO] 55 -On-butyl, [PEO] 205 -[PPO] 60 -OH, HOCH2CH2-[PEO] 205 -[PPO] 60 -On-butyl, [PEO] 217 -[PPO] 60 -OH, HOCH2CH2-[PEO] 217 -[PPO] 60 -On-butyl, [PEO] 230 -[PPO] 65 -OH, HOCH2CH2-[PEO] 230 -[PPO] 65 -On-butyl, [PEO] 240 -[PPO] 55 -OH, HOCH2CH2-[PEO] 240 -[PPO] 55 -On-butyl, [PEO] 205 -[PPO] 60 -OH, HOCH2CH2-[PEO] 205 -[PPO] 60 -On-butyl, [PEO] 314 -[PPO] 60 -OH, HOCH2CH2-[PEO] 314 -[PPO] 60 -On-butyl, [PEO] 352 -[PPO] 60 -OH, HOCH2CH2-[PEO] 352 -[PPO] 60 -On-butyl, [PEO] 409 -[PPO] 95 -OH, HOCH2CH2-[PEO] 409 -[PPO] 95 -On-butyl, [PEO] 432 -[PPO] 60 -OH, HOCH2CH2-[PEO] 432-[PPO] 60 -On-butyl, [PEO] 216 -[PPO] 60 -OH, [PEO] 216 -[PPO] 60 -n-butyl HO-[PEO] 216 -[PPO] 60 -n-butyl, HOCH2CH2-[PEO] 216 -[PPO] 50 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 50 -OH, HOCH2CH2-[PEO] 216 -[PPO] 60 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 60 -OH, HOCH2CH2-[PEO] 216 -[PPO] 70 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 70 -OH,

[0057] [ka] TIFF2026010071000003.tif241170

[0058] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 205 -[PPO] 60 -OH.

[0059] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO]205 -[PPO] 60 -On-butyl.

[0060] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 314 -[PPO] 60 -OH.

[0061] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 314 -[PPO] 60 -On-butyl.

[0062] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 352 -[PPO] 60 -OH.

[0063] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 352 -[PPO] 60 -On-butyl.

[0064] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 409 -[PPO] 95 -OH.

[0065] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 409 -[PPO] 95 -On-butyl.

[0066] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 432 -[PPO] 60 -OH.

[0067] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 432 -[PPO] 60 -On-butyl.

[0068] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 216 -[PPO] 60 -OH.

[0069] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: [PEO] 216 -[PPO] 60 -n-butyl.

[0070] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HO-[PEO] 216 -[PPO] 60 -n-butyl.

[0071] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 50 -On-butyl.

[0072] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 50 -OH.

[0073] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 60 -On-butyl.

[0074] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 60 -OH.

[0075] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 70 -On-butyl.

[0076] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure: HOCH2CH2-[PEO] 216 -[PPO] 70 -OH.

[0077] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0078] [ka]

[0079] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0080] [ka]

[0081] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0082] [ka]

[0083] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0084] [ka]

[0085] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0086] [ka]

[0087] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0088] [ka]

[0089] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0090] [ka]

[0091] In some embodiments according to any of the above aspects, the diblock copolymer has the following structure:

[0092] [ka]

[0093] In some embodiments, diblock copolymers that can be used in conjunction with the compositions and methods described herein include, for example, poly(ethylene glycol)-poly(γ-benzyl L-glutamate) PEG-PBLA, poly(ethylene glycol)-poly(D,L-lactic acid) PEG-PDLLA, poly(ethylene glycol)-poly(L-lactic acid) PEG-PLLA, poly(ethylene glycol)-poly(ε-caprolactone) PEG-PCL, poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) PEG-PLGA, poly(ethylene glycol)-poly(γ-benzyl L-glutamate) PEG-PBLG, poly(ethylene glycol)-poly(β-benzyl L-aspartate) PEG-PBLA, poly(ethylene glycol)-poly(α-benzyl carboxylate-ε-caprolactone) PEG-PBCL, and poly(ethylene glycol)-poly(δ-valerolactone) PEG-PVL. Such diblock copolymers include, for example, PEG 5000 -PCL 5000 , PEG 2000 -PCL 1400 , MPEG 5000 -PCL 5000 , MPEG 5000 -PCL 13000 , MPEG 5000 -PCL 24000 , PEG 2000 -PCL 2000 , MPEG 5000 -PCL 2500 , MPEG 5000 -PCL 5000 , MPEG 5000 -PCL 8500 , MPEG 5000 -PCL 24700 , MPEG 2000 -PCL 1200 , MPEG2000-PCL 2700 , MPEG 5000 -PCL 3800 , MPEG 5000 -PCL 18000、PEG 5000 -PCL 4000 、PEG 2000 -PCL 900 、PEG 1980 -PCL 1368 、PEG 1980 -PCL 2622 、PEG 1980 -PCL 17328 、PEG 2000 -PCL 2280 、PEG 5000 -PCL 5000 、PEG 5000 -PCL 24000 、PEG 5000 -PCL 5000 、PEG 5000 -PCL 24000 、PEG 5000 -PCL 4790 、PEG 5000 -PCL 10000 、MPEG 5333 -PCL 2638 、MPEG 5333 -PCL 4984 、MPEG 5333 -PCL 8034 、MPEG 5333 -PCL 9068 、MPEG 5000 -PCL 2166 、MPEG 2000 -PCL 1320 、MPEG 2000 -PCL 852 、MPEG 750 -PCL 464 、MPEG 750 -PCL 323 、MPEG 750 -PCL 197 、MPEG-PCL、PEG 5000 -PDLLA 4200 、PEG 5000 -PDLLA 45000 、MPEG 2000 -PDLLA 2000 、MPEG 2000 -PDLLA 1333 、MPEG 5000 -PDLLA 2143 、PEG 52000 -PDLLA 56000 、PEG 91000-PDLLA 56000 , PEG 4100 -PDLLA 1200 , PEG 6000 -PDLLA 3000 , PEG 5700 -PDLLA 5400 , PEG 6100 -PDLLA 7800 , PEG 5000 -PBCL 4700 , PEG 5000 -PBCL 4470 , PEG 12000 -PBLA 5000 , PEG 12000 -PBLA 3000 , PEG-PBLA, PEG 12000 -PBLA 5000 , MPEG 2000 -PVL 1000 , MPEG 2000 -PVL 2000 , MPEG 5000 -PVL 2600 , and MPEG 5000 -PVL 4900 These diblock copolymers are described, for example, in Hussein et al. Materials 11:1-26, 2018, the disclosure of which is incorporated herein in its entirety.

[0094] In some embodiments of any of the above aspects of the present disclosure, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is a pluripotent cell. The cell may be a CD34+ cell. In some embodiments, the cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the cell is an HSC or HPC.

[0095] In some embodiments, an agent that decreases PKC activity and / or expression activates Akt signaling. An agent that decreases PKC activity and / or expression can be a PKC inhibitor or an agent that decreases translation of a ribonucleic acid (RNA) transcript encoding PKC (i.e., a messenger RNA transcript encoding PKC).

[0096] In some embodiments, the substance that reduces the activity and / or expression of PKC is an agent that reduces the translation of an RNA transcript encoding PKC. In some embodiments, the agent comprises a nucleic acid. The nucleic acid can comprise an interfering RNA, such as a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the nucleic acid comprises an antisense oligonucleotide.

[0097] In some embodiments, the nucleic acid anneals to an endogenous RNA transcript encoding a PKC. The nucleic acid can be, for example, at least 85% complementary (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a region of an endogenous RNA transcript encoding a PKC.

[0098] In some embodiments, the substance that reduces the activity and / or expression of PKC is a PKC inhibitor. The PKC inhibitor may be staurosporine or a variant thereof. For example, the PKC inhibitor is a compound represented by formula (I):

[0099] [ka] wherein R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, 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, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6alkynyl, or optionally substituted acyl; R a and R b each independently represents 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 a and R b are joined together 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 is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 is alkynyl, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; TIFF2026010071000013.tif5132 represents an arbitrarily present bond, n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0100] In some embodiments, the PKC inhibitor is a compound of formula (II):

[0101] [ka] wherein R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, oxo, or thiocarbonyl; R2 is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted acyl; R a and R b are joined together with the atoms to which they are attached to form an optionally substituted and optionally fused heterocycloalkyl ring; R c is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0102] In some embodiments, the PKC inhibitor is a compound of formula (III):

[0103] [ka] wherein R1 is H, OH, oxo, or thiocarbonyl; R2 is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted acyl; Ring A is an optionally substituted and optionally fused heterocycloalkyl ring; R c is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0104] In some embodiments, the PKC inhibitor is a compound of formula (IV):

[0105] [ka] wherein R1 is H, OH, or oxo; Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring; R c is O or S, W is O, NH, or S; Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0106] In some embodiments, the PKC inhibitor is a compound of formula (V):

[0107] [ka] wherein R1 is H, OH, or oxo; R c is O or S, W is O, NH, or S; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; p is 0 or 1. or a salt thereof.

[0108] In some embodiments, the PKC inhibitor is a compound of formula (VI):

[0109] [ka] wherein R1 is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; s is an integer from 0 to 8. or a salt thereof.

[0110] In some embodiments, the PKC inhibitor is a compound of formula (VII):

[0111] [ka] wherein R1 is H, OH, or oxo; R2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; R3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

[0112] In some embodiments, the PKC inhibitor is a compound of formula (VIII):

[0113] [ka] wherein R1 is H, OH, or oxo; R2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; R3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

[0114] In some embodiments, the PKC inhibitor is a compound of formula (IX):

[0115] [ka] wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0116] In some embodiments, the PKC inhibitor is a compound of formula (1):

[0117] [ka] or a salt thereof.

[0118] 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-triazaoctacyclo[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).

[0119] [ka] or a salt thereof.

[0120] In some embodiments, the PKC inhibitor is a compound of formula (X):

[0121] [ka] wherein R1 is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; t is an integer from 0 to 6. or a salt thereof.

[0122] In some embodiments, the PKC inhibitor is a compound of formula (XI):

[0123] [ka] wherein R1 is H, OH, or oxo; R4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

[0124] In some embodiments, the PKC inhibitor is a compound of formula (XII):

[0125] [ka] wherein R1 is H, OH, or oxo; R4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

[0126] In some embodiments, the PKC inhibitor is a compound of formula (XIII):

[0127] [ka] wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0128] In some embodiments, the PKC inhibitor is a compound of formula (3):

[0129] [ka] or a salt thereof.

[0130] In some embodiments, the PKC inhibitor is a compound of formula (4):

[0131] [ka] or a salt thereof.

[0132] In some embodiments, the PKC inhibitor is a compound of formula (128):

[0133] [ka] or a salt thereof. This compound is also known as K252a.

[0134] In some embodiments, the PKC inhibitor is

[0135] [ka] or a salt thereof.

[0136] In some embodiments, the PKC inhibitor is a compound of formula (XIV):

[0137] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0138] In some embodiments, the PKC inhibitor is a compound represented by formula (XV):

[0139] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0140] In some embodiments, the PKC inhibitor is

[0141] [ka] or a salt thereof.

[0142] In some embodiments, the PKC inhibitor is a compound of formula (XVI):

[0143] [ka] wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

[0144] In some embodiments, the PKC inhibitor is a compound of formula (XVII):

[0145] [ka] wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

[0146] In some embodiments, the PKC inhibitor is

[0147] [ka] TIFF2026010071000038.tif204170TIFF2026010071000039.tif188170TIFF2026010071000040.tif241170TIFF2026010071000041.tif194170TIFF2026010071000042.tif194170TIFF2026010071000043.tif71170, or a salt thereof.

[0148] In some embodiments, the PKC inhibitor is a compound of formula (XVIII):

[0149] [ka] [Wherein R is H, OH, C 1-6 alkoxy, or oxo; R2 is

[0150] [ka] and optionally, the sugar moiety is derived from D-glucose, D-galactose, or D-mannose; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with 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 2-20alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which optionally contains, within the phenyl moiety, halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 is substituted with alkoxy, R5 is H or C 1-6 is alkyl, R6 is free or aliphatic C 2-22 hydroxyl esterified with a carboxylic acid or C 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, free or aliphatic C 2-22 Amino acylated with carboxylic acid, C 1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with alkoxy, R7 is free or aliphatic C 2-22 OH, C esterified with carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, azido, free or aliphatic C 2-22 Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6substituted by alkoxycarbonyl. or a salt thereof.

[0151] In some embodiments, the PKC inhibitor is a compound of formula (XIX):

[0152] [ka] [Wherein R is H, OH, C 1-6 alkoxy, or oxo; R2 is

[0153] [ka] and; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with 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 alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which may be substituted within the phenyl moiety with halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 is substituted with alkoxy, R5 is H or C 1-6 is alkyl, R6 is free or aliphatic C 2-22 hydroxyl esterified with a carboxylic acid or C 1-6 Alkoxycarbonyloxy, C1-6 Alkyl sulfonyloxy, free or aliphatic C 2-22 Amino acylated with carboxylic acid, C 1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with alkoxy, R7 is free or aliphatic C 2-22 OH, C esterified with carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, azido, free or aliphatic C 2-22 Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 substituted by alkoxycarbonyl. or a salt thereof.

[0154] 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-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-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, 1-α-anomer of N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4,6-O-triacetyl and N-(1-deoxy-6-O-methyl-2-N-acetylmuramyl)staurosporine, ... and N-(1-deoxy-6-O-methyl-2-N-acetylmuramyl)staurosporine, or a salt thereof.

[0155] In some embodiments, the PKC inhibitor is a compound of formula (XX):

[0156] [ka] wherein Z1 is H or OH; Z2 is H or OH; R1 is H, halogen, or optionally substituted alkyl; R2 is H or halogen; R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents a phenyl moiety, or a salt thereof.

[0157] In some embodiments, the PKC inhibitor is a compound of formula (XXI):

[0158] [ka] wherein Z1 is H or OH; Z2 is H or OH; R1 is H, halogen, or optionally substituted alkyl; R2 is H or halogen; R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH2-NH-serine, CO2CH3, CH2NHCO2C6H5, CONHC6H5, or CH2NHCO2CH3, where C6H5 represents a phenyl moiety, or a salt thereof.

[0159] In some embodiments, the PKC inhibitor is a compound represented by formula (XXII), (XXIII), (XXIV), or (XXV):

[0160] [ka] wherein each R1 is independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R2 is independently 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms and 9 or fewer heteroatoms, or 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 di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each n is independently an integer from 0 to 4; Each m is independently an integer from 0 to 4. or a salt thereof.

[0161] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII):

[0162] [ka] wherein each R1 is independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R2 is independently 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms and 9 or fewer heteroatoms, 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, in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms, and 9 or fewer heteroatoms; each R9 independently is 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R 10 are independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms, and in each case having 9 or fewer heteroatoms; 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.

[0163] In some embodiments, the PKC inhibitor is a compound of formula (XXVIII):

[0164] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0165] In some embodiments, the PKC inhibitor is a compound of formula (XXIX):

[0166] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0167] In some embodiments, the PKC inhibitor is a compound of formula (XXX):

[0168] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0169] In some embodiments, the PKC inhibitor is a compound of formula (XXXI):

[0170] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0171] In some embodiments, the PKC inhibitor is

[0172] [ka] The compound is selected from TIFF2026010071000057.tif231170TIFF2026010071000058.tif231170TIFF2026010071000059.tif236170TIFF2026010071000060.tif65170.

[0173] In some embodiments, the cells are further contacted with stauprimide, for example, as described in Caravatti et al. Bioorg. Medic. Chem. Letters 4:199-404, 1994, the entire disclosure of which is incorporated herein by reference.

[0174] In some embodiments of any of the above aspects or embodiments of the present disclosure, the concentration of the substance that reduces PKC activity and / or expression, when contacted with a cell, is about 100 μM to about 1 mM (e.g., 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, 230 μM, 235 μM, 240 μM, 245 μM, 250 μM, 255 μM, 260 μM, 265 μM, 270 μM, 275 μM, 280 μM, 285 μM, 290 μM, 300 μM, 305 μM, 310 μM, 315 μM, 320 μM, 330 μM, 340 μM, 345 μM, 350 μM, 355 μM, 360 μM, 365 μM, 370 μM, 375 μM, 380 μM, 385 μM, 390 μM, 400 μM, 410 μM, 420 μM, 430 μM, 440 μM, 450 μM, 460 μM, 470 μM, 480 μM, 490 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,810 μM, 815 μM, 820 μM, 825 μM, 830 μM, 835 μM, 840 μM, 845 μM, 850 μM, 855 μM, 860 μM, 865 μM, 870 μM, 875 μM, 880 μM, 885 μM, 890 μM, 895 μM, 900 μM, 905 μM, 910 μM, 915 μM, 920 μM, 925 μM, 930 μM, 935 μM, 940 μM, 945 μM, 950 μM, 955 μM, 960 μM, 965 μM, 970 μM, 975 μM, 980 μM, 985 μM, 990 μM, 995 μM, or 1 mM). In some embodiments, the concentration of the substance that reduces PKC activity and / or expression when contacted with a cell is about 200 μM to about 600 μM (e.g., about 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 In some embodiments, the concentration of the agent that reduces PKC activity and / or expression is about 400 μM when contacted with the cells.

[0175] In some embodiments of any of the above aspects, the method further comprises contacting the cell with a histone deacetylase (HDAC) inhibitor.

[0176] In some embodiments, the HDAC inhibitor is

[0177] [ka] Selected from TIFF2026010071000062.tif60170.

[0178] In some embodiments, the HDAC inhibitor is

[0179] [ka] is.

[0180] The cells can be contacted with the diblock copolymer and the HDAC inhibitor simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting them with the HDAC inhibitor. In some embodiments, the cells are contacted with the HDAC inhibitor before contacting them with the diblock copolymer.

[0181] 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 virus vector, such as a lentivirus vector, an alpharetrovirus vector, or a gammaretrovirus vector. In some embodiments, the Retroviridae family virus vector comprises a central polypurine tract, a woodchuck hepatitis virus posttranscriptional 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'-self-inactivating LTR.

[0182] In some embodiments, the viral vector is a pseudotyped viral vector containing a viral genome from one species of virus and one or more viral capsid or envelope proteins from a different species of virus, such as vesicular stomatitis virus (VSV), RD114 virus, murine leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan equine encephalitis virus (VEE), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), Semliki Forest virus (SFV), rabies virus, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), caprine arthritis and encephalitis virus (CAEV), and Sin Nombre virus. The composition may contain one or more viral envelope proteins derived from a virus selected from the group consisting of simian neoplasia virus (SNV), cherry twisted leaf virus (ChTLV), simian T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), avian carcinoma virus (MH2), avian encephalomyelitis virus (AEV), alpha mosaic virus (AMV), avian sarcoma virus CT10, and equine infectious anemia virus (EIAV).

[0183] In some embodiments, contacting the cells with one or more agents described above or herein occurs ex vivo. The cells may be freshly cultured prior to contacting, or may be cryopreserved and thawed prior to contacting.

[0184] In some embodiments, before contacting the cells with the diblock copolymer, the cells are first contacted with a substance that reduces PKC activity and / or expression. For example, the cells can be first contacted with the substance that reduces PKC activity and / or expression about 30 minutes to about 6 hours before contacting the cells with the diblock copolymer (e.g., about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours before contacting the cells with the diblock copolymer). In some embodiments, the cells are first contacted with the substance that reduces PKC activity and / or expression about 1 hour to about 3 hours before contacting the cells with the diblock copolymer (e.g., about 1 hour, 2 hours, or 3 hours before contacting the cells with the diblock copolymer). In some embodiments, the cells are first contacted with the substance that reduces PKC activity and / or expression about 2 hours before contacting the cells with the diblock copolymer.

[0185] In some embodiments, when the cells are first contacted with a substance that reduces PKC activity and / or expression before contacting the cells with the diblock copolymer, the cells are washed to remove the substance that reduces PKC activity and / or expression before contacting the cells with the diblock copolymer.

[0186] In some embodiments, the cells are contacted with the substance that reduces PKC activity and / or expression and the diblock copolymer simultaneously. For example, the cells can be contacted with the substance that reduces PKC activity and / or expression, the diblock copolymer, and the viral vector simultaneously.

[0187] In some embodiments, cells are contacted with the viral vector after exposure to a substance that reduces PKC activity and / or expression. In these examples, cells can be contacted with the viral vector and the diblock copolymer simultaneously. Alternatively, cells can be contacted with the diblock copolymer before contacting them with the viral vector. In some embodiments, cells are contacted with the viral vector before contacting them with the diblock copolymer.

[0188] Thus, in some embodiments of the present disclosure, cells are first contacted with an agent that reduces PKC activity and / or expression, then contacted with a diblock copolymer, and then contacted with a viral vector. In some embodiments, cells are first contacted with an agent that reduces PKC activity and / or expression, then contacted with a viral vector, and then contacted with a diblock copolymer.

[0189] In some embodiments, the cells are further contacted with a cyclosporine, such as cyclosporine A (CsA) or cyclosporine H (CsH). The cells can be contacted with the diblock copolymer and the cyclosporine simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the cyclosporine. In some embodiments, the cells are contacted with the cyclosporine before contacting with the diblock copolymer.

[0190] In some embodiments, the cyclosporine is CsH.

[0191] In some embodiments, the concentration of cyclosporine when contacted with cells is about 1 μM to about 10 μM (e.g., about 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, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 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, 3.9 μM, 4.0 μM, 4.1 μ 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 , 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, the cyclosporine is CsA and the concentration of the cyclosporine is about 6 μM when contacted with the cells. In some embodiments, the cyclosporine is CsH and the concentration of the cyclosporine is about 8 μM when contacted with the cells.

[0192] In some embodiments, the cells are further contacted with an activator of prostaglandin E receptor signaling. The cells can be contacted with the diblock copolymer and the activator of prostaglandin E receptor signaling simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the activator of prostaglandin E receptor signaling. In some embodiments, the cells are contacted with the activator of prostaglandin E receptor signaling before contacting with the diblock copolymer.

[0193] 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, the disclosures of each of which are incorporated herein by reference as they relate to prostaglandin E receptor signaling activators.

[0194] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of small organic molecules, prostaglandins, Wnt pathway agonists, cAMP / PI3K / AKT pathway agonists, Ca 2+ A small molecule such as a second messenger pathway agonist, a nitric oxide (NO) / angiotensin signaling agonist, or another compound known to stimulate the prostaglandin signaling pathway, such as a compound selected from mebeverine, flurandrenolide, atenolol, pindolol, gaboxadol, kynurenic acid, hydralazine, thiabendazole, bicuculline, vesamicol, peruvoside, imipramine, chlorpropamide, 1,5-pentamethylenetetrazole, 4-aminopyridine, diazoxide, benfotiamine, 12-methoxydodecenoic acid, N-formyl-Met-Leu-Phe, gallamine, IAA94, chlorotrianisene, and / or a derivative of any of these compounds.

[0195] In some embodiments, an activator of prostaglandin E receptor signaling is a naturally occurring or synthetic chemical molecule or polypeptide that binds to and / or interacts with prostaglandin E receptor and typically activates or increases one or more downstream signaling pathways associated with the prostaglandin E receptor.

[0196] 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.

[0197] In some embodiments, the activator of prostaglandin E receptor signaling is PGE2.

[0198] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of 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 butaprost), and the like. and 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).

[0199] 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 fatty acids containing 20 carbon atoms and including a 5-carbon ring, as described herein and known in the art. Examples of PGE2 "analogs" or "derivatives" include 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester, II-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-ketofluprostenol, 5-trans These include, but are not limited to, PGE2, 17-phenyl-omega-triol PGE2, PGE2 serinolamide, 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.

[0200] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin analog or derivative having a structure similar to PGE2 substituted with a halogen at position 9 (see, e.g., WO2001 / 12596, which is incorporated by reference herein in its entirety), as well as a 2-decarboxy-2-phosphinico prostaglandin derivative, such as those described in US2006 / 0247214, which is incorporated by reference herein in its entirety.

[0201] In some embodiments, the prostaglandin E receptor signaling activator is a non-PGE2-based ligand. In some embodiments, the prostaglandin E receptor signaling activator is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Further examples of non-PGE2-based EP2 agonists include carbazoles and fluorenes, as disclosed in WO2007 / 071456, the disclosure of which is incorporated herein by reference. 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 for its disclosure of such agonists.

[0202] 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 stimulating prostaglandin E 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 biologically active fragments thereof. Suitable GSK3 inhibitors for use as agents stimulating prostaglandin E receptor signaling pathway bind to GSK3a or GSK3 and reduce the activity of GSK3a or GSK3. Examples of GSK3 inhibitors include BIO(6-bromoindirubin-3), as exemplified in U.S. Pat. Nos. 6,057,117 and 6,608,063, as well as U.S. Pat. Nos. 2004 / 0092535 and 2004 / 0209878. ’ -oxime), LiCl, LiCO, or other GSK-3 inhibitors, as well as the ATP-competitive and selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also known as CT-99021 / CHIR-99021 and CT-98023 / CHIR-98023, respectively) (Chiron Corporation, Emeryville, CA). The structure of CHIR-99021 is

[0203] [ka] or a salt thereof.

[0204] The structure of CHIR-98023 is

[0205] [ka] or a salt thereof.

[0206] In some embodiments, the method further comprises contacting the cell with a GSK3 inhibitor.

[0207] In some embodiments, the GSK3 inhibitor is CHIR-99021.

[0208] In some embodiments, the GSK3 inhibitor is Li2CO3.

[0209] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the cAMP / P13K / AKT second messenger pathway, such as an agent selected from the group consisting of dibutyryl cAMP (DBcAMP), phorbol esters, forskolin, sclarerin, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4-dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, iloprost, pituitary adenylate cyclase-activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), and derivatives of these agents.

[0210] In some embodiments, the activator of prostaglandin E receptor signaling is a Ca agonist, such as an agent selected from the group consisting of Bapta-AM, fendiline, nicardipine, and derivatives of these agents. 2+ Agents that increase signaling through second messenger pathways.

[0211] 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.

[0212] In some embodiments, the cells are further contacted with a polycationic polymer. The cells can be contacted with the diblock copolymer and the polycationic polymer simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the polycationic polymer. In some embodiments, the cells are contacted with the polycationic polymer before contacting with the diblock copolymer.

[0213] In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or a polyethylene glycol / poly-L-lysine block copolymer.

[0214] In some embodiments, the polycationic polymer is protamine sulfate.

[0215] In some embodiments, the cells are further contacted with a combination of agents in addition to the diblock copolymer. For example, in some embodiments, the cells are contacted with Li2CO3 and protamine sulfate. In some embodiments, the cells are contacted with CHIR-99021 and protamine sulfate. In some embodiments, the cells are contacted with cyclosporine H and protamine sulfate.

[0216] In some embodiments, the cells are further contacted with an expansion agent during the transduction procedure. The cells may be, for example, pluripotent hematopoietic stem cells, and the expansion agent may be a pluripotent hematopoietic stem cell expansion agent, such as those known in the art or described herein.

[0217] In some embodiments, during the transduction procedure, the cells are further contacted with an agent that inhibits mTor signaling, which can be, for example, rapamycin, among other inhibitors of mTor signaling.

[0218] In some embodiments of the methods described herein, during the transduction procedure, the cells are further contacted with a transduction-enhancing agent, e.g., in addition to the diblock copolymer. Additional transduction-enhancing agents include, e.g., tacrolimus and vector fusin. In some embodiments, the additional transduction-enhancing agent is tacrolimus. In some embodiments, the additional transduction-enhancing agent is vector fusin.

[0219] In some embodiments, the cells are incubated with the viral vector (e.g., in combination with one or more agents described above) for a period of about 6 hours to about 48 hours (e.g., about 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, or 48 hours). In some embodiments, the cells are incubated with the viral vector (e.g., in combination with one or more agents described above) for a period of about 12 hours to about 24 hours (e.g., about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours). In some embodiments, the cells are incubated with the viral vector (e.g., in combination with one or more agents described above) for a period of about 16 hours to about 22 hours (e.g., about 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, or 22 hours). In some embodiments, the cells are incubated with the viral vector (e.g., in combination with one or more agents described above) for a period of about 17 hours to about 19 hours (e.g., about 17 hours, 18 hours, or 19 hours). In some embodiments, the cells are incubated with the viral vector (e.g., in combination with one or more agents described above) for a period of about 18 hours.

[0220] In some embodiments, cells are spun (e.g., by centrifugation) (i.e., "centrifuged") while in contact with the viral vector (e.g., in combination with one or more agents described above). This process, referred to herein as "spinoculation," can occur with a centripetal force of, for example, about 200 x g to about 2,000 x g. In some embodiments, cells are spun at a centripetal force of about 300 x g to about 1,200 x g while in contact with the viral vector (e.g., in combination with one or more agents described above). For example, cells can be spun at a centripetal force of about 300 x g, 400 x g, 500 x g, 600 x g, 700 x g, 800 x g, 900 x g, 1,000 x g, 1,100 x g, or 1,200 x g while in contact with the viral vector (e.g., in combination with one or more agents described above). In some embodiments, the cells are spun for about 10 minutes to about 3 hours (e.g., 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 more). In some embodiments, the cells are spun at room temperature, e.g., at a temperature of about 25°C.

[0221] In a further aspect, the disclosure features a method of expressing a transgene in a subject (e.g., a mammalian subject such as a human) by administering to the subject a population of cells, or their progeny, that have been modified according to the method of any of the above aspects or embodiments of the disclosure.

[0222] In a further aspect, the disclosure features a method of delivering a population of genetically modified cells to a subject (e.g., a mammalian subject such as a human) by administering to the subject a population of cells, or progeny thereof, that has been modified according to the method of any of the above aspects or embodiments of the disclosure.

[0223] In yet another aspect, the disclosure features a method of providing cell therapy to a subject (e.g., a mammalian subject, such as a human) in need thereof by administering to the subject a population of cells, or progeny thereof, that have been modified according to the method of any of the above aspects or embodiments of the disclosure.

[0224] In some embodiments of the foregoing three aspects of the present disclosure, the cells are allogeneic to the subject. In some embodiments, the cells are HLA-matched to the subject. In some embodiments, the cells are autologous to the subject.

[0225] In some embodiments, a population of progenitor cells is isolated from a subject (e.g., in the case of an autologous cell population) or a donor (e.g., in the case of 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, for example, by incubating the progenitor cells with one or more cell expansion agents described herein or known in the art to promote cell proliferation, thereby obtaining a population of cells that is administered to a subject. For example, the expansion agent may be StemRegenin-1, also known in the art as compound SR1, represented by the following formula (110):

[0226] [ka] SR1 and other swelling agents are described, for example, in US Pat. Nos. 8,927,281 and 9,580,426, the disclosures of each of which are incorporated herein by reference in their entireties.

[0227] Additional swelling agents that can be used in combination with the compositions and methods of the present disclosure include the compound UM-171, which is described in U.S. Patent No. 9,409,906, the disclosure of which is incorporated herein by reference in its entirety. Swelling agents that can be used herein further include structural or stereoisomeric variants of the compound UM-171, such as those described in U.S. Patent No. 2017 / 0037047, the disclosure of which is incorporated herein by reference in its entirety. The structure of the compound UM-171 is shown below in formula (111).

[0228] [ka]

[0229] In some embodiments, the swelling agent is a bromide salt of compound (111), such as the compound represented by formula (112):

[0230] [ka]

[0231] Additional expansion agents that can be used in conjunction with the compositions and methods of the present disclosure include histone deacetylase (HDAC) inhibitors, for example, as described in WO 2000 / 023567, the disclosure of which is incorporated herein by reference. Exemplary agents that can be used to expand populations of progenitor cells described herein are trichostatin A, trapoxin, trapoxin A, chlamydocin, sodium butyrate, dimethyl sulfoxide, suberanilohydroxamic acid, m-carboxycinnamic acid bishydroxamide, HC-toxin, Cyl-2, WF-3161, depudecin, and radicicol, among others.

[0232] In some embodiments, the progenitor cells are CD34+ HSCs. Using HSC expansion agents described herein and known in the art, the progenitor cells can be expanded without losing the functional capacity of HSCs.

[0233] In some embodiments, prior to isolating progenitor cells from a subject (e.g., in the case of an autologous cell population) or donor (in the case of an allogeneic cell population), the subject or donor is administered one or more mobilization agents that stimulate the movement of pluripotent cells (e.g., CD34+ HSCs and HPCs) from stem cell niches (e.g., bone marrow) into the peripheral circulation. Exemplary cell mobilization agents that can be used in combination with the compositions and methods of the present disclosure are described herein and known in the art. For example, the mobilization agent can be a C-X-C motif chemokine receptor (CXCR) 2 (CXCR2) agonist. The CXCR2 agonist can be Gro-beta or a truncated variant thereof. Gro-beta and its variants are described, for example, in U.S. Patent Nos. 6,080,398; 6,447,766; and 6,399,053, the disclosures of each of which are incorporated herein by reference in their entireties. Additionally or alternatively, the mobilizing agent can include a CXCR4 antagonist, such as plerixafor or a variant thereof. Plerixafor and structurally similar compounds are described, for example, in U.S. Patent Nos. 6,987,102; 7,935,692; and 7,897,590, the disclosures of which are each incorporated herein by reference. Additionally or alternatively, the mobilizing agent can include granulocyte colony-stimulating factor (G-CSF). The use of G-CSF as an agent for inducing the migration of pluripotent cells (e.g., CD34+ HSCs and / or HPCs) from stem cell niches to the peripheral circulation is described, for example, in US2010 / 0178271, the entire disclosure of which is incorporated herein by reference.

[0234] In some embodiments, prior to administering the population of cells to the subject, a population of endogenous pluripotent cells (e.g., a population of endogenous CD34+ HSCs or HPCs) is ablated in the subject by administering one or more conditioning agents to the subject. In some embodiments, the method comprises ablating 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 prior to administering the population of cells to the subject. The one or more conditioning agents may be myeloablative conditioning agents that deplete various hematopoietic cells from the subject's bone marrow. In some embodiments, the one or more conditioning agents are non-myeloablative conditioning agents that selectively target and ablate specific populations of endogenous pluripotent cells, such as a population of endogenous CD34+ HSCs or HPCs.

[0235] In some embodiments, upon administration of the population of cells to a subject, the administered cells, or their progeny, differentiate into one or more cell types selected from megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0236] In some embodiments, the subject has been diagnosed with a deficiency in the endogenous protein encoded by the transgene. The subject may be diagnosed with, for example, a disease described in Table 3. In some embodiments, the subject has been diagnosed with beta-thalassemia.

[0237] In some embodiments of any of the above aspects or embodiments of the present disclosure, the transgene encodes a β-globin protein. The transgene may, for example, contain a nucleic acid having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the transgene contains a nucleic acid having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the transgene contains a nucleic acid having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the transgene contains a nucleic acid having the nucleic acid sequence of SEQ ID NO:1.

[0238] 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.

[0239] In some embodiments, the β globin protein has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by one or more amino acid substitutions, insertions, and / or deletions. For example, the β globin protein can have an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by one or more conservative or non-conservative amino acid substitutions. The β globin protein can have, for example, an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by 1 to 50 conservative amino acid substitutions, 1 to 40 conservative amino acid substitutions, 1 to 30 conservative amino acid substitutions, 1 to 20 conservative amino acid substitutions, or 1 to 10 conservative amino acid substitutions, optionally in combination with one or more non-conservative amino acid substitutions.

[0240] In another aspect, the disclosure features a composition containing a mixture formed by modifying a eukaryotic cell according to the method of any of the above aspects or embodiments of the disclosure.

[0241] In a further aspect, the disclosure features a cell culture medium containing the composition of the previous aspect.

[0242] In yet another aspect, the disclosure features a population of eukaryotic cells modified according to the method of any of the above aspects or embodiments of the disclosure.

[0243] In another aspect, the disclosure features a pharmaceutical composition containing the population of cells of the preceding aspect. 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, such as a mammalian subject (e.g., a human).

[0244] In another aspect, the present disclosure features a kit containing a composition containing a mixture formed by modifying a eukaryotic cell according to the method of any of the above aspects or embodiments of the present disclosure. Additionally or alternatively, the kit may contain cell culture medium containing the composition. The kit may further contain a package insert containing instructions for transducing target cells using the contents of the kit.

[0245] In another aspect, the present disclosure features a kit containing a population of eukaryotic cells modified according to the method of any of the above aspects or embodiments of the present disclosure. Additionally or alternatively, the kit may contain a pharmaceutical composition containing a population of eukaryotic cells modified according to the method of any of the above aspects or embodiments of the present disclosure. The kit may further contain a package insert instructing a user to administer the population of cells to a subject according to any of the cell administration methods described above or herein.

[0246] definition As used herein, the terms "ablate," "ablation," "ablation," and the like refer to the depletion of one or more cells in a cell population, either in vivo or ex vivo. In some embodiments of the present disclosure, it may be desirable to ablate endogenous cells in a patient (e.g., a patient undergoing treatment for a disease described herein) before administering a therapeutic composition, such as a therapeutic cell population, to the subject. This may be beneficial, for example, to provide an environment for newly administered cells into which they can engraft. Ablation of a population of endogenous cells can be performed in a manner that selectively targets a specific cell type, for example, using an antibody or antibody-drug conjugate that binds to an antigen expressed on the target cell, followed by killing of the target cell. Additionally or alternatively, ablation can be performed in a nonspecific manner, using a cytotoxin that does not localize to a specific cell type but instead is capable of exerting a cytotoxic effect on a variety of different cells. Examples of ablation include depletion of at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more) of the cells in a cell population in vivo or in vitro. Quantifying the number of cells in a sample of cells can be performed using various cell counting techniques, such as using a counting chamber, a Coulter counter, flow cytometry, or other cell counting methods known in the art.

[0247] As used herein, the term "about" refers to an amount that varies by up to about 30% (e.g., 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) relative to a reference amount.

[0248] As used herein in the context of a protein of interest, the term "activity" refers to the biological functionality associated with the wild-type form of the protein. For example, in the context of an enzyme, the term "activity" refers to the ability of the protein to turn over a substrate in a manner that yields a product through a 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 a membrane-bound receptor, the term "activity" can refer to signal transduction initiated by the receptor, for example, upon binding to its cognate ligand. The activity level of a receptor involved in a signal transduction pathway can be detected and quantified, for example, by observing an increase in the outcome of receptor signaling, such as increased transcription of one or more genes (detectable, for example, using polymerase chain reaction techniques known in the art).

[0249] As used herein, a compound that "activates prostaglandin E receptor signal transduction" refers to a compound that has the ability to increase the signal transduction activity of prostaglandin E receptor in cells expressing prostaglandin E receptor that have been contacted with the specified compound, compared to the signal transduction activity of prostaglandin E receptor in cells expressing prostaglandin E receptor that have not been contacted with the specified compound. Assays that can be used to measure prostaglandin E receptor signal transduction are described, for example, in WO2010 / 108028, the disclosure of which is incorporated herein by reference, as this specification relates to methods for evaluating prostaglandin E receptor signal transduction.

[0250] As used herein, the terms "administering," "administration," and the like refer to providing 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)) directly to a patient by any effective route. Exemplary routes of administration are described herein and include, among others, systemic routes of administration, such as intravenous injection.

[0251] As used herein, the term "allogeneic" refers to cells, tissues, nucleic acid molecules, or other materials obtained or derived from a different subject of the same species. For example, in the context of a population of cells (e.g., a population of pluripotent cells) expressing one or more proteins described herein, allogeneic cells include cells (i) obtained from a subject not undergoing therapy and then (ii) transduced or transmitted with a vector directing the expression of one or more desired proteins. The phrase "directing expression" refers to the inclusion of one or more polynucleotides encoding one or more proteins to be expressed. The polynucleotides may contain additional sequence motifs that enhance expression of the protein of interest.

[0252] As used herein, the term "anneal" means to form a stable duplex of nucleic acids, e.g., by hybridization mediated by interstrand hydrogen bonding according to Watson-Crick base pairing. The nucleic acids of the duplex can 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 upon annealing one nucleic acid to another is a duplex structure that is not denatured by harsh washing. Exemplary harsh washing conditions are known in the art and include temperatures about 5°C below the melting temperatures of the individual strands of the duplex and low concentrations of monovalent salt, such as monovalent salt concentrations (e.g., NaCl concentrations) of less than 0.2M (e.g., 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 lower).

[0253] As used herein, the term "autologous" refers to cells, tissues, nucleic acid molecules, or other materials obtained or derived from an individual's own cells, tissues, nucleic acid molecules, etc. For example, in the context of a population of cells (e.g., a population of pluripotent cells) that express one or more proteins described herein, autologous cells include cells obtained from a patient undergoing therapy that have been transduced or transmitted with a vector that directs the expression of one or more proteins of interest.

[0254] 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 presentation properties. Cells of a common cell type may include cells isolated from a common tissue (e.g., epithelial tissue, nervous tissue, connective tissue, or muscle tissue) and / or a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0255] As used herein, the terms "conditioning" and "conditioning" refer to a process by which a subject is prepared to receive a transplant containing a population of cells (e.g., a population of pluripotent cells, such as CD34+ cells). Such manipulation facilitates the engraftment of a cell transplant, for example, by selectively depleting endogenous cells (e.g., endogenous CD34+ cells, among others) to create a void that is subsequently filled by transplanting exogenous cells. According to the methods described herein, a subject can be conditioned for a cell transplant manipulation by administering to the subject one or more agents capable of ablating endogenous cells (e.g., CD34+ cells, among others), radiation therapy, or a combination thereof. Conditioning regimens useful in combination with the compositions and methods of the present disclosure can be myeloablative or non-myeloablative. Other cell-ablative agents and methods known in the art (e.g., antibody-drug conjugates) can also be used.

[0256] As used herein, the terms "conservative mutation," "conservative substitution," "conservative amino acid substitution," and the like refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0257] [Table 1]

[0258] According to this table, the family of conservative amino acids includes: (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. Thus, a conservative variation or substitution is one that substitutes an amino acid with a member of the same amino acid family (e.g., Ser for Thr, or Lys for Arg).

[0259] As used herein, the term "diblock copolymer" refers to a nonionic polymer composed of two, and not more than two, distinct polymer regions (i.e., blocks of repeating units) covalently bonded to one another. An example of a diblock copolymer described herein is an amphiphilic copolymer, e.g., one with a region comprising a hydrophilic chain of repeating units connected to a region comprising a hydrophobic chain of repeating units, with or without a linker. Such a diblock copolymer can include a hydrophilic chain of polyoxyethylene (PEO) subunit connected to a hydrophobic chain of polyoxypropylene (PPO) subunit. A diblock copolymer of PEO and PPO subunits can be represented by the following formula: X(CHO) m -L-(C3H6O) nX2. X1 and X2 can be any chemical moiety. L can be an optional linker. In some embodiments, the PEO and PPO subunit blocks are directly covalently bonded. In some embodiments, X1 and X2 are H and OH, respectively. Other diblock copolymers include, for example, poly(ethylene glycol)-poly(γ-benzyl L-glutamate)PEG-PBLA, poly(ethylene glycol)-poly(D,L-lactic acid)PEG-PDLLA, poly(ethylene glycol)-poly(L-lactic acid)PEG-PLLA, poly(ethylene glycol)-poly(ε-caprolactone)PEG-PCL, poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)PEG-PLGA, poly(ethylene glycol)-poly(γ-benzyl L-glutamate)PEG-PBLG, poly(ethylene glycol)-poly(β-benzyl L-aspartate)PEG-PBLA, poly(ethylene glycol)-poly(α-benzyl carboxylate-ε-caprolactone)PEG-PBCL, and poly(ethylene glycol)-poly(δ-valerolactone)PEG-PVL. For clarity, as used herein, X1-[PEO]-L-[PPO]-X2 refers to a compound having the structure:

[0260] [ka] Or a structure with the opposite orientation:

[0261] [ka] means.

[0262] The length of the polymer blocks can be customized. As a result, many different diblock copolymers exist. Diblock copolymers suitable for use in combination with the compositions and methods of the present disclosure include those having a number average molecular weight of 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. Because the synthesis of diblock copolymers is associated with natural variations from batch to batch, the numerical values ​​cited above (and those used herein to characterize a given diblock copolymer) may not be precisely achievable during synthesis, and average values ​​will vary to a certain extent. Therefore, as used herein, the term "diblock copolymer" can be used interchangeably with the term "diblock copolymer" (representing several diblock copolymer entities, also referred to as a mixture of diblock copolymers), unless otherwise specified. As used herein, the term "average" in relation to the number of monomer units or molecular weight of a diblock copolymer(s) is a result of the technical inability to produce diblock copolymers that all have the same composition and therefore the same molecular weight. Diblock copolymers produced in accordance with the state of the art are referred to as mixtures of diblock copolymers, each of which exhibits variability in molecular weight, but where the mixture as a whole averages out to the molecular weight specified herein. BASF and Sigma Aldrich are suitable sources of diblock copolymers for use in conjunction with the compositions and methods of the present disclosure.

[0263] Due to variations that occur during the synthesis of diblock copolymers comprising PPO and PEO subunits, one skilled in the art will understand that the values ​​of m and n can vary, for example, by up to two-fold above and below the recited values. As used herein, therefore, a value such as n=50 represents a heterogeneous mixture of diblock copolymers where n can be from 25 to 100, e.g., 25 to 75, 26 to 74, 27 to 73, 28 to 72, 29 to 71, 30 to 70, 31 to 69, 32 to 68, 33 to 67, 34 to 66, 35 to 65, 36 to 64, 37 to 63, 38 to 62, 39 to 61, 40 to 60, 41 to 59, 42 to 58, 43 to 57, 44 to 56, 45 to 55, etc. Similarly, as used herein, a value such as n=60 represents a heterogeneous mixture of diblock copolymers where n can be from 30 to 120, e.g., from 30 to 90. Similarly, as used herein, a value such as n=70 represents a heterogeneous mixture of diblock copolymers where n can be from 35 to 140, e.g., from 35 to 105.

[0264] As used herein, the terms "embryonic stem cell" and "ES cell" refer to embryo-derived totipotent or pluripotent stem cells derived from the inner cell mass of a blastocyst that can be maintained in in vitro culture under suitable conditions. ES cells are capable of differentiating into cells of any of the three vertebrate germ layers, e.g., 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), the disclosure of which is incorporated herein by reference as it relates to the structure and functionality of embryonic stem cells.

[0265] As used herein, the term "endogenous" refers to a molecule (e.g., a 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).

[0266] As used herein, the term "expansion agent" refers to a substance capable of promoting the ex vivo expansion of a given cell type. Accordingly, a "pluripotent hematopoietic stem cell expansion agent" or "HSC expansion agent" refers to a substance capable of promoting the ex vivo expansion of a population of pluripotent hematopoietic stem cells. Pluripotent hematopoietic stem cell expansion agents include agents that expand a population of pluripotent hematopoietic stem cells such that the cells retain the functional capabilities of pluripotent hematopoietic stem cells. Exemplary pluripotent hematopoietic stem cell expansion agents that can be used in conjunction with the compositions and methods of the present disclosure include, but are not limited to, aryl hydrocarbon receptor antagonists, such as those described in U.S. Pat. Nos. 8,927,281 and 9,580,426, the entire disclosures of which are incorporated herein by reference, and in particular, the compound SR1. Additional pluripotent hematopoietic stem cell expansion agents that can be used in conjunction with the compositions and methods of the present disclosure include the compound UM-171 and other compounds described in U.S. Pat. No. 9,409,906, the disclosures of which are incorporated herein by reference in their entireties. Pluripotent hematopoietic stem cell expansion agents further include structural and / or stereoisomeric variants of the compound UM-171, such as those described in US2017 / 0037047, the disclosure of which is incorporated herein by reference in its entirety. Additional pluripotent hematopoietic stem cell expansion agents suitable for use in the present disclosure include histone deacetylase (HDAC) inhibitors such as trichostatin A, trapoxin, trapoxin A, chlamydocin, sodium butyrate, dimethyl sulfoxide, suberanilohydroxamic acid, m-carboxycinnamic acid bishydroxamide, HC-toxin, Cyl-2, WF-3161, depudecin, and radicicol, among others, as described in WO2000 / 023567, the disclosure of which is incorporated herein by reference.

[0267] As used herein, the term "express" 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 the RNA transcript (e.g., by splicing, editing, 5' capping, 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 a gene encoding a protein product, the terms "gene expression" and the like are used interchangeably with the terms "protein expression" and the like. Expression of a gene or protein of interest in a subject can be determined, for example, by detecting, in a sample obtained from the subject, an increase in the activity of the corresponding protein; an increase in the amount or concentration of mRNA encoding the corresponding protein (e.g., assessed 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., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others); and / or (e.g., in the case of an enzyme, assessed using an enzyme activity assay described herein or known in the art). As used herein, a cell is considered to "express" a gene or protein of interest if one or more, or all, of the above events are detectable within the cell or the medium in which the cell resides.For example, a gene or protein of interest is considered to be "expressed" by a cell, or population of cells, if it is possible to detect (i) the production of a corresponding RNA transcript, such as an mRNA template, by the cell, or population of cells (e.g., using the RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' capping, and / or 3' end processing, using the RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using the protein detection procedures described herein); and / or (iv) post-translational modification of the protein product (e.g., using the protein detection procedures described herein).

[0268] As used herein, the term "functional potential," when referring to multipotent cells such as pluripotent hematopoietic stem cells, refers to the functional properties of stem cells, including: 1) pluripotency (which refers to 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, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells)); 2) self-renewal (which refers to the ability of a stem cell to give rise to daughter cells that have the same potential as the mother cell and further have the ability to be generated repeatedly throughout the life of an individual without exhaustion); and 3) the ability of the stem cell or its progeny to be reintroduced into a transplant recipient, where they home to a stem cell niche and re-establish proliferative and sustained cell growth and differentiation.

[0269] As used herein, the terms "pluripotent hematopoietic stem cells" and "HSCs" refer to immature blood cells that have the ability to self-renew and differentiate into mature blood cells of diverse lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). It is known in the art that such cells may or may not contain CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are thought to comprise a subpopulation of cells with the stem cell properties described above, while in mice, HSCs are CD34-. Additionally, HSCs refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on 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), whereas 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, ST-HSCs are less quiescent (i.e., more active) and more proliferative than LT-HSCs under homeostatic conditions.However, LT-HSCs have greater self-renewal potential (i.e., they survive throughout adulthood and are serially transplantable through successive recipients), while ST-HSCs have limited self-renewal potential (i.e., they survive for only a limited period of time and do not have serial transplantation potential). Either 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 more quickly give rise to differentiated progeny.

[0270] As used herein, an agent that inhibits histone deacetylation refers to a substance or composition (e.g., a small molecule, protein, interfering RNA, messenger RNA, or other natural or synthetic compound, or a composition such as a virus or other material composed of multiple substances) that can attenuate or prevent the activity of histone deacetylase, more specifically its enzymatic activity, either through direct interaction or by indirect means, such as by causing a decrease in the amount of histone deacetylase produced in cells or by inhibiting the interaction of histone deacetylase with acetylated histone substances. Inhibiting the enzymatic activity of histone deacetylase means reducing the ability of histone deacetylase to catalyze the removal of acetyl groups from histone residues (e.g., mono-, di-, or trimethylated lysine residues; mono-methylated arginine residues; or symmetric / asymmetric dimethylated arginine residues within histone proteins). Preferably, such inhibition is specific, such that the agent that inhibits histone deacetylation reduces the ability of histone deacetylase to remove acetyl groups from histone residues at a concentration of the inhibitor that is lower than the concentration of the inhibitor required to produce another, unrelated biological effect.

[0271] As used herein, the terms "histone deacetylase" and "HDAC" refer to any member of a family of enzymes that catalyze the removal of acetyl groups from the epsilon-amino groups of lysine residues at the N-terminus of histones. Unless the context indicates otherwise, the term "histone" is meant to refer to any histone protein, including H1, H2A, H2B, H3, H4, and H5, from any species. Human HDAC proteins or gene products include, but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, and HDAC-11.

[0272] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which none of the HLA antigens are mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of multipotent hematopoietic stem cell transplantation therapy. HLA-matched (i.e., matched for all six alleles) donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells are less likely to recognize the incoming graft as foreign and, therefore, less likely to mount an immune response against the transplanted tissue.

[0273] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen is mismatched between the donor and recipient, particularly for HLA-A, HLA-B, HLA-C, and HLA-DR, such as a donor providing a hematopoietic stem cell graft to a recipient in need of multipotent hematopoietic stem cell transplantation therapy. In some embodiments, some haplotypes are matched and others are mismatched. HLA-mismatched donor-recipient pairs may be at increased risk of graft rejection compared to HLA-matched donor-recipient pairs because endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign in HLA-mismatched donor-recipient pairs, and such T cells and NK cells are therefore more likely to mount an immune response against the transplanted tissue.

[0274] As used herein, the terms "induced pluripotent stem cell," "iPS cell," and "iPSC" refer to multipotent stem cells that can be derived directly from differentiated somatic cells. Human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells, which may include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, l-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-specifying factors. Human iPS cells are characterized by their ability to differentiate into cells of any of the three vertebrate germ layers, e.g., 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), the disclosure of which is incorporated herein by reference as it relates to the structure and functionality of iPS cells.

[0275] As used herein, the term "inhibitor" refers to an agent (e.g., a small molecule, peptide fragment, protein, antibody, or antigen-binding fragment thereof) that binds to and / or otherwise suppresses the activity of a target molecule.

[0276] As used herein, the terms "interfering ribonucleic acid" and "interfering RNA" refer to RNA, such as a short interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA), that silences the expression of a target RNA transcript by (i) annealing to the target RNA transcript and forming a nucleic acid duplex, (ii) promoting nuclease-mediated degradation of the RNA transcript, and / or (iii) slowing, inhibiting, or preventing translation of the RNA transcript, for example, by sterically excluding the formation of a functional ribosome-RNA transcript complex or otherwise impairing the formation of a functional protein product from the target RNA transcript. The interfering RNA described herein can 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), the disclosures of each of which are incorporated herein by reference in their entirety.

[0277] As used herein in the context of viral transduction protocols, the term "multiplicity of infection" or "MOI" refers to the ratio of (i) virions added to a population of cells targeted for transduction to (ii) the amount of cells in the population. As an example, 1 x 10 virions are targeted for transduction. 6 A population of 1 x 10 cells 7 Transduction protocols involving contact with virions (e.g., lentiviral virions such as those described herein) are characterized by a multiplicity of infection of 10.

[0278] As used herein in the context of hematopoietic stem and / or progenitor cells, the term "mobilization" refers to the release of such cells from the stem cell niche (e.g., bone marrow) in which they typically reside, into the peripheral circulation. A "mobilizing agent" is an agent capable of inducing the release of hematopoietic stem and / or progenitor cells from the stem cell niche into the peripheral circulation.

[0279] As used herein, the term "myeloablative" or "myeloablation" refers to a conditioning regimen that substantially impairs or destroys the hematopoietic system, typically by exposure to cytotoxic agents or radiation. Myeloablation includes complete bone marrow destruction, brought about by high doses of cytotoxic agents or total body radiation, which destroys the hematopoietic system.

[0280] As used herein, the terms "non-myeloablative" or "myelosuppressive" refer to a conditioning regimen that does not eliminate substantially all hematopoietic cells from the host.

[0281] As used herein, the terms "number average molecular weight" and "Mn" refer to the statistical average molecular weight of all polymer chains within a sample and are defined as follows:

[0282]

number

[0283] As used herein, the terms "weight average molecular weight" and "Mw" refer to the weighted statistical average of all polymer chains within a sample and are defined as follows:

[0284]

number

[0285] As used herein, the term "polydispersity index" refers to a measure of the broadness of the molecular weight distribution of a polymer and is defined as follows: Polydispersity index=Mw / Mn.

[0286] As used herein, the term "pluripotent cell" refers to 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, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells)). Examples of pluripotent cells are ESCs, iPSCs, and CD34+ cells.

[0287] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase promotes transcription of the transgene. Exemplary promoters suitable for use in conjunction with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which relates to nucleic acid control elements and is incorporated herein by reference. Additionally, the term "promoter" can refer to a synthetic promoter, which is a regulatory DNA sequence that does not naturally occur in a biological system. Synthetic promoters contain portions of a naturally occurring promoter combined with a non-naturally occurring polynucleotide sequence and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

[0288] "Percent sequence complementarity" with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to nucleic acids in the reference polynucleotide sequence, aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered "complementary" to a reference nucleotide, as described herein, if the two nucleotides form a standard Watson-Crick base pair. For the avoidance of doubt, in the context of the present disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, proper Watson-Crick base pairs are referred to as "matches," while unpaired and improperly paired nucleotides are referred to as "mismatches." Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in a variety of ways within the capabilities of those skilled in the art, using publicly available computer software, such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum complementarity over the entire length of the sequences being compared. By way of illustration, the percentage sequence complementarity of a given nucleic acid sequence A to a given nucleic acid sequence B (which can alternatively be referred to as a given nucleic acid sequence A having a particular percentage complementarity to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) [where X is the number of complementary base pairs in an alignment of A and B (e.g., 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 percentage sequence complementarity of A to B will not be equal to the percentage sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be "fully complementary" to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0289] "Percent sequence identity" to a reference polynucleotide sequence or reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide sequence or reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of measuring percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, using publicly available computer software, such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. Illustratively, the percent sequence identity of a given nucleic acid or amino acid sequence A with or relative to a nucleic acid or amino acid sequence B (which can alternatively be referred to as a given nucleic acid or amino acid sequence A having a particular percent sequence identity with or relative to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic 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, then the percent sequence identity of A to B will not be considered equal to the percent sequence identity of B to A.

[0290] As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response, and other problematic adverse effects, and with a reasonable benefit / risk ratio.

[0291] As used herein, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. 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.

[0292] As used herein, the terms "stem cell" and "undifferentiated cell" refer to cells in an undifferentiated or partially differentiated state that have the developmental potential to differentiate into multiple cell types. Stem cells can proliferate and give rise to more such stem cells while maintaining functional capacity. Stem cells can divide asymmetrically. This is known as inevitable asymmetric differentiation, in which some daughter cells maintain the functional capacity of the parent stem cell, while other daughter cells express some other specific function, phenotype, and / or developmental capacity that differs from the parent cell. Daughter cells can themselves be induced to produce progeny that proliferate and differentiate into one or more mature cell types while also retaining one or more cells of the parent's developmental potential. Differentiated cells can be derived from pluripotent cells, which themselves are derived from multipotent cells, etc. Alternatively, some stem cells within a population can divide symmetrically into two stem cells. Thus, the term "stem cell" refers to any subset of cells that, under certain circumstances, have the developmental potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, maintain the ability to proliferate without undergoing substantial differentiation. In some embodiments, the term "stem cell" generally refers to a naturally occurring parent cell whose progeny (progeny cells) often specialize in different directions by differentiation, acquiring entirely separate characteristics, e.g., as occurs in the progressive diversification of embryonic cells and tissues. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such potential may be natural or may be artificially induced upon treatment with various factors. Cells that begin as stem cells can progress to a differentiated phenotype, but can then be induced to "reverse" and re-express the stem cell phenotype. This term is often also referred to by those skilled in the art as "dedifferentiation," or "reprogramming," or "retrodifferentiation."

[0293] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes a gene product (e.g., a gene product described herein). The gene product may be RNA, a peptide, or a protein. In addition to the coding region for the gene product, a transgene can include or be operably linked to one or more elements that facilitate or enhance expression, such as a promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the present disclosure can utilize any known, suitable promoter(s), enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0294] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a living organism (e.g., a mammal, e.g., a human) that has been diagnosed with and / or is undergoing treatment for a disease characterized by a gene or protein deficiency as described herein.

[0295] As used herein, the terms "transduction" and "transducing" refer to the process of introducing a viral vector construct or a portion thereof into a cell, followed by expression within the cell of a transgene encoded by the vector construct or portion thereof.

[0296] As used herein, the term "transduction efficiency" refers to the percentage of cells in a given population that are transduced with at least one copy of a vector (e.g., a viral vector such as a lentiviral vector described herein). For example, 1 x 10 6 cells are exposed to a virus (e.g., lentivirus) and after the transduction procedure, 0.5 x 10 6If cells are determined to have at least one copy in their genome, the transduction efficiency for the procedure is 50%. Exemplary methods for measuring transduction efficiency include polymerase chain reaction (PCR) procedures and flow cytometry.

[0297] As used herein, "treatment" and "treating" refer to an approach to obtaining a beneficial or desired result, e.g., a clinical result. Beneficial or desired results can include, but are not limited to, the reduction or amelioration of one or more signs or symptoms, whether detectable or undetectable; a decrease in the extent of the disease or condition; a stabilization of the state of the disease, disorder, or condition (i.e., not worsening); prevention of the spread of the disease or condition; a delay or slowing of the progression of the disease or condition; an improvement or palliation of the disease or condition; and remission (partial or complete). "Ameliorating" or "alleviating" a disease or condition means that the severity and / or undesirable clinical signs of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged compared to the severity or time course in the absence of treatment. "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disease, and those prone to have the condition or disease, as well as those in whom the condition or disease is to be prevented.

[0298] 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 foreign proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in WO 1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for expressing genes of interest. Expression vectors suitable for use in the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Vectors that can be used for 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 expressing one or more proteins described herein may contain polynucleotide sequences that increase the translation rate of the corresponding gene(s) or improve the stability or nuclear export of mRNA resulting from gene transcription. Examples of such sequence elements are 5' and 3' untranslated regions, IRES, and polyadenylation signal sites for directing efficient transcription of one or more genes carried by the expression vector. Expression vectors suitable for use with the compositions and methods described herein can also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin, among others.

[0299] As used herein, the term "vector copy number" or "VCN" refers to the number of copies of a vector, or a portion thereof (e.g., a portion encoding a transgene of interest), within the genome of a cell. The average VCN can be measured for a population of cells or for individual cell colonies. Exemplary methods for measuring VCN include PCR and flow cytometry.

[0300] As used herein, the term "beta globin," along with other gene or protein names cited in this disclosure, includes wild-type forms of the corresponding gene or protein, as well as variants thereof (e.g., splice variants, truncations, concatemers, and fusion constructs, among others). In the context of β-globin, an example of such a variant is a protein having at least 70% sequence identity (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) to any of the amino acid sequences of a wild-type β-globin protein (e.g., SEQ ID NO: 2), for example, where the β-globin variant retains the functionality of wild-type β-globin.

[0301] As used herein, the term "alkyl" refers to monovalent, optionally branched alkyl groups, such as those 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, n-hexyl, and the like.

[0302] As used herein, the term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms.

[0303] As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Preferred aryls include phenyl, naphthyl, phenanthrenyl, and the like.

[0304] As used herein, the terms "aralkyl" and "arylalkyl" are used interchangeably and refer to alkyl groups containing an aryl moiety. Similarly, the term "aryl lower alkyl" and the like refer to lower alkyl groups containing an aryl moiety.

[0305] As used herein, the term "alkylaryl" refers to an alkyl group having an aryl substituent, including benzyl, phenethyl and the like.

[0306] As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic group or a bicyclic or tricyclic fused-ring heteroaromatic group. Specific examples of heteroaromatic 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, quinolidinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, 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, pteridinyl, carbazolyl, xanthenyl, and benzoquinolyl.

[0307] As used herein, the term "alkylheteroaryl" refers to an alkyl group having a heteroaryl substituent, including 2-furylmethyl, 2-thienylmethyl, 2-(1H-indol-3-yl)ethyl, and the like.

[0308] As used herein, the term "lower alkenyl" refers to an alkenyl group preferably having from 2 to 6 carbon atoms and having at least 1 or 2 sites of alkenyl unsaturation. Exemplary alkenyl groups are ethenyl (-CH=CH), n-2-propenyl (allyl, -CHCH=CH), and the like.

[0309] As used herein, the term "alkenylaryl" refers to alkenyl groups having an aryl substituent, including 2-phenylvinyl and the like.

[0310] As used herein, the term "alkenylheteroaryl" refers to an alkenyl group having a heteroaryl substituent, including 2-(3-pyridinyl)vinyl and the like.

[0311] As used herein, the term "lower alkynyl" refers to alkynyl groups preferably having from 2 to 6 carbon atoms and having at least 1 or 2 sites of alkynyl unsaturation; preferred alkynyl groups include ethynyl (-C≡CH), propargyl (-CHC≡CH), and the like.

[0312] As used herein, the term "alkynylaryl" refers to an alkynyl group having an aryl substituent, including phenylethynyl and the like.

[0313] As used herein, the term "alkynylheteroaryl" refers to an alkynyl group having a heteroaryl substituent, including 2-thienylethynyl and the like.

[0314] As used herein, the term "cycloalkyl" refers to a monocyclic cycloalkyl group having from 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0315] As used herein, the term "lower cycloalkyl" refers to a saturated carbocyclic group of 3 to 8 carbon atoms having a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norbornyl). Preferred cycloalkyls include cyclopentyl, cyclohexyl, norbornyl, and the like.

[0316] As used herein, the term "heterocycloalkyl" refers to a cycloalkyl group in which one or more ring carbon atoms is replaced with a heteroatom such as a nitrogen atom, an oxygen atom, a sulfur atom, etc. Exemplary heterocycloalkyl groups are pyrrolidinyl, piperidinyl, oxopiperidinyl, morpholinyl, piperazinyl, oxopiperazinyl, thiomorpholinyl, azepanyl, diazepanyl, oxazepanyl, thiazepanyl, dioxothiazepinyl, azocanyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.

[0317] As used herein, the term "alkylcycloalkyl" refers to an alkyl group having a cycloalkyl substituent, including cyclohexylmethyl, cyclopentylpropyl, and the like.

[0318] As used herein, the term "alkylheterocycloalkyl" refers to a C1-C6 alkyl group having a heterocycloalkyl substituent, including 2-(1-pyrrolidinyl)ethyl, 4-morpholinylmethyl, (1-methyl-4-piperidinyl)methyl, and the like.

[0319] As used herein, the term "carboxy" refers to the group --C(O)OH.

[0320] As used herein, the term "alkylcarboxy" refers to C1-C5 alkyl groups having a carboxy substituent, including 2-carboxyethyl and the like.

[0321] As used herein, the term "acyl" refers to the group -C(O)R, where R can be, for example, C-C alkyl, aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other substituents.

[0322] As used herein, the term "acyloxy" refers to the group -OC(O)R, where R can be, for example, C-C alkyl, aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other substituents.

[0323] As used herein, the term "alkoxy" refers to the group -OR, where R is an optionally substituted alkyl group such as, for example, an optionally substituted C-C alkyl, aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other substituents. Exemplary alkoxy groups include, for example, methoxy, ethoxy, phenoxy, and the like.

[0324] As used herein, the term "alkoxycarbonyl" refers to the group -C(O)OR, where R is, for example, hydrogen, C-C alkyl, aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other possible substituents.

[0325] As used herein, the term "alkylalkoxycarbonyl" refers to alkyl groups having an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl and the like.

[0326] As used herein, the term "aminocarbonyl" refers to the group -C(O)NRR', where each of R and R' can independently be, for example, hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents.

[0327] As used herein, the term "alkylaminocarbonyl" refers to alkyl groups having an aminocarbonyl substituent, including 2-(dimethylaminocarbonyl)ethyl and the like.

[0328] As used herein, the term "acylamino" refers to the group -NRC(O)R', where each of R and R' can independently be, for example, hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, or C1-C6 alkylheteroaryl, among other substituents.

[0329] As used herein, the term "alkylacylamino" refers to alkyl groups having an acylamino substituent, including 2-(propionylamino)ethyl and the like.

[0330] As used herein, the term "ureido" refers to the group -NRC(O)NR'R'', where each of R, R', and R'' independently can be, for example, hydrogen, C1-C6 alkyl, aryl, heteroaryl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, or heterocycloalkyl, among other substituents. Exemplary ureido groups further include moieties in which R' and R'' together with the nitrogen atom to which they are attached form a 3- to 8-membered heterocycloalkyl ring.

[0331] As used herein, the term "alkylureido" refers to an alkyl group having a ureido substituent, including 2-(N'-methylureido)ethyl and the like.

[0332] As used herein, the term "amino" refers to the group -NRR', where each of R and R' can independently be, for example, hydrogen, C-C alkyl, aryl, heteroaryl, C-C alkylaryl, C-C alkylheteroaryl, cycloalkyl, or heterocycloalkyl, among other substituents. Exemplary amino groups further include moieties where R and R', together with the nitrogen atom to which they are attached, can form a 3- to 8-membered heterocycloalkyl ring.

[0333] As used herein, the term "alkylamino" refers to an alkyl group having an amino substituent, including 2-(1-pyrrolidinyl)ethyl and the like.

[0334] As used herein, the term "ammonium" refers to a positively charged group -N + RR'R'', where each of R, R', and R'' independently may be, for example, C1-C6 alkyl, C1-C6 alkylaryl, C1-C6 alkylheteroaryl, cycloalkyl, or heterocycloalkyl, among other substituents. Exemplary ammonium groups further include moieties in which R' and R'', together with the nitrogen atom to which they are attached, form a 3-8 membered heterocycloalkyl ring.

[0335] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine atoms.

[0336] As used herein, the term "sulfonyloxy" refers to the group -OSO2-R, where R is hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with halogen (e.g., -OSO2-CF3 group), aryl, heteroaryl, C1-C6 alkylaryl, and C1-C6 alkylheteroaryl.

[0337] As used herein, the term "alkylsulfonyloxy" refers to alkyl groups having a sulfonyloxy substituent, including 2-(methylsulfonyl)ethyl and the like.

[0338] As used herein, the term "sulfonyl" refers to the group "-SO2-R," where R is hydrogen, aryl, heteroaryl, C1-C6 alkyl, C1-C6 alkyl substituted with halogen (e.g., a -SO2-CF3 group), C1-C6 alkylaryl, or C1-C6 alkylheteroaryl.

[0339] As used herein, the term "alkylsulfonyl" refers to alkyl groups having a sulfonyl substituent, including 2-(methylsulfonyl)ethyl and the like.

[0340] As used herein, the term "sulfinyl" refers to the group "-S(O)-R," where R is hydrogen, C-C alkyl, C-C alkyl substituted with halogen (e.g., a -SO-CF group), aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl.

[0341] As used herein, the term "alkylsulfinyl" refers to C1-C5 alkyl groups having a sulfinyl substituent, including 2-(methylsulfinyl)ethyl and the like.

[0342] As used herein, the term "sulfanyl" refers to the group -SR, where R is, for example, alkyl, aryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other substituents. Exemplary sulfanyl groups include methylsulfanyl, ethylsulfanyl, and the like.

[0343] As used herein, the term "alkylsulfanyl" refers to alkyl groups having a sulfanyl substituent, including 2-(methylsulfanyl)ethyl and the like.

[0344] As used herein, the term "sulfonylamino" refers to the group -NRSO-R', where each of R and R' can independently be hydrogen, C-C alkylaryl, heteroaryl, C-C alkylaryl, or C-C alkylheteroaryl, among other substituents.

[0345] As used herein, the term "alkylsulfonylamino" refers to alkyl groups having a sulfonylamino substituent, including 2-(ethylsulfonylamino)ethyl and the like.

[0346] The groups described above, such as "alkyl," "alkenyl," "alkynyl," "aryl," and "heteroaryl" groups, can optionally be substituted, for example, if valence allows, with one or more substituents, such as a substituent selected from alkyl (e.g., C-C alkyl), alkenyl (e.g., C-C alkenyl), alkynyl (e.g., C-C alkynyl), cycloalkyl, heterocycloalkyl, alkylaryl (e.g., C-C alkylaryl), alkylheteroaryl (e.g., C-C alkylheteroaryl), alkylcycloalkyl (e.g., C-C alkylcycloalkyl), alkylheterocycloalkyl (e.g., C-C alkylheterocycloalkyl), amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like. In some embodiments, the substitution is one in which adjacent substituents undergo ring closure, such as situations involving vicinal functional substituents, thereby forming lactams, lactones, cyclic anhydrides, acetals, thioacetals, and aminals, among others.

[0347] As used herein, the term "optionally fused" refers to a cyclic chemical group, such as a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, that can be fused to a ring system. Exemplary ring systems that can be optionally fused to a fused chemical group include, for example, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, indazolyl, benzimidazolyl, quinolinyl, isoquinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl, cinnolinyl, indolizinyl, naphthyridinyl, pteridinyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, indolinyl, isoindolinyl, 2,3,4,5-tetrahydrobenzo[b]oxepinyl, 6,7,8,9-tetrahydro-5H-benzocycloheptenyl, chromanyl, and the like.

[0348] As used herein, the term "pharmaceutically acceptable salt" refers to a salt, such as a salt of a compound described herein, that retains the desired biological activity of the non-ionized parent compound from which the salt is formed. Examples of such salts include, but are not limited to, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts formed with 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 have the formula -NR,R',R" + Z -wherein each of R, R', and R" can independently be, for example, hydrogen, alkyl, benzyl, C-C alkyl, C-C alkenyl, C-C alkynyl, C-C alkylaryl, C-C 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, malate, fumarate, citrate, tartrate, ascorbate, cinnamoate, mandeloate, and diphenylacetate), etc.

[0349] For example, in the context of protein kinase C (PKC) inhibitors such as staurosporine, the term "variant" as used herein refers to 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., the types of cells described herein, such as CD34+ cells).In the context of small molecule PKC inhibitors such as staurosporine, structural variants of the reference compound include variants that differ from the reference compound due to the inclusion and / or placement of one or more substituents, as well as 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, variants can contain one or more nucleic acid substitutions compared to parent interfering RNA molecules.

[0350] The structural compositions described herein include tautomers, geometric isomers (e.g., E / Z and cis / trans isomers), enantiomers, diastereomers, and racemates, as well as pharmaceutically acceptable salts thereof, including, for example, acid addition salts formed with pharmaceutically acceptable acids such as hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or hydrogenphosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0351] As used herein, a chemical structural formula that does not depict the stereochemical configuration of a compound having one or more stereocenters is intended to encompass any one stereoisomer of the depicted compound, or a mixture of one or more such stereoisomers (e.g., any one enantiomer or diastereomer of the depicted compound, or a mixture of enantiomers (e.g., a racemic mixture) or diastereomers). As used herein, a chemical structural formula that does not specifically depict the stereochemical configuration of a compound having one or more stereocenters is intended to refer to a substantially pure form of the particular stereoisomer depicted. By "substantially pure" form is meant a compound having greater than 85% purity, e.g., 85% to 99%, 85% to 99.9%, 85% to 99.99%, or 85% to 100% purity, 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% purity, as assessed, for example, using chromatography and nuclear magnetic resonance techniques known in the art. [Brief explanation of the drawings]

[0352] [Figure 1]Figure 1 is a graph showing the % survival of CD34+ cells one day after transduction when treated with diblock copolymers. Six diblock copolymers were tested at concentrations ranging from 0.0001 mg / mL to 10 mg / mL (DBP1-DBP6). The table below shows the composition of each of DBP1-DBP6: [Table 2] The results show that applying diblock polymers during transduction is non-toxic to pluripotent hematopoietic stem cells. In the presence of several diblock polymers (+DBP1-6) at a range of doses (10-0.0001 mg / mL), peripherally mobilized blood CD34 cells were significantly increased. + Stem cells were transduced with lentiviral vectors for 20–24 h (vector only, multiplicity of infection of 10). Plots shown are flow cytometry (Annexin V) staining of CD34+ cells isolated from different healthy donors (○, A–D) in four independent experiments, 1 day after lentiviral transduction. - 7AAD - ) and the percentage of viable cells detected by ELISA. [Figure 2] This graph shows the fold increase in transduction efficiency one day after transduction when treated with diblock copolymers. A is DBP1, B is DBP2, C is DBP3, D is DBP4, E is DBP5, and F is DBP6. The graph demonstrates that the application of diblock polymers can improve the transduction efficiency of pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock polymers (DBP1-6, A-F) applied at final concentrations ranging from 10 to 0.0001 mg / mL. (A) The plot summarizes the fold change in the percentage of transduced cells induced by the addition of diblock polymers compared to cells treated with vector alone. The percentage of transduced cells was determined by flow cytometric detection of transgene expression 12 days after transduction in four independent experiments using CD34+ cells isolated from different healthy donors (○). [Figure 3] This graph shows the average vector copy number per cell in bone marrow aspirate cultures on day 12. DBP1 to DBP6 were tested. The graph demonstrates that application of diblock polymers improves lentiviral integration in pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock polymers (DBP1 to 6) applied at final concentrations of 0.1 to 0.0001 mg / mL. The plot summarizes the average transgene copy number (VCN) measured by droplet digital PCR detection of integrated transgene sequences in genomic DNA recovered from cell cultures 12 days after transduction. Data are shown for CD34+ cells isolated from two different healthy donors (○). [Figure 4] This graph shows the percentage of cells transduced with GFP vectors for DBP1-DBP5. Various combinations of transduction enhancer elements (TE Combo 1 or Combo 2) were tested with DBP. The figure demonstrates that the application of diblock polymers, in combination with other compounds, can improve the transduction efficiency of pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock polymers (DBP1-DBP5) applied at final concentrations of 100 to 1 μg / mL in combination with other compounds (+TE Combo 1, +TE Combo 2) that can also improve lentiviral transduction. The plot shows the percentage of transduced cells, as measured by flow cytometric detection of transgene expression 12 days after transduction. Data are the mean ± SD representing three independent experiments. [Figure 5]This graph shows the percent viability of cells treated with either DBP1 or DBP5 along with four different transduction enhancer combinations (TE Combo 1, Combo 2, Combo 3, and Combo 4). The graph demonstrates that applying diblock polymers in combination with other compounds during transduction is nontoxic to pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (vector only, multiplicity of infection of 10) for 20-24 hours in the presence of a range of doses (1-0.1 mg / mL) of diblock polymers DBP1 and DBP5 applied in combination with other compounds (TE Combos 1-4) that can also improve lentiviral transduction. The plot shown summarizes the percentage of viable cells detected by flow cytometry (Annexin V-7AAD-) 1 day after lentiviral transduction of CD34+ cells. Data are shown as mean ± SD and are representative of three independent experiments. [Figure 6] Figure 1 shows the fold change in the percentage of CD90- HSCs for cells treated with DBP1 or DBP5 along with one of the four TE combinations described above in Figure 5. The graph demonstrates that applying diblock polymers in conjunction with other compounds that enhance pluripotent hematopoietic stem cell transduction does not adversely affect the phenotype or survival of pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock polymers (DBP1 and DBP5) applied at various final concentrations in combination with other compounds that can also improve lentiviral transduction (+TE Combos 1-4). The plot shows the fold change (measured by flow cytometry) in the percentage of CD34+CD90+ stem cells detected 1 day after transduction compared to cells treated with vector alone. Data are shown as mean ± SD and are representative of three independent experiments. [Figure 7]This is a series of graphs showing that diblock copolymers of various PEO and PPO compositions result in improved lentiviral transduction of pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock copolymers of various PEO and PPO compositions (diblock PEO / PPO ratios) applied at a final concentration of 100 μg / mL. The plots show the fold change (measured by flow cytometry) in the percentage of transduced CD34+ stem cells detected 12 days after transduction compared to cells transduced in the absence of diblock copolymer, and the average VCN per cell. Each symbol represents stem cells assayed from an independent healthy donor; the healthy donors were six donors tested in two independent assays. [Figure 8] This is a series of graphs showing that diblock copolymers with a wide range of PEO and PPO block contents improve lentiviral transduction of pluripotent hematopoietic stem cells. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of diblock copolymers with various PEO and PPO compositions (diblock PEO / PPO ratios) applied at a final concentration of 100 μg / mL. The plots show the fold change (measured by flow cytometry) in the percentage of transduced CD34+ stem cells detected 12 days after transduction compared to cells transduced in the absence of diblock copolymer, and the average VCN per cell. Each symbol represents stem cells assayed from an independent healthy donor; six healthy donors were tested in two independent assays. [Figure 9]This is a series of graphs showing that diblock copolymers are compatible with RetroNectin, a recombinant human fibronectin fragment composed of three functional domains: a cell-binding domain (C domain), a heparin-binding domain (H domain), and a CS-1 domain. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of two different diblock polymer enhancer combinations (Diblock Combo 1 and 2) with or without RetroNectin (RN). Plots show the percentage of transduced CD34+ stem cells (measured by flow cytometry) and the average VCN per cell detected 12 days after transduction. At least three independent assays are shown, and each symbol represents stem cells assayed from an independent healthy donor. [Figure 10] This is a series of graphs showing that application of diblock copolymers results in improved stem cell transduction compared to that achieved with other commercial compounds. Peripherally mobilized blood CD34+ stem cells were transduced with lentiviral vectors (MOI: 10) in the presence of two different diblock polymer enhancer combinations (Diblock Combo 1 and 2) containing various diblock copolymers (PPO / PEO ratios) or an enhancer combination containing 1 mg / mL (LB) of LentiBoost (Poloxamer 338; source: Sirion Biotech). Plots show the fold change (measured by flow cytometry) in the percentage of transduced CD34+ stem cells detected 12 days after transduction compared to stem cells treated with vector alone, and the average VCN per cell. Plotted data represent three independent healthy donors, and each symbol represents stem cells assayed from an independent healthy donor. ****P<0.001, ***p<0.05 Student's paired t-test. DETAILED DESCRIPTION OF THE INVENTION

[0353] The compositions and methods described herein can be used to modify eukaryotic cells, such as pluripotent cells, including, for example, pluripotent hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). The compositions and methods of the present disclosure can be used to genetically modify such cells and engineer them to express a gene of interest and / or to propagate ex vivo. In some embodiments of the present 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 can encode a protein product or a regulatory ribonucleic acid (RNA) molecule that regulates the expression of different genes. In some embodiments, the transgene encodes a protein that is missing or non-functional in a patient (e.g., a mammalian patient, such as a human) suffering from a genetic disease, e.g., a genetic disease characterized by 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, thereby restoring gene expression in the individual.

[0354] Various viral vectors can be used in conjunction with the compositions and methods of the present disclosure. For example, the viral vector can be a retrovirus, such as a lentivirus. Other viral vectors that can be used to achieve transduction of target cells are described herein.

[0355] To enhance the degree of transduction and / or the rate at which target cells are transduced, the cells can be contacted with a diblock copolymer, e.g., a diblock copolymer composed of a hydrophilic component and a hydrophobic component. For example, the hydrophilic component can include polyoxyethylene subunits and the hydrophobic component can include polyoxypropylene subunits.

[0356] The following sections 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 for the transduced cells.

[0357] Diblock Copolymer Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include those that contain a hydrophilic block covalently bonded to a hydrophobic block. Such diblock copolymers include those that contain PEO and PPO subunits. Suitable diblock copolymers include those in which the PEO subunit of the diblock copolymer has a number average molecular weight (Mn) of about 5,000 g / mol to about 25,000 g / mol. For example, the PEO subunits of the diblock copolymer may have a molecular weight of about 5,500 g / mol, 6,000 g / mol, 6,500 g / mol, 7,000 g / mol, 7,500 g / mol, 8,000 g / mol, 8,5000 g / mol, 9,000 g / mol, 9,500 g / mol, 10,000 g / mol, 10,500 g / mol, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500 g / mol, 14,000 g / mol, 14,500 g / mol, 15,000 g / mol 1, 15,500 g / mol, 16,000 g / mol, 16,500 g / mol, 17,000 g / mol, 17,500 g / mol, 18,000 g / mol, 18,500 g / mol, 19,000 g / mol, 19,500 g / mol, 20,000 g / mol, 20,500 g / mol, 21,000 g / mol, 21,500 g / mol, 22,000 g / mol, 22,500 g / mol, 23,000 g / mol, 23,500 g / mol, 24,000 g / mol, 24,500 g / mol, or 25,000 g / mol.

[0358] For example, in some embodiments, the PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol to about 19,000 g / mol. In some specific embodiments, the PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol, 9,500 g / mol, 13,800 g / mol, 15,500 g / mol, 18,000 g / mol, or 19,000 g / mol.

[0359] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include those in which the PPO subunits of the diblock copolymer have an Mn of about 2,000 g / mol to about 10,000 g / mol (e.g., the PPO subunits of the diblock copolymer have an Mn of about 2,000 g / mol, 2,500 g / mol, 3,000 g / mol, 3,500 g / mol, 4,000 g / mol, 4,500 g / mol, 5,000 g / mol, 5,500 g / mol, 6,000 g / mol, 6,500 g / mol, 7,000 g / mol, 7,500 g / mol, 8,000 g / mol, 8,500 g / mol, 9,000 g / mol, 9,500 g / mol, or 10,000 g / mol).

[0360] For example, in some embodiments, the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol to about 5,500 g / mol, and in some particular embodiments, the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol or 5,500 g / mol.

[0361] In some embodiments, the diblock copolymer comprises more than 40% by weight (e.g., 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%, 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%, 96%, 97%, 98 %, 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).

[0362] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 50% by weight (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%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more).

[0363] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 60% by weight (e.g., 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).

[0364] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 70% by weight (e.g., 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).

[0365] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 40% to about 90% (e.g., 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%).

[0366] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 50% to about 85% (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%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%).

[0367] In some embodiments, the diblock copolymer has an average ethylene oxide content of about 60% to about 80% (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%).

[0368] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include those having an Mn of greater than about 8,000 g / mol (e.g., greater than about 8,500 g / mol, 9,000 g / mol, or 10,000 g / mol). For example, the diblock copolymer has an Mn of greater than about 10,000 g / mol (e.g., the diblock copolymer has an Mn of 10,500, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500 g / mol, 14,000 g / mol, 14,500 g / mol, 15,000 g / mol, 15,500 g / mol, 16,000 g / mol, 16,500 g / mol, 17,000 g / mol, 17,500 g / mol, 18,000 g / mol, 18,500 g / mol, 19,000 g / mol, 19,500 g / mol, 20,000 g / mol, 21,000 g / mol, 22,000 g / mol, 23,000 g / mol, 24,000 g / mol, 25,000 g / mol, 26,000 g / mol, 27,000 g / mol, 28,000 g / mol, 29,000 g / mol, 30,000 g / mol, 31,000 g / mol, 32,000 g / mol, 33,000 g / mol, 34,000 g / mol, 35,000 g / mol, 36,000 g / mol, 37,000 g / mol, 38,000 g / mol, 39,000 g / mol, 39,500 g / mol, 40,000 g / mol, 41,000 g / mol, 42,000 g / mol, (having a Mn of greater than 0,000 g / mol, 20,500 g / mol, 21,000 g / mol, 21,500 g / mol, 22,000 g / mol, 22,500 g / mol, 23,000 g / mol, 23,500 g / mol, 24,000 g / mol, 24,500 g / mol, 25,000 g / mol, 25,000 g / mol, 26,000 g / mol, 26,500 g / mol, 27,000 g / mol, 27,500 g / mol, 28,000 g / mol, 28,500 g / mol, 29,000 g / mol, 29,500 g / mol, 30,000 g / mol, or more).

[0369] In some embodiments, the diblock copolymer has a Mn of about 10,000 g / mol to about 30,000 g / mol (e.g., the diblock copolymer has a Mn of about 10,500 g / mol, 11,000 g / mol, 11,500 g / mol, 12,000 g / mol, 12,500 g / mol, 13,000 g / mol, 13,500 g / mol, 14,000 g / mol, 14,500 g / mol, 15,000 g / mol, 15,500 g / mol, 16,000 g / mol, 16,500 g / mol, 17,000 g / mol, 17,500 g / mol, 18,000 g / mol, 18,500 g / mol, 19,000 g / mol, 20,000 g / mol, 21,000 g / mol, 22,000 g / mol, 23,000 g / mol, 24,000 g / mol, 25,000 g / mol, 26,000 g / mol, 27,000 g / mol, 28,000 g / mol, 29,000 g / mol, 30,000 g / mol, 31,000 g / mol, 32,000 g / mol, 33,000 g / mol, 34,000 g / mol, 35,000 g / mol, 36,000 g / mol, 37,000 g / mol, 38,000 g / mol, 39,000 g / mol, 40,000 g / mol, 41,000 g / mol, 42,000 g / mol, 43,000 g 20,000g / mol, 20,500g / mol, 21,000g / mol, 21,500g / mol, 22,000g / mol, 22,500g / mol, 23,000g / mol, 23,500g / mol, 24,000g / mol, 24,500g / mol, 25,000g / mol, 25,000g / mol, 26,000g / mol, 26,500g / mol, 27,000g / mol, 27,500g / mol, 28,000g / mol, 28,500g / mol, 29,000g / mol, 29,500g / mol, or 30,000g / mol). For example, in some embodiments, the diblock copolymer has an Mn of about 12,000 g / mol to about 25,000 g / mol (e.g., about 12,500 g / mol to about 23,500 g / mol). In some specific embodiments, the diblock copolymer has an Mn of about 12,500 g / mol, 13,000 g / mol, 17,300 g / mol, 19,000 g / mol, 22,500 g / mol, or 23,500 g / mol.

[0370] In some embodiments, the diblock copolymer has a polydispersity index (Mw / Mn) of about 1 to about 1.2 (e.g., the diblock copolymer has a polydispersity index of about 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, or 1.20). For example, in some embodiments, the diblock copolymer has a polydispersity index of about 1.06 to about 1.17. In some particular embodiments, the diblock copolymer has a polydispersity index of from about 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, or 1.17.

[0371] Diblock copolymers that can be used in conjunction with the compositions and methods described herein can have the following structure: X1-[PEO] m -L-[PPO] n -X2 wherein m and n are integers; L is absent or a chemical linker, and X1 and X2 each independently represent an optionally present chemical substituent.

[0372] In some embodiments, the diblock copolymer has the following structure: X1-[PEO] m -[PPO] n -X2 wherein m and n are integers, and X1 and X2 each independently represent an optionally present chemical substituent.

[0373] Due to variations that occur during the synthesis of diblock copolymers containing PPO and PEO subunits, one skilled in the art will understand that the values ​​of m and n can vary, for example, by up to two-fold above and below the recited values. Thus, a value of n=50 represents a heterogeneous mixture of diblock copolymers where n can be from 25 to 100, e.g., 25 to 75, 26 to 74, 27 to 73, 28 to 72, 29 to 71, 30 to 70, 31 to 69, 32 to 68, 33 to 67, 34 to 66, 35 to 65, 36 to 64, 37 to 63, 38 to 62, 39 to 61, 40 to 60, 41 to 59, 42 to 58, 43 to 57, 44 to 56, 45 to 55, etc. Similarly, a value of n=60 represents a heterogeneous mixture of diblock copolymers where n can be from 30 to 120, e.g., 30 to 90. Similarly, a value of n=70 represents a heterogeneous mixture of diblock copolymers where n can be from 35 to 140, such as from 35 to 105.

[0374] Exemplary linkers (L) that can be used in combination with the diblock copolymers described herein are described in more detail below.

[0375] In some embodiments, X and X are each independently selected from the group consisting of: H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 It is alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido.

[0376] In some embodiments, X and X are each independently absent, H, OH, optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or optionally substituted C 1-6 It is alkylamino.

[0377] For example, in some embodiments, X1 and X2 are each independently absent, H, OH, H2N, H3CO, ethyl-O, n-butyl-O, tert-butyl-O, n-butyl, or tert-butyl.

[0378] In some embodiments of the diblock copolymer, m is from about 100 to about 500. For example, in some embodiments, m is from about 200 to about 450, e.g., from about 205 to about 432. In some embodiments, m is from 162 to 486 (e.g., 323). In some embodiments, m is from 159 to 477 (e.g., 318). In some embodiments, m is from 108 to 324 (e.g., 216). In some embodiments, m is from 103 to 309 (e.g., 205). In some embodiments, m is from 148 to 444 (e.g., 295). In some embodiments, m is from 171 to 513 (e.g., 341). In some embodiments, m is from 142 to 426 (e.g., 284). In some embodiments, m is from 100 to 300 (e.g., 200). In some embodiments, m is from 113 to 339 (e.g., 225). In some embodiments, m is 109 to 327 (e.g., 217). In some embodiments, m is 115 to 345 (e.g., 230). In some embodiments, m is 120 to 360 (e.g., 240).

[0379] In some particular embodiments, m is 200, 205, 216, 217, 225, 230, 240, 284, 314, 318, 323, 352, 409, or 432.

[0380] In some embodiments of the diblock copolymer, n is from about 10 to about 200. For example, in some embodiments, n is from about 40 to about 100, e.g., from about 50 to about 95. In some embodiments, n is from 43 to 129 (e.g., 86). In some embodiments, n is from 27 to 81 (e.g., 53). In some embodiments, n is from 29 to 87 (e.g., 57). In some embodiments, n is from 28 to 84 (e.g., 55). In some embodiments, n is from 30 to 90 (e.g., 60). In some embodiments, n is from 33 to 99 (e.g., 65). In some embodiments, n is from 28 to 84 (e.g., 55).

[0381] In some specific embodiments, n is 50, 53, 55, 57, 60, 65, 70, 86, or 95.

[0382] In some embodiments of the diblock copolymer, m is from about 100 to about 500, and n is from about 10 to about 200, e.g., from about 40 to 100, or from 50 to about 95. For example, in some embodiments, m is from about 200 to about 450, e.g., from about 205 to about 432, and n is from about 10 to about 200, e.g., from about 40 to 100, or from 50 to about 95.

[0383] In some embodiments, m is 162 to 486 (e.g., 323) and n is 43 to 129 (e.g., 86). In some embodiments, m is 162 to 486 (e.g., 323) and n is 27 to 81 (e.g., 53). In some embodiments, m is 162 to 486 (e.g., 323) and n is 29 to 87 (e.g., 57). In some embodiments, m is 162 to 486 (e.g., 323) and n is 28 to 84 (e.g., 55). In some embodiments, m is 162 to 486 (e.g., 323) and n is 30 to 90 (e.g., 60). In some embodiments, m is 162 to 486 (e.g., 323) and n is 33 to 99 (e.g., 65). In some embodiments, m is 162 to 486 (eg, 323) and n is 28 to 84 (eg, 55).

[0384] In some embodiments, m is 159 to 477 (e.g., 318) and n is 43 to 129 (e.g., 86). In some embodiments, m is 159 to 477 (e.g., 318) and n is 27 to 81 (e.g., 53). In some embodiments, m is 159 to 477 (e.g., 318) and n is 29 to 87 (e.g., 57). In some embodiments, m is 159 to 477 (e.g., 318) and n is 28 to 84 (e.g., 55). In some embodiments, m is 159 to 477 (e.g., 318) and n is 30 to 90 (e.g., 60). In some embodiments, m is 159 to 477 (e.g., 318) and n is 33 to 99 (e.g., 65). In some embodiments, m is 159 to 477 (eg, 318) and n is 28 to 84 (eg, 55).

[0385] In some embodiments, m is 108 to 324 (e.g., 216) and n is 43 to 129 (e.g., 86). In some embodiments, m is 108 to 324 (e.g., 216) and n is 27 to 81 (e.g., 53). In some embodiments, m is 108 to 324 (e.g., 216) and n is 29 to 87 (e.g., 57). In some embodiments, m is 108 to 324 (e.g., 216) and n is 28 to 84 (e.g., 55). In some embodiments, m is 108 to 324 (e.g., 216) and n is 30 to 90 (e.g., 60). In some embodiments, m is 108 to 324 (e.g., 216) and n is 33 to 99 (e.g., 65). In some embodiments, m is between 108 and 324 (eg, 216) and n is between 28 and 84 (eg, 55).

[0386] In some embodiments, m is 103 to 309 (e.g., 205) and n is 43 to 129 (e.g., 86). In some embodiments, m is 103 to 309 (e.g., 205) and n is 27 to 81 (e.g., 53). In some embodiments, m is 103 to 309 (e.g., 205) and n is 29 to 87 (e.g., 57). In some embodiments, m is 103 to 309 (e.g., 205) and n is 28 to 84 (e.g., 55). In some embodiments, m is 103 to 309 (e.g., 205) and n is 30 to 90 (e.g., 60). In some embodiments, m is 103 to 309 (e.g., 205) and n is 33 to 99 (e.g., 65). In some embodiments, m is between 103 and 309 (eg, 205) and n is between 28 and 84 (eg, 55).

[0387] In some embodiments, m is 148 to 444 (e.g., 295) and n is 43 to 129 (e.g., 86). In some embodiments, m is 148 to 444 (e.g., 295) and n is 27 to 81 (e.g., 53). In some embodiments, m is 148 to 444 (e.g., 295) and n is 29 to 87 (e.g., 57). In some embodiments, m is 148 to 444 (e.g., 295) and n is 28 to 84 (e.g., 55). In some embodiments, m is 148 to 444 (e.g., 295) and n is 30 to 90 (e.g., 60). In some embodiments, m is 148 to 444 (e.g., 295) and n is 33 to 99 (e.g., 65). In some embodiments, m is 148 to 444 (eg, 295) and n is 28 to 84 (eg, 55).

[0388] In some embodiments, m is 171 to 513 (e.g., 341) and n is 43 to 129 (e.g., 86). In some embodiments, m is 171 to 513 (e.g., 341) and n is 27 to 81 (e.g., 53). In some embodiments, m is 171 to 513 (e.g., 341) and n is 29 to 87 (e.g., 57). In some embodiments, m is 171 to 513 (e.g., 341) and n is 28 to 84 (e.g., 55). In some embodiments, m is 171 to 513 (e.g., 341) and n is 30 to 90 (e.g., 60). In some embodiments, m is 171 to 513 (e.g., 341) and n is 33 to 99 (e.g., 65). In some embodiments, m is 171 to 513 (eg, 341) and n is 28 to 84 (eg, 55).

[0389] In some embodiments, m is 142 to 426 (e.g., 284) and n is 43 to 129 (e.g., 86). In some embodiments, m is 142 to 426 (e.g., 284) and n is 27 to 81 (e.g., 53). In some embodiments, m is 142 to 426 (e.g., 284) and n is 29 to 87 (e.g., 57). In some embodiments, m is 142 to 426 (e.g., 284) and n is 28 to 84 (e.g., 55). In some embodiments, m is 142 to 426 (e.g., 284) and n is 30 to 90 (e.g., 60). In some embodiments, m is 142 to 426 (e.g., 284) and n is 33 to 99 (e.g., 65). In some embodiments, m is 142 to 426 (eg, 284) and n is 28 to 84 (eg, 55).

[0390] In some embodiments, m is 100 to 300 (e.g., 200) and n is 43 to 129 (e.g., 86). In some embodiments, m is 100 to 300 (e.g., 200) and n is 27 to 81 (e.g., 53). In some embodiments, m is 100 to 300 (e.g., 200) and n is 29 to 87 (e.g., 57). In some embodiments, m is 100 to 300 (e.g., 200) and n is 28 to 84 (e.g., 55). In some embodiments, m is 100 to 300 (e.g., 200) and n is 30 to 90 (e.g., 60). In some embodiments, m is 100 to 300 (e.g., 200) and n is 33 to 99 (e.g., 65). In some embodiments, m is 100 to 300 (eg, 200) and n is 28 to 84 (eg, 55).

[0391] In some embodiments, m is 113 to 339 (e.g., 225) and n is 43 to 129 (e.g., 86). In some embodiments, m is 113 to 339 (e.g., 225) and n is 27 to 81 (e.g., 53). In some embodiments, m is 113 to 339 (e.g., 225) and n is 29 to 87 (e.g., 57). In some embodiments, m is 113 to 339 (e.g., 225) and n is 28 to 84 (e.g., 55). In some embodiments, m is 113 to 339 (e.g., 225) and n is 30 to 90 (e.g., 60). In some embodiments, m is 113 to 339 (e.g., 225) and n is 33 to 99 (e.g., 65). In some embodiments, m is 113 to 339 (eg, 225) and n is 28 to 84 (eg, 55).

[0392] In some embodiments, m is 109 to 327 (e.g., 217) and n is 43 to 129 (e.g., 86). In some embodiments, m is 109 to 327 (e.g., 217) and n is 27 to 81 (e.g., 53). In some embodiments, m is 109 to 327 (e.g., 217) and n is 29 to 87 (e.g., 57). In some embodiments, m is 109 to 327 (e.g., 217) and n is 28 to 84 (e.g., 55). In some embodiments, m is 109 to 327 (e.g., 217) and n is 30 to 90 (e.g., 60). In some embodiments, m is 109 to 327 (e.g., 217) and n is 33 to 99 (e.g., 65). In some embodiments, m is 109 to 327 (eg, 217) and n is 28 to 84 (eg, 55).

[0393] In some embodiments, m is 115 to 345 (e.g., 230) and n is 43 to 129 (e.g., 86). In some embodiments, m is 115 to 345 (e.g., 230) and n is 27 to 81 (e.g., 53). In some embodiments, m is 115 to 345 (e.g., 230) and n is 29 to 87 (e.g., 57). In some embodiments, m is 115 to 345 (e.g., 230) and n is 28 to 84 (e.g., 55). In some embodiments, m is 115 to 345 (e.g., 230) and n is 30 to 90 (e.g., 60). In some embodiments, m is 115 to 345 (e.g., 230) and n is 33 to 99 (e.g., 65). In some embodiments, m is 115 to 345 (eg, 230) and n is 28 to 84 (eg, 55).

[0394] In some embodiments, m is 120 to 360 (e.g., 240) and n is 43 to 129 (e.g., 86). In some embodiments, m is 120 to 360 (e.g., 240) and n is 27 to 81 (e.g., 53). In some embodiments, m is 120 to 360 (e.g., 240) and n is 29 to 87 (e.g., 57). In some embodiments, m is 120 to 360 (e.g., 240) and n is 28 to 84 (e.g., 55). In some embodiments, m is 120 to 360 (e.g., 240) and n is 30 to 90 (e.g., 60). In some embodiments, m is 120 to 360 (e.g., 240) and n is 33 to 99 (e.g., 65). In some embodiments, m is 120 to 360 (eg, 240) and n is 28 to 84 (eg, 55).

[0395] In some embodiments of the diblock polymer, m is 205, 216, 314, 352, 409, or 432, and n is 50, 60, 70, or 95. In some embodiments, m is 205 and n is 60. In some embodiments, m is 216 and n is 60. In some embodiments, m is 216 and n is 50. In some embodiments, m is 216 and n is 70. In some embodiments, m is 314 and n is 60. In some embodiments, m is 352 and n is 60. In some embodiments, m is 409 and n is 95. In some embodiments, m is 432 and n is 60.

[0396] In some embodiments of the diblock copolymer, the ratio of m:n is from about 1 to about 12. For example, in some embodiments, the ratio of m:n is from about 2 to about 8, e.g., from about 3.4 to about 7.2. In some embodiments, the ratio of m:n is from about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 ...8, 5.9, 5.1, 5.2, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.1, 5.2, 5.3, 5.4, 5.5 .4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 9.5, 9.6, 8.7, 8.8, 8.9, 9 or higher. In some particular embodiments, the ratio of m:n is about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, or more.

[0397] In some embodiments, the diblock copolymer has the following structure:

[0398] [ka]

[0399] In some embodiments, the diblock copolymer has a structure selected from the following species: In each structure, the listed values ​​of n and m should be understood to represent a heterogeneous mixture of diblock copolymers in which n and m can vary from up to two-fold below the listed value to two-fold above the listed value. [PEO] 323 -[PPO]86 -OH, HOCH2CH2-[PEO] 323 -[PPO] 86 -On-butyl, [PEO] 318 -[PPO] 53 -OH, HOCH2CH2-[PEO] 318 -[PPO] 53- On-butyl, [PEO] 216 -[PPO] 53 -OH, HOCH2CH2-[PEO] 216 -[PPO] 53 -On-butyl, [PEO] 205 -[PPO] 53 -OH, HOCH2CH2-[PEO] 205 -[PPO] 53 -On-butyl, [PEO] 295 -[PPO] 57 -OH, HOCH2CH2-[PEO] 295 -[PPO] 57 -On-butyl, [PEO] 341 -[PPO] 57 -OH, HOCH2CH2-[PEO] 341 -[PPO] 57 -On-butyl, [PEO] 284 -[PPO] 57 -OH, HOCH2CH2-[PEO] 284 -[PPO] 57 -On-butyl, [PEO] 200 -[PPO] 55 -OH, HOCH2CH2-[PEO] 200 -[PPO] 55 -On-butyl, [PEO] 205 -[PPO] 60 -OH, HOCH2CH2-[PEO] 205 -[PPO] 60 -On-butyl, [PEO] 217 -[PPO] 60 -OH, HOCH2CH2-[PEO] 217 -[PPO] 60 -On-butyl, [PEO] 230 -[PPO] 65 -OH, HOCH2CH2-[PEO] 230 -[PPO] 65 -On-butyl, [PEO] 240 -[PPO] 55 -OH, HOCH2CH2-[PEO] 240 -[PPO] 55 -On-butyl, [PEO] 205 -[PPO] 60 -OH, HOCH2CH2-[PEO] 205 -[PPO] 60 -On-butyl, [PEO] 314 -[PPO] 60 -OH, HOCH2CH2-[PEO] 314 -[PPO] 60 -On-butyl, [PEO] 352 -[PPO] 60 -OH, HOCH2CH2-[PEO] 352 -[PPO] 60 -On-butyl, [PEO] 409 -[PPO] 95 -OH, HOCH2CH2-[PEO] 409 -[PPO] 95 -On-butyl, [PEO] 432 -[PPO] 60 -OH, HOCH2CH2-[PEO] 432-[PPO] 60 -On-butyl, [PEO] 216 -[PPO] 60 -OH, [PEO] 216 -[PPO] 60 -n-butyl, HO-[PEO] 216 -[PPO] 60 -n-butyl, HOCH2CH2-[PEO] 216 -[PPO] 50 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 50 -OH, HOCH2CH2-[PEO] 216 -[PPO] 60 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 60 -OH, HOCH2CH2-[PEO] 216 -[PPO] 70 -On-butyl, HOCH2CH2-[PEO] 216 -[PPO] 70 -OH,

[0400] [ka] Showing TIFF2026010071000077.tif220170.

[0401] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 205 -[PPO] 60 -OH.

[0402] This diblock copolymer has the approximate chemical formula H(C2H4O) 205 (C3H6O) 60OH. The Mn of this diblock copolymer is about 12,500 g / mol. The polydispersity index of this diblock copolymer is about 1.1.

[0403] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 216 -[PPO] 60 -OH.

[0404] This diblock copolymer has the approximate chemical formula H(C2H4O) 216 (C3H6O) 60 OH. The Mn of this diblock copolymer is about 13,000 g / mol. The polydispersity index of this diblock copolymer is about 1.08.

[0405] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 314 -[PPO] 60 -OH.

[0406] This diblock copolymer has the approximate chemical formula H(C2H4O) 314 (C3H6O) 60 OH. The Mn of this diblock copolymer is about 17,300 g / mol. The polydispersity index of this diblock copolymer is about 1.13.

[0407] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 352 -[PPO] 60 -OH.

[0408] This diblock copolymer has the approximate chemical formula H(C2H4O) 352 (C3H6O) 60OH. The Mn of this diblock copolymer is about 19,000 g / mol. The polydispersity index of this diblock copolymer is about 1.13.

[0409] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 409 -[PPO] 95 -OH.

[0410] This diblock copolymer has the approximate chemical formula H(C2H4O) 409 (C3H6O) 95 OH. The Mn of this diblock copolymer is about 23,500 g / mol. The polydispersity index of this diblock copolymer is about 1.17.

[0411] Diblock copolymers that can be used in conjunction with the compositions and methods of the present disclosure include diblock copolymers having the following structure: [PEO] 432 -[PPO] 60 -OH.

[0412] This diblock copolymer has the approximate chemical formula H(C2H4O) 432 (C3H6O) 60 OH. The Mn of this diblock copolymer is about 22,500 g / mol. The polydispersity index of this diblock copolymer is about 1.11.

[0413] The ethylene oxide content and propylene oxide content of the diblock copolymers described herein can be measured using the method disclosed in Alexandridis and Hatton, Colloids and Surfaces A: Physicochemical and Engineering Aspects 96:1-46 (1995), the entire disclosure of which is incorporated herein by reference. The diblock copolymers described herein can be synthesized, for example, according to the method described in Feng et al., Polymers 9:1-31, 2017, the entire disclosure of which is incorporated herein by reference.

[0414] In some embodiments, diblock copolymers that can be used in conjunction with the compositions and methods described herein include, for example, poly(ethylene glycol)-poly(γ-benzyl L-glutamate) PEG-PBLA, poly(ethylene glycol)-poly(D,L-lactic acid) PEG-PDLLA, poly(ethylene glycol)-poly(L-lactic acid) PEG-PLLA, poly(ethylene glycol)-poly(ε-caprolactone) PEG-PCL, poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) PEG-PLGA, poly(ethylene glycol)-poly(γ-benzyl L-glutamate) PEG-PBLG, poly(ethylene glycol)-poly(β-benzyl L-aspartate) PEG-PBLA, poly(ethylene glycol)-poly(α-benzyl carboxylate-ε-caprolactone) PEG-PBCL, and poly(ethylene glycol)-poly(δ-valerolactone) PEG-PVL. Such diblock copolymers include, for example, PEG 5000 -PCL 5000 , PEG 2000 -PCL 1400 , MPEG 5000 -PCL 5000 , MPEG 5000 -PCL 13000 , MPEG 5000 -PCL 24000 , PEG 2000 -PCL 2000 , MPEG5000 -PCL 2500 、MPEG 5000 -PCL 5000 、MPEG 5000 -PCL 8500 、MPEG 5000 -PCL 24700 、MPEG 2000 -PCL 1200 、MPEG2000-PCL 2700 、MPEG 5000 -PCL 3800 、MPEG 5000 -PCL 18000 、PEG 5000 -PCL 4000 、PEG 2000 -PCL 900 、PEG 1980 -PCL 1368 、PEG 1980 -PCL 2622 、PEG 1980 -PCL 17328 、PEG 2000 -PCL 2280 、PEG 5000 -PCL 5000 、PEG 5000 -PCL 24000 、PEG 5000 -PCL 5000 、PEG 5000 -PCL 24000 、PEG 5000 -PCL 4790 、PEG 5000 -PCL 10000 、MPEG 5333 -PCL 2638 、MPEG 5333 -PCL 4984 、MPEG 5333 -PCL 8034 、MPEG 5333 -PCL 9068 、MPEG 5000 -PCL 2166 、MPEG 2000 -PCL 1320 、MPEG 2000 -PCL 852 、MPEG 750 -PCL 464 、MPEG 750 -PCL 323 、MPEG750 -PCL 197 , MPEG-PCL, PEG 5000 -PDLLA 4200 , PEG 5000 -PDLLA 45000 , MPEG 2000 -PDLLA 2000 , MPEG 2000 -PDLLA 1333 , MPEG 5000 -PDLLA 2143 , PEG 52000 -PDLLA 56000 , PEG 91000 -PDLLA 56000 , PEG 4100 -PDLLA 1200 , PEG 6000 -PDLLA 3000 , PEG 5700 -PDLLA 5400 , PEG 6100 -PDLLA 7800 , PEG 5000 -PBCL 4700 , PEG 5000 -PBCL 4470 , PEG 12000 -PBLA 5000 , PEG 12000 -PBLA 3000 , PEG-PBLA, PEG 12000 -PBLA 5000 , MPEG 2000 -PVL 1000 , MPEG 2000 -PVL 2000 , MPEG 5000 -PVL 2600 , and MPEG 5000 -PVL 4900 These diblock copolymers are described, for example, in Hussein et al. Materials 11:1-26, 2018, the disclosure of which is incorporated herein in its entirety.

[0415] Linker The diblock copolymers described herein can optionally include a linker connecting the PEO and PPO subunit blocks of the polymer. The PEO and PPO components of the diblock copolymer can be directly bonded to each other, for example, without an intervening linker. The linker can be a peptide linker or a synthetic linker.

[0416] Synthetic Linkers Various linkers can be used to covalently bond the PEO component to the PPO component, for example, to form the diblock copolymers described herein. Exemplary linkers include those that can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference for its disclosure regarding linkers suitable for chemical coupling). Examples of linkers useful in synthesizing the conjugates described herein include those containing Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, among others, which are suitable for reacting with electrophiles, such as nucleophilic substituents present in antibodies, antigen-binding fragments, proteins, peptides, and small molecules (such as amine and thiol moieties). For example, suitable linkers for synthesizing diblock copolymers include, but are not limited to, alkyl, cycloalkyl, and heterocycloalkyl linkers, such as open-chain ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl chains, cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, piperidinyl, morpholino, or others containing two reactive moieties (e.g., a halogen atom, an aldehyde group, an ester group, an acyl chloride group, an acyl anhydride group, a tosyl group, a mesyl group, or a brosyl group, among others, which can be displaced by reactive nucleophilic atoms present in the PEO or PPO polymer), aryl or heteroaryl linkers, such as a benzyl, naphthyl, or pyridyl group, which contain two halomethyl groups displaceable by reactive nucleophilic atoms present in the PEO or PPO polymer.Exemplary linkers include succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others, as described in, for example, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference for its disclosure regarding chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linker described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference for its disclosure regarding linkers for chemical conjugation.

[0417] As described herein, additional linkers that can connect one block of the copolymer to another include linkers that are covalently attached to one block of the copolymer (e.g., PEO or PPO) at one end of the linker and contain a chemical moiety formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the other component of the diblock copolymer (e.g., PEO or PPO) at the other end of the linker. Exemplary reactive substituents that can be used to form linkers include, but are not limited to, the hydroxyl moieties of serine, threonine, and tyrosine residues; the amino moieties of lysine residues; the carboxyl moieties of aspartic acid and glutamic acid residues; and the thiol moieties of cysteine ​​residues, as well as propargyl, azide, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of unnatural amino acids. Linkers useful in combination with the diblock copolymers described herein include, but are not limited to, linkers containing chemical moieties formed by coupling reactions as described in Table 2 below. The curved lines represent the points of attachment to each component of the conjugate.

[0418] [Table 3] TIFF2026010071000079.tif203170TIFF2026010071000080.tif193170TIFF2026010071000081.tif229170TIFF2026010071000082.tif229170

[0419] Peptide Linker In addition to the synthetic linkers described above, attachment of PEO polymers to PPO polymers can be achieved via peptide linkers. Exemplary peptide linkers include those containing one or more glycine residues. Such linkers can be sterically flexible due to the ability of glycine to access various torsion angles. For example, peptide linkers useful in combination with the compositions and methods described herein include polyglycine, polyserine, or combinations thereof. Additional examples of peptide linkers include those that also contain one or more polar amino acids, such as serine and threonine. For example, linkers useful in combination with the compositions and methods described herein include those containing one or more repeats of glycine and serine. Additional linkers include those containing one or more cationic or anionic residues, such as lysine, arginine, aspartate, or glutamate residues.

[0420] PKC regulators Various agents can be used to reduce PKC activity and / or expression. Without being limited by mechanism, such agents can complement viral transduction by stimulating Akt signaling and / or maintaining cofilin in a dephosphorylated state, promoting actin depolymerization. This actin depolymerization event may serve to remove a physical barrier that inhibits viral vector entry into the nucleus of target cells.

[0421] 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 staurosporine or a variant thereof. For example, the PKC inhibitor is a compound represented by formula (I):

[0422] [ka] wherein R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, 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, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted acyl; R a and R b each independently represents 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 a and R b are joined together 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 dis H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 is alkynyl, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; TIFF2026010071000084.tif4169 represents an arbitrarily present bond, n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0423] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO 1991 / 009034, the entire disclosure of which is incorporated herein by reference. Examples of such staurosporine variants include those represented by formula (II):

[0424] [ka] wherein R1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, oxo, or thiocarbonyl; R2 is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted acyl; R a and R b are joined together with the atoms to which they are attached to form an optionally substituted and optionally fused heterocycloalkyl ring; R c is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0425] Further examples of such staurosporine variants include those of formula (III):

[0426] [ka] wherein R1 is H, OH, oxo, or thiocarbonyl; R2 is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted acyl; Ring A is an optionally substituted and optionally fused heterocycloalkyl ring; R c is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0427] Further examples of such staurosporine variants include those of formula (IV):

[0428] [ka] wherein R1 is H, OH, or oxo; Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring; R c is O or S, W is O, NH, or S; Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0429] Further examples of such staurosporine variants include those of formula (V):

[0430] [ka] wherein R1 is H, OH, or oxo; R c is O or S, W is O, NH, or S; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; p is 0 or 1. or a salt thereof.

[0431] Further examples of such staurosporine variants include those of formula (VI):

[0432] [ka] wherein R1 is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; s is an integer from 0 to 8. or a salt thereof.

[0433] Further examples of such staurosporine variants include those of formula (VII):

[0434] [ka] wherein R1 is H, OH, or oxo; R2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; R3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

[0435] Further examples of such staurosporine variants include those of formula (VIII):

[0436] [ka] wherein R1 is H, OH, or oxo; R2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; R3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

[0437] Further examples of such staurosporine variants include those of formula (IX):

[0438] [ka] wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0439] Further examples of such staurosporine variants include those represented by formula (1):

[0440] [ka] or a salt thereof.

[0441] 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-triazaoctacyclo[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).

[0442] [ka] or a salt thereof.

[0443] Further examples of such staurosporine variants include those of formula (X):

[0444] [ka] wherein R1 is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; t is an integer from 0 to 6. or a salt thereof.

[0445] Further examples of such staurosporine variants include those of formula (XI):

[0446] [ka] wherein R1 is H, OH, or oxo; R4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

[0447] An example of such a staurosporine variant is represented by formula (XII):

[0448] [ka] wherein R1 is H, OH, or oxo; R4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

[0449] Further examples of such staurosporine variants include those of formula (XIII):

[0450] [ka] wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, m is an integer from 0 to 4. or a salt thereof.

[0451] Further examples of such staurosporine variants include those represented by formula (3):

[0452] [ka] or a salt thereof.

[0453] Further examples of such staurosporine variants include those represented by formula (4):

[0454] [ka] or a salt thereof.

[0455] Further examples of such staurosporine variants are:

[0456] [ka] or a salt thereof.

[0457] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO 1993 / 007153, the entire disclosure of which is incorporated herein by reference. An example of such a staurosporine variant is represented by formula (XIV):

[0458] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0459] In some embodiments, the PKC inhibitor is a compound represented by formula (XV):

[0460] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0461] In some embodiments, the PKC inhibitor is

[0462] [ka] or a salt thereof.

[0463] 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 herein by reference. An example of such a staurosporine variant is represented by formula (XVI):

[0464] [ka] wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

[0465] In some embodiments, the PKC inhibitor is a compound of formula (XVII):

[0466] [ka] wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

[0467] In some embodiments, the PKC inhibitor is

[0468] [ka] TIFF2026010071000108.tif204170TIFF2026010071000109.tif188170TIFF2026010071000110.tif189170TIFF2026010071000111.tif194170TIFF2026010071000112.tif188170TIFF2026010071000113.tif124170, or a salt thereof.

[0469] 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 herein by reference. An example of such a staurosporine variant is represented by formula (XVIII):

[0470] [ka] [Wherein R is H, OH, C 1-6 alkoxy, or oxo; R2 is

[0471] [ka] and optionally, the sugar moiety is derived from D-glucose, D-galactose, or D-mannose; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with 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 2-20alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which may be substituted within the phenyl moiety with halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 is substituted with alkoxy, R5 is H or C 1-6 is alkyl, R6 is free or aliphatic C 2-22 hydroxyl esterified with a carboxylic acid or C 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, free or aliphatic C 2-22 Amino acylated with carboxylic acid, C 1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with alkoxy, R7 is free or aliphatic C 2-22 OH, C esterified with carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, azido, free or aliphatic C 2-22 Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 substituted by alkoxycarbonyl. or a salt thereof.

[0472] In some embodiments, the PKC inhibitor is a compound of formula (XIX):

[0473] [ka] [Wherein R is H, OH, C 1-6 alkoxy, or oxo; R2 is

[0474] [ka] and; R3 is H, OH, C 1-6 Alkanoyloxy, C 1-6 alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with 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 alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which may be substituted within the phenyl moiety with halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 is substituted with alkoxy, R5 is H or C 1-6 is alkyl, R6 is free or aliphatic C 2-22 hydroxyl esterified with a carboxylic acid or C 1-6 Alkoxycarbonyloxy, C 1-6Alkyl sulfonyloxy, free or aliphatic C 2-22 Amino acylated with carboxylic acid, C 1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, or C 1-6 is substituted with alkoxy, R7 is free or aliphatic C 2-22 OH, C esterified with carboxylic acid 1-6 Alkoxycarbonyloxy, C 1-6 Alkyl sulfonyloxy, azido, free or aliphatic C 2-22 Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C 1-6 Alkyl, C 1-6 Alkoxy, or C 1-6 substituted by alkoxycarbonyl. or a salt thereof.

[0475] 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-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-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, 1-α-anomer of N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4,6-O-triacetyl and N-(1-deoxy-6-O-methyl-2-N-acetylmuramyl)staurosporine, ... and N-(1-deoxy-6-O-methyl-2-N-acetylmuramyl)staurosporine, or a salt thereof.

[0476] 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 herein by reference. An example of such a staurosporine variant is represented by formula (XX):

[0477] [ka] wherein Z1 is H or OH; Z2 is H or OH; R1 is H, halogen, or optionally substituted alkyl; R2 is H or halogen; R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally represented by the formula: wherein X is CH-NH-serine, COCH, CHNHCOCH, CONHCH, or CHNHCOCH, where CH represents a phenyl moiety; or a salt thereof.

[0478] 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 herein by reference. An example of such a staurosporine variant is represented by formula (XXI):

[0479] [ka] wherein Z1 is H or OH; Z2 is H or OH; R1 is H, halogen, or optionally substituted alkyl; R2 is H or halogen; R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally represented by the formula: wherein X is CH-NH-serine, COCH, CHNHCOCH, CONHCH, or CHNHCOCH, where CH represents a phenyl moiety; or a salt thereof.

[0480] In some embodiments, the PKC inhibitor is a staurosporine variant described in US2005 / 0020570, the entire disclosure of which is incorporated herein by reference. Examples of such staurosporine variants are represented by formula (XXII), (XXIII), (XXIV), or (XXV):

[0481] [ka] wherein each R1 is independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R2 is independently 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms and 9 or fewer heteroatoms, or 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 di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each n is independently an integer from 0 to 4; Each m is independently an integer from 0 to 4. or a salt thereof.

[0482] In some embodiments, the PKC inhibitor is a compound of formula (128):

[0483] [ka] or a salt thereof. This compound is also known as K252a.

[0484] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII):

[0485] [ka] wherein each R1 is independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R2 is independently 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each R5 is independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms and 9 or fewer heteroatoms, 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, in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms, and 9 or fewer heteroatoms; each R9 independently is 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R 10are independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms, and in each case having 9 or fewer heteroatoms; 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.

[0486] In some embodiments, the PKC inhibitor is a staurosporine variant described in US 5,624,949, the entire disclosure of which is incorporated herein by reference. An example of such a staurosporine variant is represented by formula (XXVIII):

[0487] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0488] In some embodiments, the PKC inhibitor is a compound of formula (XXIX):

[0489] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0490] In some embodiments, the PKC inhibitor is a compound of formula (XXX):

[0491] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0492] In some embodiments, the PKC inhibitor is a compound of formula (XXXI):

[0493] [ka] wherein R1 is H or optionally substituted C 1-6 is alkyl, R2 is an optionally substituted C 1-6 It is alkyl. or a salt thereof.

[0494] In some embodiments, the PKC inhibitor is

[0495] [ka] The compound is selected from TIFF2026010071000128.tif225170TIFF2026010071000129.tif167170TIFF2026010071000130.tif183170TIFF2026010071000131.tif241170.

[0496] In some embodiments, the cells are further contacted with stauprimide, for example, as described in Caravatti et al. Bioorg. Medic. Chem. Letters 4:199-404, 1994, the entire disclosure of which is incorporated herein by reference.

[0497] Interfering RNA The exemplary PKC regulator that can be used in combination with the compositions and methods of the present disclosure includes interfering RNA molecules, such as short interfering RNA (siRNA), short hairpin RNA (shRNA) and / or microRNA (miRNA), that reduce PKC gene expression.The method for producing interfering RNA molecules is known in the art, and is described in detail in, for example, WO2004 / 044136 and US Patent No. 9,150,605, the disclosures of which are each incorporated herein by reference in their entirety.

[0498] HDAC inhibitors To increase transgene expression during viral transduction, various agents can be used to inhibit histone deacetylase. Without being bound by theory, reduced transgene expression from viral vectors can be caused by epigenetic silencing of the vector genome via histone deacetylation. Therefore, the methods described herein can further include contacting cells with an HDAC inhibitor, for example, before, simultaneously with, or after contacting the cells with the diblock copolymer, to improve viral transduction and / or increase transgene expression. Hydroxamic acids represent a particularly robust class of HDAC inhibitors that inhibit these enzymes via a hydroxamate functionality that binds to cationic zinc within the active site of these enzymes. Exemplary inhibitors include trichostatin A, as well as vorinostat (N-hydroxy-N'-phenyl-octanediamide, described in Marks et al., Nature Biotechnology 25, 84 to 90 (2007); Stenger, Community Oncology 4, 384-386 (2007), the disclosures of which are incorporated herein by reference). Other HDAC inhibitors include panobinostat, described in Drugs of the Future 32(4):315-322 (2007), the disclosures of which are incorporated herein by reference.

[0499] [ka] Panobinostat

[0500] Further examples of hydroxamic acid inhibitors of histone deacetylase include the compounds shown below, which are described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference.

[0501] [ka] Trichostatin A

[0502] [ka] SAHA

[0503] [ka] Tubasin

[0504] [ka] LAQ824

[0505] [ka] Sulfonamides

[0506] [ka] Scriptide

[0507] [ka] CBHA

[0508] [ka] Oxamflatin

[0509] Other HDAC inhibitors that do not contain hydroxamate substituents have also been developed, including valproic acid (Gottlicher, et al., EMBO J. 20(24):6969-6978 (2001)) and mocetinostat (N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide, described in Balasubramanian et al., Cancer Letters 280:211-221 (2009)), the disclosures of each of which are incorporated herein by reference. Other small molecule inhibitors that utilize chemical functionality different from hydroxamate include those described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosures of which are incorporated herein by reference.

[0510] [ka] Phenylbutyric acid

[0511] [ka] MS-275

[0512] [ka] C1-994

[0513] [ka] Trifluoromethyl ketone

[0514] [ka] α-ketoamide

[0515] Further examples of chemical modulators of histone acetylation useful with the compositions and methods of the present invention include modulators of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, Sirt1, Sirt2, and / or HATs, such as butyrylhydroxamic acid, M344, LAQ824 (dacinostat), AR-42, belinostat (PXD101), CUDC-101, scriptaid, sodium phenylbutyrate, tasquinimod, xinostat (JNJ-26481585), pracinostat (SB939), CUDC-907, entinostat (MS-275), mocetinostat (MGCD0103), tubastatin A HCl, PCI-34051, droxinostat, PCI-24781 (abexinostat), RGFP966, rosirinostat (ACY-1215), CI994 (tacedinaline), Tubacin, RG2833 (RGFP109), resminostat, tubastatin A, BRD73954, BG45, 4SC-202, CAY10603, LMK-235, nextulastat A, TMP269, HPOB, cambinol, and anacardic acid.

[0516] In some particular embodiments, the HDAC inhibitor is scriptaid.

[0517] The cells can be contacted with the diblock copolymer and the HDAC inhibitor simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting them with the HDAC inhibitor. In some embodiments, the cells are contacted with the HDAC inhibitor before contacting them with the diblock copolymer.

[0518] Cyclosporine In some embodiments, the cells are further contacted with a cyclosporine, such as cyclosporine A (CsA) or cyclosporine H (CsH), during viral transduction. The cells can be contacted with the diblock copolymer and the cyclosporine simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the cyclosporine. In some embodiments, the cells are contacted with the cyclosporine before contacting with the diblock copolymer.

[0519] In some embodiments, the cyclosporine is CsH.

[0520] In some embodiments, the concentration of cyclosporine when contacted with cells is about 1 μM to about 10 μM (e.g., about 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, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 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, 3.9 μM, 4.0 μM, 4.1 μ 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 , 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, the cyclosporine is CsA and the concentration of the cyclosporine is about 6 μM when contacted with the cells. In some embodiments, the cyclosporine is CsH and the concentration of the cyclosporine is about 8 μM when contacted with the cells.

[0521] Activators of prostaglandin E receptor signaling In some embodiments, the cells are further contacted with an activator of prostaglandin E receptor signaling. The cells can be contacted with the diblock copolymer and the activator of prostaglandin E receptor signaling simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the activator of prostaglandin E receptor signaling. In some embodiments, the cells are contacted with the activator of prostaglandin E receptor signaling before contacting with the diblock copolymer.

[0522] 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, the disclosures of each of which are incorporated herein by reference as they relate to prostaglandin E receptor signaling activators.

[0523] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of small organic molecules, prostaglandins, Wnt pathway agonists, cAMP / PI3K / AKT pathway agonists, Ca 2+ A small molecule such as a second messenger pathway agonist, a nitric oxide (NO) / angiotensin signaling agonist, or another compound known to stimulate the prostaglandin signaling pathway, such as a compound selected from mebeverine, flurandrenolide, atenolol, pindolol, gaboxadol, kynurenic acid, hydralazine, thiabendazole, bicuculline, vesamicol, peruvoside, imipramine, chlorpropamide, 1,5-pentamethylenetetrazole, 4-aminopyridine, diazoxide, benfotiamine, 12-methoxydodecenoic acid, N-formyl-Met-Leu-Phe, gallamine, IAA94, chlorotrianisene, and / or a derivative of any of these compounds.

[0524] In some embodiments, an activator of prostaglandin E receptor signaling is a naturally occurring or synthetic chemical molecule or polypeptide that binds to and / or interacts with prostaglandin E receptor and typically activates or increases one or more downstream signaling pathways associated with the prostaglandin E receptor.

[0525] 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.

[0526] In some embodiments, the activator of prostaglandin E receptor signaling is PGE2.

[0527] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of 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 butaprost), and the like. and 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).

[0528] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin E receptor ligand, such as prostaglandin E2 (PGE2), or an analog or derivative thereof. Prostaglandins generally refer to hormone-like molecules derived from fatty acids containing 20 carbon atoms and including a 5-carbon ring, as described herein and known in the art. Examples of PGE2 "analogs" or "derivatives" include 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester, II-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-ketofluprostenol, 5-trans These include, but are not limited to, PGE2, 17-phenyl-omega-triol PGE2, PGE2 serinolamide, 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.

[0529] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin analog or derivative having a structure similar to PGE2 substituted with a halogen at position 9 (see, e.g., WO2001 / 12596, which is incorporated by reference herein in its entirety), as well as a 2-decarboxy-2-phosphinico prostaglandin derivative, such as those described in US2006 / 0247214, which is incorporated by reference herein in its entirety.

[0530] In some embodiments, the prostaglandin E receptor signaling activator is a non-PGE2-based ligand. In some embodiments, the prostaglandin E receptor signaling activator is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Further examples of non-PGE2-based EP2 agonists include carbazoles and fluorenes, as disclosed in WO2007 / 071456, the disclosure of which is incorporated herein by reference. 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 for its disclosure of such agonists.

[0531] 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 stimulating prostaglandin E 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 biologically active fragments thereof. Suitable GSK3 inhibitors for use as agents stimulating prostaglandin E receptor signaling pathway bind to GSK3a or GSK3 and reduce the activity of GSK3a or GSK3. Examples of GSK3 inhibitors include BIO(6-bromoindirubin-3), as exemplified in U.S. Pat. Nos. 6,057,117 and 6,608,063, as well as U.S. Pat. Nos. 2004 / 0092535 and 2004 / 0209878. ’ -oxime), LiCl, Li2CO3, or other GSK-3 inhibitors, as well as the ATP-competitive and selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also known as CT-99021 / CHIR-99021 and CT-98023 / CHIR-98023, respectively) (Chiron Corporation, Emeryville, CA). The structure of CHIR-98023 is

[0532] [ka] or a salt thereof.

[0533] In some embodiments, the method further comprises contacting the cell with a GSK3 inhibitor.

[0534] In some embodiments, the GSK3 inhibitor is CHIR-99021.

[0535] In some embodiments, the GSK3 inhibitor is Li2CO3.

[0536] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the cAMP / P13K / AKT second messenger pathway, such as an agent selected from the group consisting of dibutyryl cAMP (DBcAMP), phorbol esters, forskolin, sclarerin, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4-dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, iloprost, pituitary adenylate cyclase-activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), and derivatives of these agents.

[0537] In some embodiments, the activator of prostaglandin E receptor signaling is a Ca agonist, such as an agent selected from the group consisting of Bapta-AM, fendiline, nicardipine, and derivatives of these agents. 2+ Agents that increase signaling through second messenger pathways.

[0538] 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.

[0539] Polycationic Polymers In some embodiments of the methods described herein, the cells are further contacted with a polycationic polymer. The cells can be contacted with the diblock copolymer and the polycationic polymer simultaneously. Alternatively, the cells can be contacted with the diblock copolymer before contacting with the polycationic polymer. In some embodiments, the cells are contacted with the polycationic polymer before contacting with the diblock copolymer.

[0540] In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or a polyethylene glycol / poly-L-lysine block copolymer.

[0541] In some embodiments, the polycationic polymer is protamine sulfate.

[0542] In some embodiments, the cells are further contacted with an expansion agent during the transduction procedure. The cells may be, for example, pluripotent hematopoietic stem cells, and the expansion agent may be a pluripotent hematopoietic stem cell expansion agent, such as those known in the art or described herein.

[0543] Additional transduction enhancers In some embodiments of the methods described herein, during the transduction procedure, the cells are further contacted with an agent that inhibits mTor signaling, which can be, for example, rapamycin, among other inhibitors of mTor signaling.

[0544] In some embodiments of the methods described herein, during the transduction procedure, the cells are further contacted with a transduction-enhancing agent, e.g., in addition to the diblock copolymer. Additional transduction enhancers include, e.g., tacrolimus and vector fusin. In some embodiments, the additional transduction-enhancing agent is tacrolimus. In some embodiments, the additional transduction-enhancing agent is vector fusin.

[0545] Spinoculation In some embodiments of the present disclosure, cells targeted for transduction can be spun, e.g., by centrifugation, while being cultured with a viral vector (e.g., in combination with one or more additional agents described herein). This "spinoculation" process can occur with a centripetal force of, e.g., about 200 x g to about 2,000 x g. The centripetal force can be, e.g., about 300 x g to about 1,200 x g (e.g., about 300 x g, 400 x g, 500 x g, 600 x g, 700 x g, 800 x g, 900 x g, 1,000 x g, 1,100 x g, or 1,200 x g, or higher). In some embodiments, the cells are spun for about 10 minutes to about 3 hours (e.g., 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 more). In some embodiments, the cells are spun at room temperature, e.g., at a temperature of about 25°C.

[0546] 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), the disclosures of each of which are incorporated herein by reference.

[0547] 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 cells of the ectoderm, which gives rise to the skin and nervous system; the endoderm, which forms the gastrointestinal and respiratory tract, endocrine glands, liver, and pancreas; and the mesoderm, which forms bone, cartilage, muscle, connective tissue, and most of the circulatory system.

[0548] Cells that can be used in combination with the compositions and methods described herein include multipotent hematopoietic stem cells and hematopoietic progenitor cells. Hematopoietic stem cells (HSCs) are immature blood cells that have the ability to self-renew and differentiate into mature blood cells, including various lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Human HSCs are CD34+. In addition, HSCs refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). Either of these HSCs can be used in combination with the compositions and methods described herein.

[0549] HSCs and other multipotent progenitor cells can be obtained from blood products. Blood products are products obtained from the body or bodily organs that contain cells of hematopoietic origin. 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 for cells with characteristics of HSCs or myeloid progenitors in various ways. For example, more mature, differentiated cells can be selected based on the cell surface molecules they express. Blood products can be fractionated by positively selecting for CD34+ cells to contain a subpopulation of self-renewing, multipotent, multipotent hematopoietic stem cells that home to the multipotent hematopoietic stem cell niche and can be reintroduced into the transplant recipient to reestablish proliferative and sustained hematopoiesis. Such selection can be achieved, for example, using commercially available magnetic anti-CD34 beads (Dynal, Lake Success, NY). Myeloid progenitor cells can also be isolated based on the markers they express. Unfractionated blood products can be obtained directly from donors or retrieved from cryopreservation. HSCs and myeloid progenitor cells can also be obtained by differentiation of ES cells, iPS cells, or other reprogrammed mature cell types.

[0550] Cells that can be used in conjunction with the compositions and methods described herein include allogeneic and autologous cells. When allogeneic cells are used, the cells may optionally be HLA-matched to the subject receiving the cell treatment.

[0551] Cells that can be used in combination with the compositions and methods described herein include CD34+ / CD90+ cells and CD34+ / CD164+ cells, which may contain a higher percentage of HSCs. These cells are described in Radtke et al. Sci. Transl. Med. 9:1-10, 2017 and Pellin et al. Nat. Comm. 1:2395, 2019, the entire disclosures of each of which are incorporated herein by reference.

[0552] Viral vectors for transgene expression Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes into mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized 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 viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and 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, mutant 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 virus. Examples of retroviruses include avian leukosis sarcoma, avian C virus, mammalian C, B, and D viruses, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor viruses, bovine leukemia viruses, feline leukemia viruses, feline sarcoma viruses, avian leukemia viruses, human T-cell leukemia viruses, baboon endogenous viruses, gibbon leukemia viruses, Mason-Pfizer monkey viruses, simian immunodeficiency viruses, simian sarcoma viruses, Rous sarcoma viruses, and lentiviruses. 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.

[0553] Retroviral vectors The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that can be used in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, transduce a wide range of dividing and non-dividing cell types with high efficiency and confer stable, long-term transgene expression. An overview of optimized methods for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

[0554] The use of lentivirus-based gene transfer technology relies on the in vitro generation of recombinant lentiviral particles that harbor a highly deleted viral genome and harbor a transgene of interest. Specifically, recombinant lentiviruses are recovered by co-expression in permissive cell lines of (1) a packaging construct, i.e., a vector that expresses (or is expressed in trans) the Gag-Pol precursor along with Rev; (2) a vector that generally expresses a heterologous envelope receptor; and (3) a transcription vector that consists of viral cDNA with all open reading frames removed but that maintains the sequences necessary for replication, encapsidation, and expression of the inserted and expressed sequences.

[0555] The LV used in the methods and compositions described herein can include one or more of a 5'-long terminal repeat (LTR), an HIV signal sequence, an HIV Psi signal 5'-splice site (SD), a delta-GAG element, a Rev response element (RRE), a 3'-splice site (SA), an elongation element (EF) 1-alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). Lentiviral vectors optionally include a central polypurine tract (cPPT) and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), as described in U.S. Pat. No. 6,136,597, the disclosure of which is incorporated herein by reference for its disclosure regarding the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Lentiviral vectors can further include a pHR' backbone, which can include, for example, as shown below.

[0556] Lentigen LV, as described in Lu et al., Journal of Gene Medicine 6:963 (2004), can be used to express DNA molecules and / or transduce cells. LVs used in the methods and compositions described herein can include a 5'-long terminal repeat (LTR), an HIV signal sequence, an HIV Psi signal 5'-splice site (SD), a delta-GAG element, a Rev response element (RRE), a 3'-splice site (SA), an elongation element (EF) 1-alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). It will be readily apparent to one skilled in the art that, optionally, one or more of these regions can be replaced with other regions that perform similar functions.

[0557] Enhancer elements can be used to increase expression of modified DNA molecules or to increase the efficiency of lentiviral integration. LVs used in the methods and compositions described herein can include a nef sequence. LVs used in the methods and compositions described herein can include a cPPT sequence, which improves vector integration. cPPT acts as a second origin of (+)-strand DNA synthesis, introducing partial strand overlap at the center of the endogenous HIV genome. Introduction of a cPPT sequence into the transcription vector backbone significantly increased nuclear transport and the total amount of genome integrated into target cell DNA. LVs used in the methods and compositions described herein can include a woodchuck posttranscriptional regulatory element (WPRE). WPREs act at the transcription level, increasing the total amount of mRNA in cells by promoting nuclear export of transcripts and / or increasing the efficiency of polyadenylation of nascent transcripts. Addition of a WPRE to an LV results in substantial improvements in the level of transgene expression from several different promoters both in vitro and in vivo. The LVs used in the methods and compositions described herein can include both a cPPT sequence and a WPRE sequence. The vectors can also include an IRES sequence, which allows for the expression of multiple polypeptides from a single promoter.

[0558] In addition to IRES sequences, other elements that allow for the expression of multiple polypeptides are useful. Vectors used in the methods and compositions described herein can include multiple promoters that allow for the expression of more than one polypeptide. Vectors used in the methods and compositions described herein can include proteolytic cleavage sites that allow for the expression of more than one polypeptide. Examples of proteolytic cleavage sites that allow for the expression of more than one polypeptide 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), the disclosures of which are incorporated herein by reference as they relate to proteolytic cleavage sites that allow for the expression of more than one polypeptide. It will be readily apparent to one of skill in the art that other elements that allow for the expression of multiple polypeptides that are identified in the future will be useful and can be utilized in vectors suitable for use with the compositions and methods described herein.

[0559] The vectors used in the methods and compositions described herein may be clinical grade vectors.

[0560] Treatment methods Exemplary diseases treatable using the compositions and methods of the present disclosure Transgenes that can be introduced into target cells using the compositions and methods of the present disclosure and ultimately delivered to a patient (e.g., by administration of the target cells to the patient) include those encoding therapeutic proteins. Recipients of the transgene (e.g., recipients of cells transduced to express the transgene) may suffer from a disease characterized by a deficiency in the encoded protein. For example, transgenes that can be expressed in target cells and delivered to a patient according to the compositions and methods of the present disclosure include transgenes encoding β-globin, which are particularly useful for treating patients with β-thalassemia. Exemplary nucleic acid and amino acid sequences of human β-globin cDNA and protein are provided below.

[0561] Exemplary wild-type human β-globin cDNA sequence: ATGGTGCATCTGACCCCGGAAGAAAAAAGCGCGGTGACCGCGCTGGGGCAAAGTGAACGTGGATGAAGTGGGCGGCGAAGCGCTGGGCCGCCTGCTGGTGGTGTATCCGTGGACCCAGCCTTTTTGAAAGCTTTGGCGATCTGAGCACCCCGGATGCGGTGATGGGCAACCCGAAAGTGAAAGCGCATGGCAAAAAAGTGCTGGGCGCGTTTAGCG ATGGCCTGGCGCATCTGGATAACCTGAAAGGCACCTTTGCGACCCTGAGCGAACTGCATTGCGATAAACTGCATGTGGATCCGGAAAACTTTCGCCTGCTGGGCAACGTGCTGGTGTGCGTGCTGGCGCATCATTTTGGCAAAGAATTTACCCCGCCGGTGCAGGCGGCGTATCAGAAAGTGGTGGCGGGCGTGGCGAACGCGCTGGCGCATAAATATCAT (SEQ ID NO: 1) An exemplary wild-type human beta-globin amino acid sequence: MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH (SEQ ID NO: 2) Further examples of transgenes that can be used in conjunction with the compositions and methods of the present 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, corticotropin-releasing hormone, thyrotropin-releasing hormone (TRH), follicle-stimulating hormone (FSH), 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, acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (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 (BMP) BMP1 15, any one of the transforming growth factor beta (TGFβ), any one of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BNDF), neurotrophins NT-3, NT-4 / 5, and NT-6, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0562] Further examples of transgenes that can be used in combination with the compositions and methods of the present disclosure include thrombopoietin (TPO), interleukins (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, monocyte chemoattractant protein (MCP-1), leukemia inhibitory factor (LIF), granulocyte myocardium (GMM), and leukemia-associated protein (LAP). Transgenes encoding proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines, such as phage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), Fas ligand, tumor necrosis factors α and β (TNFα and TNFβ), interferons (IFN) IFN-α, IFN-β, and IFN-γ, stem cell factor, and flk-2 / flt3 ligand, are also encompassed by the present 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, as well as genetically engineered MHC molecules, including single-chain MHC molecules. Useful gene products further include complement control proteins such as membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2, and CD59.

[0563] Further examples of suitable transgenes include those encoding any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Examples of such receptors include, inter alia, the trk family of receptors, such as flt-1, flk-1, TIE-2; TrkA, MuSK, Eph, PDGF receptor, EGF receptor, HER2, insulin receptor, IGF-1 receptor, the FGF family of receptors, TGFβ receptor, interleukin receptor, interferon receptor, serotonin receptor, α-adrenergic receptor, β-adrenergic receptor, GDNF receptor, and p75 neurotrophin receptor. Further examples are 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 (VCAMs), and selectins, i.e., 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 are transgenes encoding apolipoprotein ligands for these receptors, including ApoAI, ApoAIV, and ApoE.Additional transgenes include antimicrobial peptides such as defensins and majinin; transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, and NF-κB; members of 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 These 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 regulatory factor 1 (IRF-1); Wilms tumor protein; ETS-binding proteins; STATs; GATA box-binding proteins, such as GATA-3; and those encoding the forkhead family of winged helix proteins.

[0564] Other useful transgenes include carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-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, glutathione peroxidase and reductase, heme oxygenase, angiotensin-converting enzyme, endothelin-1, atrial natriuretic peptide, prourokinase, urokinase, plasminogen activator, heparin cofactor 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, insulin Surfactants, β-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also called P-protein), H-protein, T-protein, Menkes disease protein, tumor suppressors (e.g., p53), cystic fibrosis transmembrane conductance regulator (CFTR), product of the Wilson disease gene PWD, Cu / Zn superoxide dismutase, aromatic amino acid decarboxylase, tyrosine hydroxylase, acetylcholine synthetase, prohormone convertase, protease inhibitors, lactate These include those encoding any one or more of the individual chains or species of collagen, elastin, fibronectin, thrombospondin, vitronectin, and tenascin, as well as suicide genes such as thymidine kinase and cytosine deaminase.Other useful proteins include acid β-glucosidase, α-galactosidase a, α-1-iduronidase, iduroate sulfatase, lysosomal acid α-glucosidase, sphingomyelinase, hexosaminidase A, hexosaminidase A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, α-fucosidase, α- and β-mannosidase, aspartylglycosamidase, neuramidase, galactosylglycos ... These include those involved in lysosomal storage diseases, including 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.

[0565] Other useful transgenes include those encoding non-naturally occurring polypeptides, such as one or more chimeric or hybrid polypeptides with non-natural amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, single-chain recombinant immunoglobulins may be useful in certain immunocompromised patients. Other useful proteins include truncated receptors lacking transmembrane and cytoplasmic domains. These truncated receptors can be used to antagonize the function of their corresponding ligands by binding without accompanying receptor signaling. Other types of non-naturally occurring gene sequences include sense and antisense molecules, such as ribozymes, that can be used to regulate gene expression, as well as catalytic nucleic acids.

[0566] Exemplary transgenes that can be expressed in target cells and then administered to a patient for treatment of a disease characterized by a deficiency or dysfunction of the encoded product include those encoding the protein products listed in Table 3 below.

[0567] [Table 4] TIFF2026010071000148.tif213170TIFF2026010071000149.tif205170TIFF2026010071000150.tif20617 0TIFF2026010071000151.tif229170TIFF2026010071000152.tif229170TIFF2026010071000153.tif38170

[0568] Donor cell selection In some embodiments, the subject receiving treatment is a donor who provides cells (e.g., pluripotent cells such as CD34+ HSCs or HPCs) that are subsequently modified to express one or more therapeutic proteins of the present disclosure before being readministered to the patient. In such cases, the derived cells (e.g., CD34+ HSCs or HPCs) may be reinfused into the subject, e.g., after incorporation of a transgene encoding one or more therapeutic proteins of the present disclosure and / or destruction of an allelic variant carrying a deleterious mutation, allowing the cells to subsequently home to hematopoietic tissues and establish proliferative hematopoiesis, thereby restoring transgene expression in the patient. In cases where the cells receiving treatment also serve as the cell donor, the transplanted cells (e.g., HSCs or HPCs) are less likely to undergo graft rejection. This results from the fact that the infused cells are derived from the patient and express the same HLA class I and class II antigens expressed by the patient. Alternatively, the subject and donor may be different. In some embodiments, the subject and donor may be related, e.g., HLA-matched. As described herein, HLA-matched donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells within the transplant recipient are less likely to recognize the incoming hematopoietic or progenitor cell graft as foreign and therefore less likely to mount an immune response against the transplanted tissue. An exemplary HLA-matched donor-recipient pair is a genetically related donor and recipient, such as a familial donor-recipient pair (e.g., a sibling donor-recipient pair). 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.

[0569] Pharmaceutical Compositions and Dosages When a subject is administered a population of cells that co-express one or more therapeutic proteins of the present disclosure, the number of cells administered will depend, for examp...

Claims

1. 1. A method of transducing a eukaryotic cell to express a transgene, the method comprising contacting the cell with (i) a viral vector encoding the transgene and (ii) a diblock copolymer comprising polyoxyethylene (PEO) subunits and polyoxypropylene (PPO) subunits.

2. A method for expressing a transgene in a eukaryotic cell, the method comprising contacting the cell with (i) a viral vector encoding the transgene, and (ii) a diblock copolymer comprising a PEO subunit and a PPO subunit.

3. A method for promoting the transfer of a viral vector encoding an introduced gene into the nucleus of a eukaryotic cell, the method comprising contacting the cell with (i) the viral vector and (ii) a diblock copolymer comprising a PEO subunit and a PPO subunit.

4. The diblock copolymer has the following structure: X １ -[PEO] ｍ -L-[PPO] ｎ -X ２ wherein m and n are integers; L is absent or is a chemical linker, and X １ and X ２ each independently represents an optionally present chemical substituent. The method according to any one of claims 1 to 3, comprising:

5. The diblock copolymer has the following structure: X １ -[PEO] ｍ -[PPO] ｎ -X ２ wherein m and n are integers, and X １ and X ２ each independently represents an optionally present chemical substituent. The method of claim 4, comprising:

6. X １ and X ２ are each independently absent, H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ 6. The method of claim 4 or 5, wherein the aryl group is alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido.

7. X １ and X ２ are each independently absent, H, OH, optionally substituted C １－６ Alkyl, optionally substituted C １－６ Alkoxy or optionally substituted C １－６ The method of claim 6, wherein the alkylamino is alkylamino.

8. X １ and X ２ are each independently absent or H, OH, H ２ N.H. ３ 8. The method of claim 7, wherein the alkyl group is CO, ethyl-O, n-butyl-O, tert-butyl-O, n-butyl, or tert-butyl.

9. 9. The method of any one of claims 1 to 8, wherein the PEO subunits of the diblock copolymer have a number average molecular weight (Mn) of about 5,000 g / mol to about 25,000 g / mol.

10. 10. The method of claim 9, wherein the PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol to about 19,000 g / mol.

11. 11. The method of claim 10, wherein the PEO subunits of the diblock copolymer have an Mn of about 9,000 g / mol, 9,500 g / mol, 13,800 g / mol, 15,500 g / mol, 18,000 g / mol, or 19,000 g / mol.

12. 12. The method of any one of claims 1 to 11, wherein the PPO subunits of the diblock copolymer have an Mn of about 2,000 g / mol to about 10,000 g / mol.

13. 13. The method of claim 12, wherein the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol to about 5,500 g / mol.

14. 14. The method of claim 13, wherein the PPO subunits of the diblock copolymer have an Mn of about 3,500 g / mol or 5,500 g / mol.

15. The method of any one of claims 1 to 14, wherein the diblock copolymer has an average ethylene oxide content of greater than 40% by weight.

16. 16. The method of claim 15, wherein the diblock copolymer has an average ethylene oxide content of greater than 50% by weight.

17. 17. The method of claim 16, wherein the diblock copolymer has an average ethylene oxide content of greater than 60% by weight.

18. 18. The method of claim 17, wherein the diblock copolymer has an average ethylene oxide content of greater than 70% by weight.

19. 19. The method of any one of claims 1 to 18, wherein the diblock copolymer has a Mn greater than about 8,000 g / mol.

20. 20. The method of claim 19, wherein the diblock copolymer has a Mn greater than about 10,000 g / mol.

21. 21. The method of claim 19, wherein the diblock copolymer has an Mn of about 10,000 g / mol to about 30,000 g / mol.

22. 22. The method of claim 21, wherein the diblock copolymer has an Mn of about 12,000 g / mol to about 25,000 g / mol.

23. 23. The method of claim 22, wherein the diblock copolymer has an Mn of about 12,500 g / mol to about 23,500 g / mol.

24. 24. The method of claim 23, wherein the diblock copolymer has an Mn of about 12,500 g / mol, 13,000 g / mol, 17,300 g / mol, 19,000 g / mol, 22,500 g / mol, or 23,500 g / mol.

25. 25. The method of any one of claims 1 to 24, wherein the diblock copolymer has a polydispersity index (Mw / Mn) of from about 1 to about 1.

2.

26. 26. The method of claim 25, wherein the diblock copolymer has a polydispersity index of from about 1.06 to about 1.

17.

27. 27. The method of claim 26, wherein the diblock copolymer has a polydispersity index of from about 1.08, 1.10, 1.11, 1.13, or 1.

17.

28. 28. The method of any one of claims 4 to 27, wherein m is from about 100 to about 500.

29. 29. The method of claim 28, wherein m is from about 200 to about 450.

30. 30. The method of claim 29, wherein m is from about 162 to about 486, from about 159 to about 477, from about 108 to about 324, from about 103 to about 309, from about 148 to about 444, from about 171 to about 513, from about 142 to about 426, from about 100 to about 300, from about 113 to about 339, from about 109 to about 327, from about 115 to about 345, or from about 120 to about 360.

31. 31. The method of claim 30, wherein m is about 200, 205, 216, 217, 225, 230, 240, 284, 314, 318, 323, 352, 409, or 432.

32. 32. The method of any one of claims 4 to 31, wherein n is from about 10 to about 200.

33. 33. The method of claim 32, wherein n is from about 40 to about 100.

34. 34. The method of claim 33, wherein n is from about 43 to about 129, from about 27 to about 81, from about 29 to about 87, from about 28 to about 84, from about 30 to about 90, from about 33 to about 99, or from about 28 to about 84.

35. 35. The method of claim 34, wherein n is about 50, 53, 55, 57, 60, 65, 70, 86, or 95.

36. 36. The method of any one of claims 4 to 35, wherein the ratio of m:n is from about 1 to about 12.

37. 37. The method of claim 36, wherein the ratio of m:n is from about 2 to about 8.

38. 38. The method of claim 37, wherein the ratio of m:n is from about 2 to about 7.

2.

39. 39. The method of claim 38, wherein the ratio of m:n is about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, or 7.

2.

40. The method of any one of claims 4 to 39, wherein the diblock copolymer has a structure selected from the following: [PEO] ３２３ -[PPO] ８６ -OH、 HOCH ２ CH ２ -[PEO] ３２３ - [PPO] ８６ —O-n-butyl, [PEO] ３１８ -[PPO] ５３ -OH、 HOCH ２ CH ２ -[PEO] ３１８ - [PPO] ５３ —O-n-butyl, [PEO] ２１６ -[PPO] ５３ -OH、 HOCH ２ CH ２ -[PEO] ２１６ - [PPO] ５３ —O-n-butyl, [PEO] ２０５ -[PPO] ５３ -OH、 HOCH ２ CH ２ -[PEO] ２０５ - [PPO] ５３ —O-n-butyl, [PEO] ２９５ -[PPO] ５７ -OH、 HOCH ２ CH ２ -[PEO] ２９５ - [PPO] ５７ —O-n-butyl, [PEO] ３４１ -[PPO] ５７ -OH、 HOCH ２ CH ２ -[PEO] ３４１ - [PPO] ５７ —O-n-butyl, [PEO] ２８４ -[PPO] ５７ -OH、 HOCH ２ CH ２ -[PEO] ２８４ - [PPO] ５７ —O-n-butyl, [PEO] ２００ -[PPO] ５５ -OH、 HOCH ２ CH ２ -[PEO] ２００ - [PPO] ５５ —O-n-butyl, [PEO] ２０５ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ２０５ - [PPO] ６０ —O-n-butyl, [PEO] ２１７ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ２１７ - [PPO] ６０ —O-n-butyl, [PEO] ２３０ -[PPO] ６５ -OH、 HOCH ２ CH ２ -[PEO] ２３０ - [PPO] ６５ —O-n-butyl, [PEO] ２４０ -[PPO] ５５ -OH、 HOCH ２ CH ２ -[PEO] ２４０ - [PPO] ５５ —O-n-butyl, [PEO] ２０５ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ２０５ - [PPO] ６０ —O-n-butyl, [PEO] ３１４ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ３１４ - [PPO] ６０ —O-n-butyl, [PEO] ３５２ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ３５２ - [PPO] ６０ —O-n-butyl, [PEO] ４０９ -[PPO] ９５ -OH、 HOCH ２ CH ２ -[PEO] ４０９ - [PPO] ９５ —O-n-butyl, [PEO] ４３２ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ４３２ - [PPO] ６０ —O-n-butyl, [PEO] ２１６ -[PPO] ６０ -OH、 [PEO] ２１６ - [PPO] ６０ -n-butyl, HO-[PEO] ２１６ - [PPO] ６０ -n-butyl, HOCH ２ CH ２ -[PEO] ２１６ - [PPO] ５０ —O-n-butyl, HOCH ２ CH ２ -[PEO] ２１６ -[PPO] ５０ -OH、 HOCH ２ CH ２ -[PEO] ２１６ - [PPO] ６０ —O-n-butyl, HOCH ２ CH ２ -[PEO] ２１６ -[PPO] ６０ -OH、 HOCH ２ CH ２ -[PEO] ２１６ - [PPO] ７０ —O-n-butyl, HOCH ２ CH ２ -[PEO] ２１６ -[PPO] ７０ -OH、 【Chemistry 1】 【change】

41. The method of any one of claims 1 to 40, wherein the cell is a mammalian cell.

42. 42. The method of claim 41, wherein the mammalian cell is a human cell.

43. The method of any one of claims 1 to 42, wherein the cell is a pluripotent cell.

44. The method of any one of claims 1 to 43, wherein the cells are CD34+ cells.

45. 44. The method of any one of claims 1 to 43, wherein the cells are embryonic stem cells.

46. The method of any one of claims 1 to 43, wherein the cells are induced pluripotent stem cells.

47. 44. The method of any one of claims 1 to 43, wherein the cells are pluripotent hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs).

48. 48. The method of any one of claims 1 to 47, wherein the method further comprises contacting the cell with an agent that decreases the activity and / or expression of protein kinase C (PKC).

49. 49. The method of claim 48, wherein the substance that reduces the activity and / or expression of PKC activates Akt signal transduction.

50. 50. The method of claim 48 or 49, wherein the substance that decreases the activity and / or expression of PKC is a PKC inhibitor or an agent that decreases the translation of a ribonucleic acid (RNA) transcript encoding PKC.

51. 51. The method of claim 50, wherein the substance that decreases the activity and / or expression of PKC is a PKC inhibitor.

52. 51. The method of claim 50, wherein the substance that decreases the activity and / or expression of PKC is an agent that decreases the translation of an RNA transcript encoding PKC.

53. 53. The method of claim 52, wherein the agent comprises a nucleic acid.

54. 54. The method of claim 53, wherein the nucleic acid comprises an interfering RNA.

55. 55. The method of claim 54, wherein the interfering RNA is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

56. 54. The method of claim 53, wherein the nucleic acid comprises an antisense oligonucleotide.

57. 57. The method of any one of claims 53 to 56, wherein the nucleic acid anneals to an endogenous RNA transcript encoding PKC.

58. 58. The method of claim 57, wherein the nucleic acid is at least 85% complementary to an endogenous RNA transcript encoding PKC.

59. 59. The method of claim 58, wherein the nucleic acid is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an endogenous RNA transcript encoding PKC.

60. The PKC inhibitor is a compound represented by formula (I) 【Chemistry 2】 [In the formula, R １ is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, 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; R ２ is H, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ 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 ａ and R ｂ are joined together 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 is H, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ alkenyl, or optionally substituted C ２－６ is alkynyl, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; 【change】 represents an optionally present bond, n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

61. 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 amido, halogen, oxo, or thiocarbonyl; R ２ is H, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ alkynyl, or optionally substituted acyl; R ａ and R ｂ are joined together with the atoms to which they are attached to form an optionally substituted and optionally fused heterocycloalkyl ring; R ｃ is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

62. The compound is a compound represented by formula (III) 【Chemistry 4】 [In the formula, R １ is H, OH, oxo, or thiocarbonyl; R ２ is H, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ alkynyl, or optionally substituted acyl; Ring A is an optionally substituted and optionally fused heterocycloalkyl ring; R ｃ is O or S, Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

63. 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 O or S, W is O, NH, or S; Each X is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

64. The compound is a compound represented by formula (V): 【Transformation 6】 [In the formula, R １ is H, OH, or oxo; R ｃ is O or S, W is O, NH, or S; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; p is 0 or 1. or a salt thereof.

65. The compound is a compound represented by formula (VI) 【Transformation 7】 [In the formula, R １ is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; and s is an integer from 0 to 8. or a salt thereof.

66. 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, optionally substituted alkoxy, or optionally substituted acyloxy; R ３ is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

67. 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, optionally substituted alkoxy, or optionally substituted acyloxy; R ３ is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido. or a salt thereof.

68. The compound is a compound represented by formula (IX): 【Chemistry 10】 wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

69. The compound is a compound represented by formula (1) 【Chemistry 11】 or a salt thereof.

70. The compound is staurosporine, that is, (2S,3R,4R,6R)-3-methoxy-2-methyl-4-(methylamino)-29-oxa-1,7,17-triazaoctacyclo[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】 or a salt thereof.

71. The compound is a compound represented by formula (X): 【Chemistry 13】 [In the formula, R １ is H, OH, or oxo; Each Z is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; and t is an integer from 0 to 6. or a salt thereof.

72. The compound is a compound represented by formula (XI): 【Chemistry 14】 [In the formula, R １ is H, OH, or oxo; R ４ is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

73. The compound is a compound represented by formula (XII): 【Chemistry 15】 [In the formula, R １ is H, OH, or oxo; R ４ is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy. or a salt thereof.

74. The compound is a compound represented by formula (XIII) 【Chemistry 16】 wherein each X is independently 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 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; Each Y is independently 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, 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 optionally fused aryl, optionally substituted optionally fused heteroaryl, optionally substituted optionally fused cycloalkyl, or optionally substituted optionally fused heterocycloalkyl; n is an integer from 0 to 4, and m is an integer of 0 to 4. or a salt thereof.

75. The compound is a compound represented by formula (3): 【Chemistry 17】 or a salt thereof.

76. The compound is a compound represented by formula (4): [Chemistry 18] or a salt thereof.

77. The compound is a compound represented by formula (128): 【Chemistry 19】 or a salt thereof.

78. The compound is 【Chemistry 20】 or a salt thereof.

79. The compound is a compound represented by formula (XIV): 【Chemistry 21】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

80. The compound is a compound represented by formula (XV): 【Chemistry 22】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

81. The compound is 【Chemistry 23】 or a salt thereof.

82. The compound is a compound represented by formula (XVI): 【Chemistry 24】 wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

83. The compound is a compound represented by formula (XVII): 【Chemistry 25】 wherein R is H, optionally substituted alkyl, optionally substituted acyl, optionally substituted sulfonyl, optionally substituted sulfinyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. or a salt or quaternized variant thereof.

84. The compound is 【Chemistry 26】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 or a salt thereof.

85. The compound is a compound represented by formula (XVIII): 【Chemistry 27】 [Wherein R is H, OH, C １－６ alkoxy, or oxo; R ２ teeth, 【Chemistry 28】 and optionally wherein the sugar moiety is derived from D-glucose, D-galactose, or D-mannose; R ３ is H, OH, C １－６ Alkanoyloxy, C １－６ alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, or C １－６ is substituted with alkoxy, R ４ OH, C １－６ Alkanoyloxy, benzoyloxy, benzyloxy, amino, C １－６ Alkylamino, di-C １－６ Alkylamino, C １－６ Alkoxycarbonylamino, C ２－２０ alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which may be selected from the group consisting of halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, C １－６ is substituted with alkoxy, R ５ is H or C １－６ is alkyl, R ６ is free or aliphatic C ２－２２ a hydroxyl esterified with a carboxylic acid, or １－６ Alkoxycarbonyloxy, C １－６ Alkylsulfonyloxy, free or aliphatic C ２－２２ Amino acylated with carboxylic acid, C １－６ alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, or C １－６ is substituted with alkoxy, R ７ is free or aliphatic C ２－２２ OH, C esterified with carboxylic acid １－６ Alkoxycarbonyloxy, C １－６ Alkyl sulfonyloxy, azido, free or aliphatic C ２－２２ Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, C １－６ Alkoxy, or C １－６ is substituted with alkoxycarbonyl. or a salt thereof.

86. The compound is a compound represented by formula (XIX): 【Chemistry 29】 [Wherein R is H, OH, C １－６ alkoxy, or oxo; R ２ teeth, 【Transformation 30】 and R ３ is H, OH, C １－６ Alkanoyloxy, C １－６ alkoxy, benzyloxy, benzoyloxy, or phenyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, or C １－６ is substituted with alkoxy, R ４ OH, C １－６ Alkanoyloxy, benzoyloxy, benzyloxy, amino, C １－６ Alkylamino, di-C １－６ Alkylamino, C １－６ Alkoxycarbonylamino, C ２－２０ alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which may be selected from the group consisting of halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, C １－６ is substituted with alkoxy, R ５ is H or C １－６ is alkyl, R ６ is free or aliphatic C ２－２２ a hydroxyl esterified with a carboxylic acid, or １－６ Alkoxycarbonyloxy, C １－６ Alkylsulfonyloxy, free or aliphatic C ２－２２ Amino acylated with carboxylic acid, C １－６ alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, or C １－６ is substituted with alkoxy, R ７ is free or aliphatic C ２－２２ OH, C esterified with carboxylic acid １－６ Alkoxycarbonyloxy, C １－６ Alkyl sulfonyloxy, azido, free or aliphatic C ２－２２ Amino acylated with 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 may contain halogen, hydroxyl, trifluoromethyl, C １－６ Alkyl, C １－６ Alkoxy, or C １－６ is substituted with alkoxycarbonyl. or a salt thereof.

87. The compound 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-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 -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-2-N-acetyl 87. The method of claim 86, wherein the staurosporine is selected from N-(1-deoxy-6-O-acetyl-2-N-acetylmuramyl)staurosporine, N-(1-deoxy-6-O-toluenesulfonyl-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.

88. The compound is a compound represented by formula (XX): 【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 a halogen, R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH ２ -NH-Serine, CO ２ CH ３ , C.H. ２ NHCO ２ C ６ H ５ , CONHC ６ H ５ , or C.H. ２ NHCO ２ CH3, wherein C ６ H ５ represents a phenyl moiety.] or a salt thereof.

89. The compound is a compound represented by formula (XXI): 【Chemistry 32】 [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 a halogen, R is OH or optionally substituted alkoxy; X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH ２ -NH-Serine, CO ２ CH ３ , C.H. ２ NHCO ２ C ６ H ５ , CONHC ６ H ５ , or C.H. ２ NHCO ２ CH3, wherein C ６ H ５ represents a phenyl moiety.] or a salt thereof.

90. The compound is a compound represented by formula (XXII), (XXIII), (XXIV), or (XXV) 【Transformation 33】 [In the formula, each R １ are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R ２ are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R ５ are independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having 29 or fewer carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having 20 or fewer carbon atoms and 9 or fewer heteroatoms, or 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-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each Q' is independently H, OH, halogen, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; each n is independently an integer from 0 to 4; Each m is independently an integer from 0 to 4. or a salt thereof.

91. The compound is a compound represented by formula (XXVI) or (XXVII) 【Transformation 34】 [In the formula, each R １ are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R ２ are independently optionally substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or di-substituted amino, cyano, nitro, mercapto, substituted mercapto, carboxy, esterified carboxy, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R ５ are independently H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical, in each case having up to 29 carbon atoms, or a heterocyclic or heterocyclic-aliphatic radical, in each case having up to 20 carbon atoms and up to 9 heteroatoms, or up to 30 carbon atoms; Each R ８ are independently acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical in each case having 20 or fewer carbon atoms, and 9 or fewer heteroatoms; Each R ９ are independently 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-disubstituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-disubstituted aminosulfonyl; Each R １０ are independently an acyl having 30 or fewer carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical in each case having 29 or fewer carbon atoms, a heterocyclic or heterocyclic-aliphatic radical in each case having 20 or fewer carbon atoms, and a heteroatom in each case having 9 or fewer heteroatoms; 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.

92. The compound is a compound represented by formula (XXVIII) 【Chemistry 35】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

93. The compound is a compound represented by formula (XXIX) 【Transformation 36】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

94. The compound is a compound represented by formula (XXX): 【Chemistry 37】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

95. The compound is a compound represented by formula (XXXI): 【Transformation 38】 [In the formula, R １ is H or optionally substituted C １－６ is alkyl, R ２ is an optionally substituted C １－６ is alkyl. or a salt thereof.

96. The compound is 【Chemistry 39】 【change】 【change】 【change】 【change】 61. The method of claim 60, wherein the

97. 97. The method of any one of claims 1 to 96, wherein the method further comprises contacting the cell with a histone deacetylase (HDAC) inhibitor.

98. The HDAC inhibitor is 【Chemistry 40】 【change】 98. The method of claim 97, wherein the

99. the HDAC inhibitor 【Chemistry 41】 99. The method of claim 98, wherein:

100. 100. The method of any one of claims 1 to 99, wherein the method further comprises contacting the cell with a glycogen synthase kinase 3 (GSK3) inhibitor.

101. The GSK3 inhibitor is 6-bromoindirubin-3'-oxime (BIO), LiCl, Li ２ CO ３ , CHIR-99021, and CHIR-98023.

102. 102. The method of claim 101, wherein the GSK3 inhibitor is CHIR-99021.

103. The GSK3 inhibitor is Li ２ CO ３ The method of claim 101, wherein

104. 104. The method of any one of claims 1 to 103, wherein the viral vector is selected from the group consisting of a virus of the Retroviridae family, an adeno-associated virus, an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpesvirus, and a poxvirus.

105. The method of claim 104, wherein the viral vector is a Retroviridae family viral vector.

106. The method of claim 105, wherein the Retroviridae family viral vector is a lentiviral vector.

107. 107. The method of claim 106, wherein the Retroviridae family viral vector is an alpharetroviral vector or a gammaretroviral vector.

108. The method of any one of claims 104 to 107, wherein the Retroviridae family viral vector comprises a central polypurine tract, 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'-self-inactivating LTR.

109. The method according to any one of claims 1 to 108, wherein the viral vector is a pseudotyped viral vector.

110. The pseudotyped viral vector is selected from the group consisting of vesicular stomatitis virus (VSV), RD114 virus, murine leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan equine encephalitis virus (VEE), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), Semliki Forest virus (SFV), rabies virus, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), caprine arthritis encephalitis virus (CAEV), Sin Nombru virus (SNV), and rabies virus.

110. The method of claim 109, wherein the virus comprises one or more envelope proteins from a virus selected from: SNV, cherry twisted leaf virus (ChTLV), simian T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), avian carcinoma virus (MH2), avian encephalomyelitis virus (AEV), alpha mosaic virus (AMV), avian sarcoma virus CT10, and equine infectious anemia virus (EIAV).

111. The method of claim 110, wherein the pseudotyped viral vector comprises a VSV-G envelope protein.

112. The method of any one of claims 1 to 111, wherein said contacting occurs ex vivo.

113. 113. The method of claim 112, wherein the cells are freshly cultured or cryopreserved prior to said contacting.

114. 114. The method of any one of claims 1 to 113, wherein the cells are further contacted with cyclosporine.

115. 115. The method of claim 114, wherein the cyclosporin is cyclosporin A or cyclosporin H.

116. 116. The method of claim 115, wherein the cyclosporin is cyclosporin H.

117. 117. The method of any one of claims 1 to 116, wherein the cell is further contacted with an activator of prostaglandin E receptor signaling.

118. 118. The method of claim 117, wherein the activator of prostaglandin E receptor signaling is prostaglandin E2.

119. The method of any one of claims 1 to 118, wherein the cells are further contacted with a polycationic polymer.

120. 120. The method of claim 119, wherein the polycationic polymer is polybrene, protamine sulfate, polyethyleneimine, or a polyethylene glycol / poly-L-lysine block copolymer.

121. 121. The method of claim 120, wherein the polycationic polymer is protamine sulfate.

122. 122. The method of any one of claims 1 to 121, wherein the cells are spun by centrifugation while in contact with the viral vector.

123. 123. The method of claim 122, wherein the cells are spun at a centripetal force of about 300 x g to about 1,200 x g.

124. 124. The method of claim 122 or 123, wherein the cells are spun at a temperature of about 25°C.

125. 125. A method of expressing a transgene in a subject, said method comprising administering to said subject a population of cells, or their progeny, which have been modified according to the method of any of claims 1 to 124.

126. 125. A method for delivering a population of genetically modified cells to a subject, said method comprising administering to said subject a population of cells, or a progeny thereof, which has been modified according to the method of any of claims 1 to 124.

127. 125. A method of providing cell therapy to a subject in need thereof, said method comprising administering to said subject a population of cells, or their progeny, that have been modified according to the method of any of claims 1-124.

128. 128. The method of any one of claims 125 to 127, wherein the cells are allogeneic to the subject.

129. 129. The method of claim 128, wherein the cells are HLA-matched to the subject.

130. 128. The method of any one of claims 125 to 127, wherein the cells are autologous to the subject.

131. 131. The method of any one of claims 125-130, wherein prior to said contacting, a population of progenitor cells is isolated from said subject or donor, and said progenitor cells are expressed ex vivo to obtain said population of cells that are administered to said subject.

132. 132. The method of claim 131, wherein the progenitor cells are CD34+ HSCs and the progenitor cells are expanded without losing the functional capacity of HSCs.

133. 133. The method of claim 131 or 132, wherein one or more pluripotent cell mobilizing agents are administered to the subject or donor prior to isolation of the progenitor cells from the subject or donor.

134. 134. The method of any one of claims 125-133, wherein the population of endogenous pluripotent cells is ablated in the subject by administering one or more conditioning agents to the subject prior to administering the population of cells to the subject.

135. 134. The method of any one of claims 125-133, wherein the method comprises ablating a population of endogenous pluripotent cells in the subject by administering one or more conditioning agents to the subject prior to administering the population of cells to the subject.

136. 136. The method of claim 134 or 135, wherein the one or more conditioning agents are non-myeloablative conditioning agents.

137. 137. The method of any one of claims 125-136, wherein upon administration of the population of cells to the subject, the administered cells, or their progeny, differentiate into one or more cell types selected from megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

138. 138. The method of any one of claims 125 to 137, wherein the subject is a mammal.

139. 139. The method of claim 138, wherein the subject is a human.

140. 140. The method of any one of claims 125 to 139, wherein the subject has been diagnosed with a deficiency in the endogenous protein encoded by the transgene.

141. 141. The method of claim 140, wherein the subject has been diagnosed with a disease listed in Table 3.

142. 141. The method of claim 140, wherein the subject has been diagnosed with beta thalassemia.

143. 143. The method of any one of claims 1 to 142, wherein the transgene encodes a beta globin protein.

144. 144. The method of claim 143, wherein the transgene comprises a nucleic acid having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

145. 145. The method of claim 144, wherein the transgene comprises a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

146. 146. The method of claim 145, wherein the transgene comprises a nucleic acid having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

147. 147. The method of claim 146, wherein the transgene comprises a nucleic acid having the nucleic acid sequence of SEQ ID NO:

1.

148. 148. The method of any one of claims 143 to 147, wherein the beta-globin protein has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

2.

149. 149. The method of claim 148, wherein the beta globin protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

2.

150. 150. The method of claim 149, wherein the beta globin protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

2.

151. 151. The method of claim 150, wherein the beta globin protein has the amino acid sequence of SEQ ID NO:

2.

152. A composition comprising a mixture formed by the method of any one of claims 1 to 124.

153. 153. A cell culture medium comprising the composition of claim 152.

154. A population of eukaryotic cells that has been modified according to the method of any one of claims 1 to 124.

155. 155. A pharmaceutical composition comprising the population of eukaryotic cells of claim 154, wherein the pharmaceutical composition further comprises one or more excipients, diluents, or carriers.

156. 156. The pharmaceutical composition of claim 155, wherein the pharmaceutical composition is formulated for administration to a subject.

157. 157. The pharmaceutical composition of claim 156, wherein the subject is a mammal.

158. 158. The pharmaceutical composition of claim 157, wherein the subject is a human.

159. 159. The pharmaceutical composition of any one of claims 156 to 158, wherein the pharmaceutical composition is formulated for intravenous injection into the subject.

160. 154. A kit comprising the composition of claim 152 or the cell culture medium of claim 153.

161. 161. The kit of claim 160, wherein the kit further comprises a package insert containing instructions for transducing the cells.

162. 165. A kit comprising a population of cells according to claim 164 or a pharmaceutical composition according to any one of claims 155 to 159.

163. 163. The kit of claim 162, wherein the kit further comprises a package insert instructing a user to administer the population of cells to a subject according to the method of any one of claims 125 to 151.

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