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COMPOSITIONS AND METHODS FOR MODIFYING EUKARYOTIC CELLS

20250290097 ยท 2025-09-18

    Inventors

    Cpc classification

    International classification

    Abstract

    Described herein are compositions and methods for modifying eukaryotic cells, for example, to express a transgene of interest and/or to produce an expanded population of cells ex vivo. Using the compositions and methods of the disclosure, a population of eukaryotic cells, such as a population of pluripotent cells (e.g., CD34+ hematopoietic stem or progenitor cells) may be transduced to express a gene of interest by contacting the cells with a viral vector, such as a lentiviral vector, and a diblock copolymer, such as a diblock copolymer composed of a hydrophilic region and a hydrophobic region. For example, the diblock copolymer may be composed of polyoxyethylene (PEO) subunits and polyoxypropylene (PRO) subunits. Additionally, the compositions and methods described herein can be used to promote the proliferation or survival of a population of pluripotent cells (e.g., CD34+ hematopoietic stem or progenitor cells) ex vivo, for example, by contacting the cells with a diblock copolymer.

    Claims

    1. A method of; (i) transducing a eukaryotic cell to express a transgene; or (ii) 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 polyoxyethylene (PEO) subunits and polyoxypropylene (PPO) subunits.

    2. (canceled)

    3. A method of promoting migration of a viral vector encoding a transgene to the nucleus of a eukaryotic cell, the method comprising contacting the cell with (i) the viral vector and (ii) a diblock copolymer comprising PEO subunits and PPO subunits.

    4. The method of claim 1, wherein: (i) the diblock copolymer comprises a structure: X.sub.1-[PEO].sub.m-L-[PPO].sub.n-X.sub.2 wherein m and n are integers; L is not present or is a chemical linker; and X.sub.1 and X.sub.2 each, independently, represent optionally present chemical substituents; (ii) the PEO subunits of the diblock copolymer have a number average molecular weight (Mn) of from about 5,000 g/mol to about 25,000 g/mol; (iii) the PPO subunits of the diblock copolymer have a Mn of from about 2,000 g/mol to about 10,000 g/mol; (iv) the diblock copolymer has an average ethylene oxide content of greater than 40% by mass; (v) the diblock copolymer has a Mn of greater than about 8,000 g/mol; (vi) the diblock copolymer has a polydispersity index (Mw/Mn) of from about 1 to about 1.2; (vii) the cell is a mammalian cell; (viii) the cell is a pluripotent cell; (ix) the cell is a CD34+ cell, an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell (HSC), or a hematopoietic progenitor cell (HPC); (x) the method further comprises contacting the cell with a substance that reduces activity and/or expression of protein kinase C (PKC); (xi) the method further comprises contacting the cell with a glycogen synthase kinase 3 (GSK3) inhibitor; (xii) the viral vector is selected from the group consisting of a Retroviridae family virus, an adeno-associated virus, an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, and a poxvirus; (xiii) the viral vector is a pseudotyped viral vector; (xiv) the contacting occurs ex vivo; (xv) the cell is further contacted with a cyclosporine; (xvi) the cell is further contacted with an activator of prostaglandin E receptor signaling; (xvii) the cell is further contacted with a polycationic polymer; (xviii) the cell is spun by centrifugation while being contacted with the viral vector; (xix) the transgene encodes a beta-globin protein; or (xx) the method further comprises contacting the cell with a histone deacetylase (HDAC) inhibitor.

    5. The method of claim 4, wherein: (i) the diblock copolymer comprises a structure:
    X.sub.1-[PEO].sub.m-[PPO].sub.n-X.sub.2 wherein m and n are integers; and X.sub.1 and X.sub.2 each, independently, represent optionally present chemical substituents; (ii) X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido; (iii) the PEO subunits of the diblock copolymer have a Mn of from about 9,000 g/mol to about 19,000 g/mol; (iv) the PPO subunits of the diblock copolymer have a Mn of from about 3,500 g/mol to about 5,500 g/mol; (v) the diblock copolymer has an average ethylene oxide content of greater than 50% by mass; (vi) the diblock copolymer has a Mn of greater than about 10,000 g/mol or the diblock copolymer has a Mn of from about 10,000 g/mol to about 30,000 g/mol; (vii) the diblock copolymer has a polydispersity index of from about 1.06 to about 1.17; (viii) m is from about 100 to about 500; (ix) n is from about 10 to about 200; (x) a ratio of m:n is from about 1 to about 12; (xi) the mammalian cell is a human cell; (xii) the substance that reduces activity and/or expression of PKC activates Akt signal transduction; (xiii) the GSK3 inhibitor is selected from the group consisting of 6-bromoindirubin-3-oxime (BIO), LiCl, Li.sub.2CO.sub.3, CHIR-99021, and CHIR-98023; (xiv) the viral vector is a Retroviridae family viral vector; (xv) the pseudotyped viral vector comprises one or more envelope proteins from a virus selected from 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 Nombre 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), Alfa mosaic virus (AMV), avian sarcoma virus CT10, and equine infectious anemia virus (EIAV); (xvi) the cell has been freshly cultured or has been cryopreserved prior to the contacting; (xvii) the cyclosporine is cyclosporine A or cyclosporine H; (xviii) the activator of prostaglandin E receptor signaling is prostaglandin E2; (xix) the polycationic Polymer is polybrene, protamine sulfate, polyethylenimine, or a Polyethylene glycol/poly-L-lysine block copolymer; (xx) the cell is spun at a centripetal force of from about 300q to about 1,200q; (xxi) the cell is spun at a temperature of about 25 C.; (xxii) the transgene comprises a nucleic acid having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; (xxiii) 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; or (xxiv) the substance that reduces activity and/or expression of PKC is a PKC inhibitor or an agent that reduces translation of a ribonucleic acid (RNA) transcript encoding PKC.

    6. (canceled)

    7. The method of claim 5, wherein; (i) X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.1-6 alkoxy, or optionally substituted C.sub.1-6 alkylamino, (ii) the PEO subunits of the diblock copolymer have a 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; (iii) the PPO subunits of the diblock copolymer have a Mn of about 3,500 g/mol or 5,500 g/mol; (iv) the diblock copolymer has an average ethylene oxide content of greater than 60% by mass; (v) the diblock copolymer has a Mn of from about 12,000 g/mol to about 25,000 g/mol; (vi) the diblock copolymer has a polydispersity index of from about 1.08, 1.10, 1.11, 1.13, or 1.17; (vii) m is from about 200 to about 450; (viii) n is from about 40 to about 100; (ix) the ratio of m:n is from about 2 to about 8; (x) the agent comprises a nucleic acid: (xi) the Retroviridae family viral vector is a lentiviral vector, an alpharetroviral vector, or a gammaretroviral vector; (xii) the Retroviridae family viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5-LTR, HIV signal sequence, HIV Psi signal 5-splice site, delta-GAG element, 3-splice site, and a 3-self inactivating LTR; (xiii) the pseudotyped viral vector comprises a VSV-G envelope protein; (xiv) the transgene comprises a nucleic acid having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; or (xv) 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.

    8. The method of claim 7, wherein: (i) X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, H.sub.2N, H.sub.3CO, ethyl-O, n-butyl-O, tert-butyl-O, n-butyl, or tert-butyl, (ii) the diblock copolymer has an average ethylene oxide content of greater than 70% by mass; (iii) the diblock copolymer has a Mn of from about 12,500 g/mol to about 23,500 g/mol; (iv) m is from about 162 to about 486, about 159 to about 477, about 108 to about 324, about 103 to about 309, about 148 to about 444, about 171 to about 513, about 142 to about 426, about 100 to about 300, about 113 to about 339, about 109 to about 327, about 115 to about 345, or about 120 to about 360; (v) n is from about 43 to about 129, about 27 to about 81, about 29 to about 87, about 28 to about 84, about 30 to about 90, about 33 to about 99, or about 28 to about 84; (vi) the ratio of m:n is from about 2 to about 7.2; (vii) the nucleic acid comprises an interfering RNA or an antisense oligonucleotide; (viii) the nucleic acid anneals to an endogenous RNA transcript encoding PKC; (ix) the transgene comprises a nucleic acid having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 1; or (x) 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.

    9.-23. (canceled)

    24. The method of claim 8, wherein; (i) the diblock copolymer has a 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; (ii) m is about 200, 205, 216, 217, 225, 230, 240, 284, 314, 318, 323, 352, 409, or 432; (iii) n is about 50, 53, 55, 57, 60, 65, 70, 86, or 95; (iv) 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; (v) the interfering RNA is a short interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA): (vi) the nucleic acid is at least 85% complementary to an endogenous RNA transcript encoding PKC; (vii) the transgene comprises a nucleic acid having the nucleic acid sequence of SEQ ID NO: 1; or (viii) the beta-globin protein has the amino acid sequence of SEQ ID NO: 2.

    25.-39. (canceled)

    40. The method of claim 4, wherein the diblock copolymer has a structure selected from: [PEO].sub.323-[PPO].sub.86-OH, HOCH.sub.2CH.sub.2-[PEO].sub.323-[PPO].sub.86-O-n-butyl, [PEO].sub.318-[PPO].sub.53-OH, HOCH.sub.2CH.sub.2-[PEO].sub.318-[PPO].sub.53-O-n-butyl, [PEO].sub.216-[PPO].sub.53-OH, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.53-O-n-butyl, [PEO].sub.205-[PPO].sub.53-OH, HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.53-O-n-butyl, [PEO].sub.295-[PPO].sub.57-OH, HOCH.sub.2CH.sub.2-[PEO].sub.295-[PPO].sub.57-O-n-butyl, [PEO].sub.341-[PPO].sub.57-OH, HOCH.sub.2CH.sub.2-[PEO].sub.341-[PPO].sub.57-O-n-butyl, [PEO].sub.284-[PPO].sub.57-OH, HOCH.sub.2CH.sub.2-[PEO].sub.284-[PPO].sub.57-O-n-butyl, [PEO].sub.200-[PPO].sub.55-OH, HOCH.sub.2CH.sub.2-[PEO].sub.200-[PPO].sub.55-O-n-butyl, [PEO].sub.205-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.60-O-n-butyl, [PEO].sub.217-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.217-[PPO].sub.60-O-n-butyl, [PEO].sub.230-[PPO].sub.65-OH, HOCH.sub.2CH.sub.2-[PEO].sub.230-[PPO].sub.65-O-n-butyl, [PEO].sub.240-[PPO].sub.55-OH, HOCH.sub.2CH.sub.2-[PEO].sub.240-[PPO].sub.55-O-n-butyl, [PEO].sub.205-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.60-O-n-butyl, [PEO].sub.314-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.314-[PPO].sub.60-O-n-butyl, [PEO].sub.352-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.352-[PPO].sub.60-O-n-butyl, [PEO].sub.409-[PPO].sub.95-OH, HOCH.sub.2CH.sub.2-[PEO].sub.409-[PPO].sub.95-O-n-butyl, [PEO].sub.432-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.432-[PPO].sub.60-O-n-butyl, [PEO].sub.216-[PPO].sub.60-OH, [PEO].sub.216-[PPO].sub.60-n-butyl, HO-[PEO].sub.216-[PPO].sub.60-n-butyl, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.50-O-n-butyl, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.50-OH, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.60-O-n-butyl, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.60-OH, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.70-O-n-butyl, HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.70-OH, ##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186##

    41.-58. (canceled)

    59. The method of claim 24, wherein the nucleic acid is at least 90%, 95%, 96%, 97%, 98%, 99% complementary, or is 100% complementary, to an endogenous RNA transcript encoding PKC.

    60.-124. (canceled)

    125. A method of: (i) expressing a transgene in a subject, (ii) delivering a population of genetically modified cells to a subject; or (iii) providing cell therapy to a subject in need thereof, the method comprising administering to the subject a population of cells that have been modified in accordance with the method of claim 1 or progeny thereof.

    126.-127. (canceled)

    128. The method of claim 125, wherein: (i) the cells are allogeneic with respect to the subject; (ii) the cells are autologous with respect to the subject; (iii) prior to the contacting, a population of precursor cells is isolated from the subject or a donor, and wherein the precursor cells are expanded ex vivo to yield the population of cells being administered to the subject; (iv) prior to administering the population of cells to the subject, a population of endogenous pluripotent cells is ablated in the subject by administration of one or more conditioning agents to the subject; (v) the method comprises ablating a population of endogenous pluripotent cells in the subject by administering to the subject one or more conditioning agents prior to administering to the subject the population of cells; (vi) upon administration of the population of cells to the subject, the administered cells, or Progeny thereof, differentiate into one or more cell types selected from megakaryocytes, thrombocytes, Platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes; (vii) the subject is a mammal; or (viii) the subject has been diagnosed as having a deficiency of an endogenous protein encoded by the transgene.

    129. The method of claim 128, wherein; (i) the cells are HLA-matched to the subject; (ii) the precursor cells are CD34+ HSCs, and wherein the precursor cells are expanded without loss of HSC functional potential; (iii) prior to isolation of the precursor cells from the subject or donor, the subject or donor is administered one or more pluripotent cell mobilization agents; (iv) the one or more conditioning agents are non-myeloablative conditioning agents; (v) the subject is a human; (vi) the subject has been diagnosed as having a disease set forth in Table 3; or (vii) the subject has been diagnosed as having beta thalassemia.

    130.-151. (canceled)

    152. A composition comprising a mixture formed by the method of claim 1.

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

    154. A population of eukaryotic cells that have been modified in accordance with the method of claim 1.

    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.-159. (canceled)

    160. A kit comprising the composition of claim 152.

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

    162. A kit comprising the population of cells of claim 154.

    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 in accordance with the method of: (i) expressing a transgene in a subject; (ii) delivering a population of genetically modified cells to a subject; or (iii) providing cell therapy to a subject in need thereof, the method comprising administering to the subject (a) a population of cells that have been modified in accordance with the method of: (a1) transducing a eukaryotic cell to express a transgene; or (a2) expressing a transgene in a eukaryotic cell, the method comprising contacting the cell with (x) a viral vector encoding the transgene and (y) a diblock copolymer comprising polyoxyethylene (PEO) subunits and polyoxypropylene (PPO) subunits; or (b) Progeny thereof.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0503] FIG. 1 is a graph showing % viability of CD34+ cells one day post transduction when treated with a diblock copolymer. Six diblock copolymers were tested (DBP1-DBP6) at a concentration of from 0.0001 mg/mL to 10 mg/mL. The following table illustrates the composition for each of DBP1-DBP6:

    TABLE-US-00002 Approximate Approximate DBP # Mn of PEO (kDa) Mn of PPO (kDa) m n DBP1 9.5 5 108 to 324 43 to 129 (average of 216) (average of 86) DBP2 19 5 216 to 648 43 to 129 (average of 432) (average of 86) DBP3 9 5 103 to 309 43 to 129 (average of 205) (average of 86) DBP4 15.5 5 176 to 528 43 to 129 (average of 352) (average of 86) DBP5 13.8 5 157 to 471 43 to 129 (average of 314) (average of 86) DBP6 18 5.5 205 to 615 48 to 143 (average of 409) (average of 95)
    The results indicate that application of diblock polymers during transduction is non-toxic to hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector for 20-24 hours, (Vector only, multiplicity of infection 10), in the presence of dose ranges (10-0.0001 mg/mL) of several diblock polymers (+DBP1-6). Plot shown summarises the percentage of viable cells detected by flow cytometry (AnnexinV.sup.7AAD.sup.) 1 day after lentiviral transduction of CD34+ cells isolated from different healthy donors (, A-D) in 4 independent experiments.

    [0504] FIGS. 2A-2F are graphs showing fold increase in transduction efficiency on day post transduction when treated with a diblock copolymer. FIG. 2A is DBP1, FIG. 2B is DBP2, FIG. 2C is DBP3, FIG. 2D is DBP4, FIG. 2E is DBP5, and FIG. 2F is DBP6. The graphs indicate that application of diblock polymers can enhance transduction efficiency of hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock polymers (DBP1-6, A-F) applied at final concentrations of 10-0.0001 mg/mL. (A) Plots summarise the fold change in percentage of transduced cells induced by the addition of diblock polymers, relative to cells treated with vector alone. Percentage of transduced cells was determined by flow cytometry detection of transgene expression 12 days post-transduction in 4 independent experiments, using CD34+ cells isolated from different healthy donors ().

    [0505] FIG. 3 is a graph showing mean vector copy number per cell in day 12 myeloid liquid cultures. DBP1-DBP6 were tested. The graph indicates that application of diblock polymers improves lentiviral integration of hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock polymers (DBP1-6) applied at final concentrations of 0.1-0.0001 mg/mL. Plot summarises the mean transgene copy number (VCN), determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA harvested from cell cultures 12 days post-transduction. Data shown for CD34+ cells isolated from 2 different healthy donors ().

    [0506] FIG. 4 is a graph showing percent transduced cells using a GFP vector for DBP1-DBP5. Various combination of transduction enhancer elements (TE combo 1 or combo 2) were tested with the DBP. The figure indicates that application of diblock polymers can enhance transduction efficiency of hematopoietic stem cells in concert with other compounds. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock polymers (DBP1-5) applied at final concentrations of 100-1 g/mL in combination with other compounds which can also improve lentiviral transduction (+TE combo 1, +TE combo 2). Plot shows the percentage of transduced cells, determined by flow cytometry detection of transgene expression 12 days post-transduction. Data shows meanSD, in a representative of 3 independent experiments.

    [0507] FIG. 5 is a graph showing percent viability of cells treated with either DBP1 or DBP5 with four different transduction enhancer combinations, TE combo 1, combo 2, combo 3, and combo 4. The graph indicates that application of diblock polymers in combination with other compounds during transduction is non-toxic to hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector for 20-24 hours, (Vector only, multiplicity of infection 10), in the presence of dose ranges (1-0.1 mg/mL) of diblock polymers DBP1 and DBP5, applied in combination with other compounds which can also improve lentiviral transduction (TE combo 1-4). Plot shown summarises the percentage of viable cells detected by flow cytometry (AnnexinV.sup.7AAD.sup.) 1 day after lentiviral transduction of CD34+ cells. Data shows meanSD, and is representative of 3 independent experiments.

    [0508] FIG. 6 is a graph showing fold change in percent CD90-HSC for cells treated with DBP1 or DBP5 with one of the four TE combinations noted above in FIG. 5. The graph indicates that application of diblock polymers in concert with other compounds which enhance transduction of hematopoietic stem cells does not adversely affect hematopoietic stem cell phenotype or survival. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock polymers (DBP1 and DPB5) applied at various final concentrations in combination with other compounds which can also improve lentiviral transduction (+TE combo 1-4). Plot shows the fold change in percentage of CD34+CD90+ stem cells (determined by flow cytometry) detected 1 day post transduction, relative to cells treated with vector alone. Data shows meanSD, and is representative of 3 independent experiments.

    [0509] FIG. 7 is a series of graphs showing that diblock copolymers with varying PEO and PPO compositions result in improvements in lentiviral transduction of hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock copolymers of various PEO and PPO compositions (Diblock PEO/PPO ratio), applied at a final concentration of 100 g/mL. Plots show the fold change in percentage of transduced CD34+ stem cells (determined by flow cytometry) and mean VCN per cell detected 12 days post transduction, relative to cells transduced in the absence of diblock copolymer. Each symbol represents stem cells assayed from an independent healthy donor, were 6 donors were tested in 2 independent assays.

    [0510] FIG. 8 is a series of graphs showing that diblock copolymers having broad ranges of PEO and PPO block content enhance lentiviral transduction of hematopoietic stem cells. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of diblock copolymers of various PEO and PPO compositions (Diblock PEO/PPO ratio), applied at a final concentration of 100 g/mL. Plots show the fold change in percentage of transduced CD34+ stem cells (determined by flow cytometry) and mean VCN per cell detected 12 days post transduction, relative to cells transduced in the absence of diblock copolymer. Each symbol represents stem cells assayed from an independent healthy donor, were 6 donors were tested in 2 independent assays.

    [0511] FIG. 9 is a series of graphs showing that diblock copolymers are compatible with RetroNectin, a recombinant human fibronectin fragment composed of three functional domains: the cell-binding domain (C-domain), heparin-binding domain (H-domain), and CS-1 domain. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of 2 different diblock polymer enhancer combinations (Diblock combo 1 & 2) with or without RetroNectin (RN). Plots show the percentage of transduced CD34+ stem cells (determined by flow cytometry) and mean VCN per cell detected 12 days post transduction and are representative of at least 3 independent assays, where each symbol represents stem cells assayed from an independent healthy donor.

    [0512] FIG. 10 is a series of graphs showing that application of diblock copolymers effectuates improved enhancement of stem cell transduction relative to that achieved by other commercial compounds. Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector (MOI 10), in the presence of 2 different diblock polymer enhancer combinations (Diblock combo 1 & 2), with various diblock copolymers (PPO/PEO ratio), or an enhancer combination containing 1 mg/mL (LB) LentiBoost (poloxamer 338; source Sirion Biotech). Plots show the fold change in percentage of transduced CD34+ stem cells (determined by flow cytometry) and mean VCN per cell detected 12 days post transduction compared to stem cell treatment with vector only. Data plotted shows 3 independent healthy donors, where each symbol represents stem cells assayed from an independent healthy donor. ****P<0.001, ***P<0.05 paired students t-test.

    DETAILED DESCRIPTION

    [0513] The compositions and methods described herein can be used, for example, to modify eukaryotic cells, such as pluripotent cells, including hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Using the compositions and methods of the disclosure, such cells may be engineered to express a gene of interest, and/or manipulated so as to proliferate ex vivo. In some embodiments of the disclosure, a population of pluripotent cells, such as a population of HSCs and/or HPCs, is contacted with a viral vector encoding a transgene. The transgene may encode a protein product or a regulatory ribonucleic acid (RNA) molecule that modulates the expression of a different gene. In some embodiments, the transgene encodes a protein that is deficient or non-functional in a patient (e.g., a mammalian patient, such as a human) suffering from a genetic disease for example, a genetic disease characterized by a loss-of-function mutation. The cell may be contacted with the virus in a manner that promotes transduction of the cell so as to express the desired transgene. In some embodiments, the cell is then administered to a patient suffering from a disease described above, thereby restoring gene expression in the individual.

    [0514] A variety of viral vectors can be used in conjunction with the compositions and methods of the disclosure. For examples, the viral vector may be a retrovirus, such as a lentivirus. Other viral vectors that may be used to achieve transduction of a target cell are described herein.

    [0515] To augment the extent of transduction and/or the rate at which the target cell is transduced, the cell may be contacted with a diblock copolymer, such as a diblock copolymer composed of a hydrophilic component and a hydrophobic component. For example, the hydrophilic component may include polyoxyethylene subunits and the hydrophobic component may include polyoxypropylene subunits.

    [0516] The sections that follow describe the use of various viral vectors and agents that can be used to augment viral transduction of a target cell and an array of therapeutic uses of the transduced cells.

    Diblock Copolymers

    [0517] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include those that include a hydrophilic block covalently connected to a hydrophobic block. Such diblock copolymers include those with PEO and PPO subunits. Suitable diblock copolymers include those in which the PEO subunits of the diblock copolymer have a number average molecular weight (Mn) of from about 5,000 g/mol to about 25,000 g/mol. For example, the PEO subunits of the diblock copolymer may have a 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, 13,500 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, 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).

    [0518] For example, in some embodiments, the PEO subunits of the diblock copolymer have a Mn of from about 9,000 g/mol to about 19,000 g/mol. In some particular embodiments, the PEO subunits of the diblock copolymer have a 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.

    [0519] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include those in which the PPO subunits of the diblock copolymer have a Mn of from about 2,000 g/mol to about 10,000 g/mol (e.g., the PPO subunits of the diblock copolymer have a 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).

    [0520] For example, in some embodiments, the PPO subunits of the diblock copolymer have a Mn of from about 3,500 g/mol to about 5,500 g/mol. In some particular embodiments, the PPO subunits of the diblock copolymer have a Mn of about 3,500 g/mol or 5,500 g/mol.

    [0521] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 40% by mass (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%, or more).

    [0522] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 50% by mass (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).

    [0523] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 60% by mass (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).

    [0524] In some embodiments, the diblock copolymer has an average ethylene oxide content of greater than 70% by mass (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).

    [0525] In some embodiments, the diblock copolymer has an average ethylene oxide content of from 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%).

    [0526] In some embodiments, the diblock copolymer has an average ethylene oxide content of from 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%).

    [0527] In some embodiments, the diblock copolymer has an average ethylene oxide content of from 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%).

    [0528] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include those with a 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 may have a Mn of greater than about 10,000 g/mol (e.g., the diblock copolymer has a Mn of greater than 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, 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, 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).

    [0529] In some embodiments, the diblock copolymer has a Mn of from 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, 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, 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, or 30,000 g/mol). For example, in some embodiments, the diblock copolymer has a Mn of from 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 particular embodiments, the diblock copolymer has a 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.

    [0530] In some embodiments, the diblock copolymer has a polydispersity index (Mw/Mn) of from 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 from 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.

    [0531] A diblock copolymer that can be used in conjunction with the compositions and methods described herein may have a structure:


    X.sub.1-[PEO].sub.m-L-[PPO].sub.n-X.sub.2 [0532] wherein m and n are integers; [0533] L is not present or is a chemical linker; and [0534] X.sub.1 and X.sub.2 each, independently, represent optionally present chemical substituents.

    [0535] In some embodiments, the diblock copolymer has a structure:


    X.sub.1-[PEO].sub.m-[PPO].sub.n-X.sub.2 [0536] wherein m and n are integers; and [0537] X.sub.1 and X.sub.2 each, independently, represent optionally present chemical substituents.

    [0538] Due to variation that occurs during synthesis of diblock copolymers that include PPO and PEO subunits, one of skill in the art will appreciate that values of m and n can vary, for example, by up to 2-fold above and 2-fold below the value recited. Therefore, a value of n=50 represents a heterogeneous mixture of diblock copolymers in which n may be from 25 to 100, such as a value of from 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, and the like. Similarly, a value of n=60 represents a heterogeneous mixture of diblock copolymers in which n may be from 30 to 120, such as from 30 to 90. Similarly, a value of n=70 represents a heterogeneous mixture of diblock copolymers in which n may be from 35 to 140, such as from 35 to 105.

    [0539] Exemplary linkers (L) that may be used in conjunction with the diblock copolymers described herein are described in more detail below.

    [0540] In some embodiments, X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido.

    [0541] In some embodiments, X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.1-6 alkoxy, or optionally substituted C.sub.1-6 alkylamino.

    [0542] For example, in some embodiments, X.sub.1 and X.sub.2 are each, independently, not present or are H, OH, H.sub.2N, H.sub.3CO, ethyl-O, n-butyl-O, tert-butyl-O, n-butyl, or tert-butyl.

    [0543] 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, such as 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).

    [0544] 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 from 109 to 327 (e.g., 217). In some embodiments, m is from 115 to 345 (e.g., 230). In some embodiments, m is from 120 to 360 (e.g., 240).

    [0545] In some particular embodiments, m is 200, 205, 216, 217, 225, 230, 240, 284, 314, 318, 323, 352, 409, or 432.

    [0546] 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, such as 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).

    [0547] In some particular embodiments, n is 50, 53, 55, 57, 60, 65, 70, 86, or 95.

    [0548] In some embodiments of the diblock copolymer, m is from about 100 to about 500 and n is from about 10 to about 200, such as from about 40 to 100 or 50 to about 95. For example, in some embodiments, m is from about 200 to about 450, such as from about 205 to about 432, and n is from about 10 to about 200, such as from about 40 to 100 or 50 to about 95.

    [0549] In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 162 to 486 (e.g., 323) and n is from 28 to 84 (e.g., 55).

    [0550] In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 159 to 477 (e.g., 318) and n is from 28 to 84 (e.g., 55).

    [0551] In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 108 to 324 (e.g., 216) and n is from 28 to 84 (e.g., 55).

    [0552] In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 103 to 309 (e.g., 205) and n is from 28 to 84 (e.g., 55).

    [0553] In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 148 to 444 (e.g., 295) and n is from 28 to 84 (e.g., 55).

    [0554] In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 171 to 513 (e.g., 341) and n is from 28 to 84 (e.g., 55).

    [0555] In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 142 to 426 (e.g., 284) and n is from 28 to 84 (e.g., 55).

    [0556] In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 100 to 300 (e.g., 200) and n is from 28 to 84 (e.g., 55).

    [0557] In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 113 to 339 (e.g., 225) and n is from 28 to 84 (e.g., 55).

    [0558] In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 109 to 327 (e.g., 217) and n is from 28 to 84 (e.g., 55).

    [0559] In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 115 to 345 (e.g., 230) and n is from 28 to 84 (e.g., 55).

    [0560] In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 43 to 129 (e.g., 86). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 27 to 81 (e.g., 53). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 29 to 87 (e.g., 57). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 28 to 84 (e.g., 55). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 30 to 90 (e.g., 60). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 33 to 99 (e.g., 65). In some embodiments, m is from 120 to 360 (e.g., 240) and n is from 28 to 84 (e.g., 55).

    [0561] 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.

    [0562] In some embodiments of the diblock copolymer, a 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, such as from about 3.4 to about 7.2. In some 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, 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 more. 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.

    [0563] In some embodiments, the diblock copolymer has the structure:

    ##STR00084##

    [0564] In some embodiments, the diblock copolymer has a structure selected from the following species. In each structure, it is to be understood that the indicated values of n and m denote heterogenous mixtures of diblock copolymers in which n and m may vary from up to 2-fold below the indicated value to 2-fold above the indicated value: [0565] [PEO].sub.323-[PPO].sub.86-OH, [0566] HOCH.sub.2CH.sub.2-[PEO].sub.323-[PPO].sub.86-O-n-butyl, [0567] [PEO].sub.318-[PPO].sub.53-OH, [0568] HOCH.sub.2CH.sub.2-[PEO].sub.318-[PPO].sub.53-O-n-butyl, [0569] [PEO].sub.216-[PPO].sub.53-OH, [0570] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.53-O-n-butyl, [0571] [PEO].sub.205-[PPO].sub.53-OH, [0572] HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.53-O-n-butyl, [0573] [PEO].sub.295-[PPO].sub.57-OH, [0574] HOCH.sub.2CH.sub.2-[PEO].sub.295-[PPO].sub.57-O-n-butyl, [0575] [PEO].sub.341-[PPO].sub.57-OH, [0576] HOCH.sub.2CH.sub.2-[PEO].sub.341-[PPO].sub.57-O-n-butyl, [0577] [PEO].sub.284-[PPO].sub.57-OH, [0578] HOCH.sub.2CH.sub.2-[PEO].sub.284-[PPO].sub.57-O-n-butyl, [0579] [PEO].sub.200-[PPO].sub.55-OH, [0580] HOCH.sub.2CH.sub.2-[PEO].sub.200-[PPO].sub.55-O-n-butyl, [0581] [PEO].sub.205-[PPO].sub.60-OH, [0582] HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.60-O-n-butyl, [0583] [PEO].sub.217-[PPO].sub.60-OH, [0584] HOCH.sub.2CH.sub.2-[PEO].sub.217-[PPO].sub.60-O-n-butyl, [0585] [PEO].sub.230-[PPO].sub.55-OH, [0586] HOCH.sub.2CH.sub.2-[PEO].sub.230-[PPO].sub.55-O-n-butyl, [0587] [PEO].sub.240-[PPO].sub.55-OH, [0588] HOCH.sub.2CH.sub.2-[PEO].sub.240-[PPO].sub.55-O-n-butyl, [0589] [PEO].sub.205-[PPO].sub.60-OH, [0590] HOCH.sub.2CH.sub.2-[PEO].sub.205-[PPO].sub.60-O-n-butyl, [0591] [PEO].sub.314-[PPO].sub.60-OH, [0592] HOCH.sub.2CH.sub.2-[PEO].sub.314-[PPO].sub.60-O-n-butyl, [0593] [PEO].sub.352-[PPO].sub.60-OH, [0594] HOCH.sub.2CH.sub.2-[PEO].sub.352-[PPO].sub.60-O-n-butyl, [0595] [PEO].sub.409-[PPO].sub.95-OH, [0596] HOCH.sub.2CH.sub.2-[PEO].sub.409-[PPO].sub.95-O-n-butyl, [0597] [PEO].sub.432-[PPO].sub.60-OH, [0598] HOCH.sub.2CH.sub.2-[PEO].sub.432-[PPO].sub.60-O-n-butyl, [0599] [PEO].sub.216-[PPO].sub.60-OH, [0600] [PEO].sub.216-[PPO].sub.60-n-butyl, [0601] HO-[PEO].sub.216-[PPO].sub.60-n-butyl, [0602] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.50-O-n-butyl, [0603] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.50-OH, [0604] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.60-O-n-butyl, [0605] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.60-OH, [0606] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.70-O-n-butyl, [0607] HOCH.sub.2CH.sub.2-[PEO].sub.216-[PPO].sub.70-OH,

    ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##

    [0608] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0609] [PEO].sub.205-[PPO].sub.60-OH.

    [0610] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.205(C.sub.3H.sub.6O).sub.60OH. The Mn of this diblock copolymer is about 12,500 g/mol. The polydispersity index of this diblock copolymer is about 1.1.

    [0611] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0612] [PEO].sub.216-[PPO].sub.60-OH.

    [0613] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.216(C.sub.3H.sub.6O).sub.60OH. The Mn of this diblock copolymer is about 13,000 g/mol. The polydispersity index of this diblock copolymer is about 1.08.

    [0614] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0615] [PEO].sub.314-[PPO].sub.60-OH.

    [0616] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.314(C.sub.3H.sub.6O).sub.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.

    [0617] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0618] [PEO].sub.352-[PPO].sub.60-OH.

    [0619] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.352(C.sub.3H.sub.6O).sub.60OH. The Mn of this diblock copolymer is about 19,000 g/mol. The polydispersity index of this diblock copolymer is about 1.13.

    [0620] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0621] [PEO].sub.409-[PPO].sub.95-OH.

    [0622] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.409(C.sub.3H.sub.6O).sub.95OH. The Mn of this diblock copolymer is about 23,500 g/mol. The polydispersity index of this diblock copolymer is about 1.17.

    [0623] Diblock copolymers that may be used in conjunction with the compositions and methods of the disclosure include a diblock copolymer having the structure: [0624] [PEO].sub.432-[PPO].sub.60-OH.

    [0625] This diblock copolymer has the approximate chemical formula H(C.sub.2H.sub.4O).sub.432(C.sub.3H.sub.6O).sub.60OH. The Mn of this diblock copolymer is about 22,500 g/mol. The polydispersity index of this diblock copolymer is about 1.11.

    [0626] The ethylene oxide content and propylene oxide content of a diblock copolymer, as described herein, can be determined using methods disclosed in Alexandridis and Hatton, Colloids and Surfaces A: Physicochemical and Engineering Aspects 96:1-46 (1995), the disclosure of which is incorporated herein by reference in its entirety. The diblock copolymers described herein may be synthesized according to the methods described in, e.g., Feng et al. Polymers 9: 1-31, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

    [0627] 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(-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(s-caprolactone) PEG-PCL, poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) PEG-PLGA, poly(ethylene glycol)-poly (-benzyl L-glutamate) PEG-PBLG, poly(ethylene glycol)-poly(-benzyl L-aspartate) PEG-PBLA, poly(ethylene glycol)-poly(-benzyl carboxylate--caprolactone) PEG-PBCL, and poly(ethylene glycol)-poly(5-valerolactone) PEG-PVL. Such diblock copolymers include, for example PEG.sub.5000-PCL.sub.5000, PEG.sub.2000-PCL.sub.1400, MPEG.sub.5000-PCL.sub.5000, MPEG.sub.5000-PCL.sub.13000, MPEG.sub.5000-PCL.sub.24000, PEG.sub.2000-PCL.sub.2000, MPEG.sub.5000-PCL.sub.2500, MPEG.sub.5000-PCL.sub.5000, MPEG.sub.5000-PCL.sub.8500, MPEG.sub.5000-PCL.sub.24700, MPEG.sub.2000-PCL.sub.1200, MPEG.sub.2000-PCL.sub.2700, MPEG.sub.5000-PCL.sub.3800, MPEG.sub.5000-PCL.sub.18000, PEG.sub.5000-PCL.sub.4000, PEG.sub.2000-PCL.sub.900, PEG.sub.1980-PCL.sub.1368, PEG.sub.1980-PCL.sub.2622, PEG.sub.1980-PCL.sub.17328, PEG.sub.2000-PCL.sub.2280, PEG.sub.5000-PCL.sub.5000, PEG.sub.5000-PCL.sub.24000, PEG.sub.5000-PCL.sub.5000, PEG.sub.5000-PCL.sub.24000, PEG.sub.5000-PCL.sub.4790, PEG.sub.5000-PCL.sub.10000, MPEG.sub.5333-PCL.sub.2638, MPEG.sub.5333-PCL.sub.4984, MPEG.sub.5333-PCL.sub.8034, MPEG.sub.5333-PCL.sub.9068, MPEG.sub.5000-PCL.sub.2166, MPEG.sub.2000-PCL.sub.1320, MPEG.sub.2000-PCL.sub.852, MPEG.sub.750-PCL.sub.464, MPEG.sub.750-PCL.sub.323, MPEG.sub.750-PCL.sub.197, MPEG-PCL, PEG.sub.5000-PDLLA.sub.4200, PEG.sub.5000-PDLLA.sub.45000, MPEG.sub.2000-PDLLA.sub.2000, MPEG.sub.2000-PDLLA.sub.1333, MPEG.sub.5000-PDLLA.sub.2143, PEG.sub.52000-PDLLA.sub.5000, PEG.sub.91000-PDLLA.sub.5000, PEG.sub.4100-PDLLA.sub.1200, PEG.sub.6000-PDLLA.sub.3000, PEG.sub.5700-PDLLA.sub.5400, PEG.sub.6100-PDLLA.sub.7800, PEG.sub.5000-PBCL.sub.4700, PEG.sub.5000-PBCL.sub.4470, PEG.sub.12000-PBLA.sub.5000, PEG.sub.12000-PBLA.sub.3000, PEG-PBLA, PEG.sub.12000-PBLA.sub.5000, MPEG.sub.2000-PVL.sub.1000, MPEG.sub.2000-PVL.sub.2000, MPEG.sub.5000-PVL.sub.2600, and MPEG.sub.5000-PVL.sub.4900. These diblock copolymers are described, e.g., in Hussein et al. Materials 11: 1-26, 2018, the disclosure of which is hereby incorporated in its entirety.

    Linkers

    [0628] The diblock copolymers described herein may optionally include a linker that connects the PEO subunit block and PPO subunit block of the polymer. The PEO and PPO components of the diblock copolymer may be directly bound to one another, for instance, without an intervening linker. The linker may be a peptidic linker or a synthetic linker.

    Synthetic Linkers

    [0629] A variety of linkers can be used to covalently couple the PEO component with the PPO component, for instance, so as to form a diblock copolymer as described herein. Exemplary linkers include those that may be cleaved, for instance, by 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 as it pertains to linkers suitable for chemical coupling). Examples of linkers useful for the synthesis of conjugates described herein include those that contain electrophiles, such as Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, among others, suitable for reaction with nucleophilic substituents present within antibodies, antigen-binding fragments, proteins, peptides, and small molecules, such as amine and thiol moieties. For instance, linkers suitable for the synthesis of diblock copolymers include, without limitation, alkyl, cycloalkyl, and heterocycloalkyl linkers, such as open-chain ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl chains, cyclohexyl groups, cyclopentyl groups, cyclobutyl groups, cyclopropyl groups, piperidinyl groups, morpholino groups, or others containing two reactive moieties (e.g., halogen atoms, aldehyde groups, ester groups, acyl chloride groups, acyl anhydride groups, tosyl groups, mesyl groups, or brosyl groups, among others, that can be displaced by reactive nucleophilic atoms present within a PEO or PPO polymer), aryl or heteroaryl linkers, such as benzyl, napthyl, or pyridyl groups containing two halomethyl groups that can be displaced by reactive nucleophilic atoms present within a 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 described, for instance, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linkers, which are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.

    [0630] Additional linkers through which one block of the copolymer may be bound to another as described herein include linkers that are covalently bound to one block of the copolymer (e.g., PEO or PPO) on one end of the linker and, on the other end of the linker, contain a chemical moiety formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the other component of the diblock copolymer (e.g., PEO or PPO). Exemplary reactive substituents that may be used to form linkers include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. Linkers useful in conjunction with the diblock copolymers described herein include, without limitation, linkers containing chemical moieties formed by coupling reactions as depicted in Table 2 below. Curved lines designate points of attachment to each component of the conjugate.

    TABLE-US-00003 TABLE 2 Exemplary chemical moieties formed by coupling reactions in the formation of diblock copolymers Exemplary Coupling Reaction Chemical Moiety Formed by Coupling Reaction [3 + 2] Cycloaddition [00090]embedded image [3 + 2] Cycloaddition [00091]embedded image [3 + 2] Cycloaddition, Esterification [00092]embedded image [3 + 2] Cycloaddition, Esterification [00093]embedded image [3 + 2] Cycloaddition, Esterification [00094]embedded image [3 + 2] Cycloaddition, Esterification [00095]embedded image [3 + 2] Cycloaddition, Esterification [00096]embedded image [3 + 2] Cycloaddition, Esterification [00097]embedded image [3 + 2] Cycloaddition, Esterification [00098]embedded image [3 + 2] Cycloaddition, Esterification [00099]embedded image [3 + 2] Cycloaddition, Esterification [00100]embedded image [3 + 2] Cycloaddition, Esterification [00101]embedded image [3 + 2] Cycloaddition, Esterification [00102]embedded image [3 + 2] Cycloaddition, Etherification [00103]embedded image [3 + 2] Cycloaddition [00104]embedded image Michael addition [00105]embedded image Michael addition [00106]embedded image Imine condensation, Amidation [00107]embedded image Imine condensation [00108]embedded image Disulfide formation [00109]embedded image Thiol alkylation [00110]embedded image Condensation, Michael addition [00111]embedded image

    Peptidic Linkers

    [0631] In addition to the synthetic linkers described above, the binding of a PEO polymer to a PPO polymer can be effectuated by way of a peptidic linker. Exemplary peptide linkers include those that contain one or more glycine residues. Such linkers may be sterically flexible due to the ability of glycine to access a variety of torsional angles. For instance, peptide linkers useful in conjunction with the compositions and methods described herein include polyglycine, polyserine, or a combination thereof. Additional examples of peptidic linkers include those that also contain one or more polar amino acids, such as serine threonine. For instance, linkers useful in conjunction with the compositions and methods described herein include those that contain one or more repeats of glycine and serine. Additional linkers include those that contain one or more cationic or anionic residues, such as a lysine, arginine, aspartate, or glutamate residue.

    PKC Modulating Agents

    [0632] A variety of agents can be used to reduce PKC activity and/or expression. Without being limited by mechanism, such agents can augment viral transduction by stimulating Akt signal transduction and/or maintaining cofilin in a dephosphorylated state, thereby promoting actin depolymerization. This actin depolymerization event may serve to remove a physical barrier that hinders entry of a viral vector into the nucleus of a target cell.

    Staurosporine and Variants Thereof

    [0633] In some embodiments, the substance that reduces 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 may be a compound represented by formula (1)

    ##STR00112## [0634] wherein R.sub.1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido; [0635] R.sub.2 is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, or optionally substituted acyl; [0636] R.sub.a and R.sub.b are each, independently, H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, or optionally substituted C.sub.2-6 alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl, or R.sub.a and R.sub.b, together with the atoms to which they are bound, are joined to form an optionally substituted and optionally fused heterocycloalkyl ring; [0637] R.sub.c is O, NR.sub.d, or S; [0638] R.sub.d is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, or optionally substituted C.sub.2-6 alkynyl; [0639] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0640] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0641] - - - represents a bond that is optionally present; [0642] n is an integer from 0-4; and [0643] m is an integer from 0-4; [0644] or a salt thereof.

    [0645] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO 1991/009034, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (II)

    ##STR00113## [0646] wherein R.sub.1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, oxo, or thiocarbonyl; [0647] R.sub.2 is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, or optionally substituted acyl; [0648] R.sub.a and R.sub.b, together with the atoms to which they are bound, are joined to form an optionally substituted and optionally fused heterocycloalkyl ring; [0649] R.sub.c is O or S; [0650] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0651] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0652] n is an integer from 0-4; and [0653] m is an integer from 0-4; [0654] or a salt thereof.

    [0655] Additional examples of such staurosporine variants are represented by formula (III)

    ##STR00114## [0656] wherein R.sub.1 is H, OH, oxo, or thiocarbonyl; [0657] R.sub.2 is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, or optionally substituted acyl; [0658] Ring A is an optionally substituted and optionally fused heterocycloalkyl ring; [0659] R.sub.c is O or S; [0660] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0661] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0662] n is an integer from 0-4; and [0663] m is an integer from 0-4; [0664] or a salt thereof.

    [0665] Further examples of such staurosporine variants are represented by formula (IV)

    ##STR00115## [0666] wherein R.sub.1 is H, OH, or oxo; [0667] Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring; [0668] R.sub.c is O or S; [0669] W is O, NH, or S; [0670] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0671] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0672] n is an integer from 0-4; and [0673] m is an integer from 0-4; [0674] or a salt thereof.

    [0675] Additional examples of such staurosporine variants are represented by formula (V)

    ##STR00116## [0676] wherein R.sub.1 is H, OH, or oxo; [0677] R.sub.c is O or S; [0678] W is O, NH, or S; [0679] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; and [0680] p is O or 1; [0681] or a salt thereof.

    [0682] Additional examples of such staurosporine variants are represented by formula (VI)

    ##STR00117## [0683] wherein R.sub.1 is H, OH, or oxo; [0684] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; and [0685] s is an integer from 0-8; [0686] or a salt thereof.

    [0687] Further examples of such staurosporine variants are represented by formula (VII)

    ##STR00118## [0688] wherein R.sub.1 is H, OH, or oxo; [0689] R.sub.2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; and [0690] R.sub.3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido [0691] or a salt thereof.

    [0692] Additional examples of such staurosporine variants are represented by formula (VIII)

    ##STR00119## [0693] wherein R.sub.1 is H, OH, or oxo; [0694] R.sub.2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; and [0695] R.sub.3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido [0696] or a salt thereof.

    [0697] Further examples of such staurosporine variants are represented by formula (IX)

    ##STR00120## [0698] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0699] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0700] n is an integer from 0-4; and [0701] m is an integer from 0-4; [0702] or a salt thereof.

    [0703] Additional examples of such staurosporine variants are represented by formula (1)

    ##STR00121## [0704] or a salt thereof.

    [0705] In some embodiments, the PKC inhibitor is staurosporine, (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)

    ##STR00122## [0706] or a salt thereof.

    [0707] Further examples of such staurosporine variants are represented by formula (X)

    ##STR00123## [0708] wherein R.sub.1 is H, OH, or oxo; [0709] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; and [0710] t is an integer from 0-6; [0711] or a salt thereof.

    [0712] Additional examples of such staurosporine variants are represented by formula (XI)

    ##STR00124## [0713] wherein R.sub.1 is H, OH, or oxo; and [0714] R.sub.4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; [0715] or a salt thereof.

    [0716] Further examples of such staurosporine variants are represented by formula (XII)

    ##STR00125## [0717] wherein R.sub.1 is H, OH, or oxo; and [0718] R.sub.4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; [0719] or a salt thereof.

    [0720] Additional examples of such staurosporine variants are represented by formula (XIII)

    ##STR00126## [0721] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0722] 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 alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted 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; [0723] n is an integer from 0-4; and [0724] m is an integer from 0-4; [0725] or a salt thereof.

    [0726] Additional examples of such staurosporine variants are represented by formula (3)

    ##STR00127## [0727] or a salt thereof.

    [0728] Additional examples of such staurosporine variants are represented by formula (4)

    ##STR00128## [0729] or a salt thereof.

    [0730] Additional examples of such staurosporine variants are:

    ##STR00129## ##STR00130## ##STR00131## [0731] or a salt thereof.

    [0732] In some embodiments, the PKC inhibitor is a staurosporine variant described in WO 1993/007153, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XIV)

    ##STR00132## [0733] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0734] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0735] or a salt thereof.

    [0736] In some embodiments, the PKC inhibitor is a compound represented by formula (XV)

    ##STR00133## [0737] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0738] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0739] or a salt thereof.

    [0740] In some embodiments, the PKC inhibitor is a compound selected from:

    ##STR00134##

    or a salt thereof.

    [0741] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Pat. No. 5,093,330, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XVI)

    ##STR00135## [0742] 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; [0743] or a salt or quaternized variant thereof.

    [0744] In some embodiments, the PKC inhibitor is a compound represented by formula (XVII)

    ##STR00136## [0745] 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; [0746] or a salt or quaternized variant thereof.

    [0747] In some embodiments, the PKC inhibitor is a compound selected from:

    ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150##

    or a salt thereof.

    [0748] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Pat. No. 5,264,431, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XVIII)

    ##STR00151## [0749] wherein R is H, OH, C.sub.1-6 alkoxy, or oxo; and [0750] R.sub.2 is

    ##STR00152##

    optionally wherein the configuration of the sugar moiety is derived from D-glucose, D-galactose, or D-mannose; [0751] R.sub.3 is H, OH, C.sub.1-6 alkanoyloxy, C.sub.1-6 alkoxy, benzyloxy, benzoyloxy or phenyloxy, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, or C.sub.1-6 alkoxy; [0752] R.sub.4 is OH, C.sub.1-6 alkanoyloxy, benzoyloxy, benzyloxy, amino, C.sub.1-6 alkylamino, di-C.sub.1-6 alkylamino, C.sub.1-6 alkoxycarbonylamino, C.sub.2-20 alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy; [0753] R.sub.5 is H or C.sub.1-6 alkyl; [0754] R.sub.6 is hydroxyl which is free or esterified with an aliphatic C.sub.2-22 carboxylic acid, or is C.sub.1-6 alkoxycarbonyloxy, C.sub.1-6 alkylsulfonyloxy, amino which is free or acylated with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, or C.sub.1-6 alkoxy; and [0755] R.sub.7 is OH which is free or esterified with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkoxycarbonyloxy, C.sub.1-6 alkylsulfonyloxy, azido, amino which is free or acylated with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkylamino, di-C.sub.1-6 alkylamino, C.sub.1-6 alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino or benzyloxycarbonylamino, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, or C.sub.1-6 alkoxycarbonyl; [0756] or a salt thereof.

    [0757] In some embodiments, the PKC inhibitor is a compound represented by formula (XIX)

    ##STR00153## [0758] wherein R is H, OH, C.sub.1-6 alkoxy, or oxo; and [0759] R.sub.2 is

    ##STR00154## [0760] R.sub.3 is H, OH, C.sub.1-6 alkanoyloxy, C.sub.1-6 alkoxy, benzyloxy, benzoyloxy or phenyloxy, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, or C.sub.1-6 alkoxy; [0761] R.sub.4 is OH, C.sub.1-6 alkanoyloxy, benzoyloxy, benzyloxy, amino, C.sub.1-6 alkylamino, di-C.sub.1-6 alkylamino, C.sub.1-6 alkoxycarbonylamino, C.sub.2-20 alkanoylamino, benzoylamino, benzyloxycarbonylamino, or phenyloxycarbonylamino, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy; [0762] R.sub.5 is H or C.sub.1-6 alkyl; [0763] R.sub.6 is hydroxyl which is free or esterified with an aliphatic C.sub.2-22 carboxylic acid, or is C.sub.1-6 alkoxycarbonyloxy, C.sub.1-6 alkylsulfonyloxy, amino which is free or acylated with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkoxycarbonylamino, azido, benzoyloxy, benzyloxycarbonyloxy, benzoylamino, benzyloxycarbonylamino, or phenylsulfonyloxy, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, or C.sub.1-6 alkoxy; and [0764] R.sub.7 is OH which is free or esterified with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkoxycarbonyloxy, C.sub.1-6 alkylsulfonyloxy, azido, amino which is free or acylated with an aliphatic C.sub.2-22 carboxylic acid, C.sub.1-6 alkylamino, di-C.sub.1-alkylamino, C.sub.1-6 alkoxycarbonylamino, carbamoylamino, benzoyloxy, benzyloxycarbonyloxy, phenylsulfonyloxy, benzoylamino, benzylamino or benzyloxycarbonylamino, each of which is optionally substituted in the phenyl moiety by halogen, hydroxyl, trifluoromethyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, or C.sub.1-6 alkoxycarbonyl; [0765] or a salt thereof.

    [0766] In some embodiments, the PKC inhibitor is a compound selected from N-(1--O-Benzyl-2-N-acetylmuramyl)staurosporine, N-(2-N-Acetyl-muramyl)staurosporine, N-(6-O-Mesyl-1--O-benzyl-2-N-acetylmuramyl)staurosporine, N-(6-Azido-1--O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-Amino-1--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--O-Benzyl-2-N-acetylhomomuramyl)staurosporine, N-(1--O-Benzyl-2-N-acetyl-L-homomuramyl)staurosporine, the 1--anomer of N-(2-N-acetyl-L-homomuramyl)staurosporine, N-(1--O-Benzyl-4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1--O-Benzyl-4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporin, N-(1-Deoxy-2-N-acetylmuramyl)staurosporine, the 1--anomer of N-(4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, the 1--anomer of N-(4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-,4,6-O-Triacetyl-2-N-acetylmuramyl)staurosporine, N-(1-Deoxy-6-O-acetyl-2-N-acetylmuramyl)staurosporine, N-(1-Deoxy-6-O-mesyl-2-N-acetylmuramyl)staurosporine, N-(1-Deoxy-6-O-toluolsulfonyl-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.

    [0767] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Pat. No. 5,461,146, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XX)

    ##STR00155## [0768] wherein Z.sub.1 is H or OH; [0769] Z.sub.2 is H or OH; [0770] R.sub.1 is H, halogen, or optionally substituted alkyl; [0771] R.sub.2 is H or halogen; [0772] R is OH or optionally substituted alkoxy; and [0773] X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH.sub.2NH-serine, CO.sub.2CH.sub.3, CH.sub.2NHCO.sub.2C.sub.6H.sub.5, CONHC.sub.6H.sub.5, or CH.sub.2NHCO.sub.2CH.sub.3, wherein C.sub.6H.sub.5 denotes a phenyl moiety; or a salt thereof.

    [0774] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Pat. No. 5,756,494, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XXI)

    ##STR00156## [0775] wherein Z.sub.1 is H or OH; [0776] Z.sub.2 is H or OH; [0777] R.sub.1 is H, halogen, or optionally substituted alkyl; [0778] R.sub.2 is H or halogen; [0779] R is OH or optionally substituted alkoxy; and [0780] X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH.sub.2NH-serine, CO.sub.2CH.sub.3, CH.sub.2NHCO.sub.2C.sub.6H.sub.5, CONHC.sub.6H.sub.5, or CH.sub.2NHCO.sub.2CH.sub.3, wherein C.sub.6H.sub.5 denotes a phenyl moiety; [0781] or a salt thereof.

    [0782] In some embodiments, the PKC inhibitor is a staurosporine variant described in US 2005/0020570, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XXII), (XXIII), (XXIV), or (XXV)

    ##STR00157## [0783] wherein each R.sub.1 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-di-substituted aminosulfonyl; [0784] each R.sub.2 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl, or N-mono- or N,N-di-substituted aminosulfonyl; [0785] each R.sub.5 is, independently, H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical with up to 29 carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical with up to 20 carbon atoms in each case, and in each case up to 9 heteroatoms, or acyl with up to 30 carbon atoms; and [0786] each X is, independently, O, OH and H, or a pair of hydrogen atoms; [0787] 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl; [0788] 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl; [0789] each n is, independently, an integer from 0-4; and [0790] each m is, independently, an integer from 0-4; [0791] or a salt thereof.

    [0792] In some embodiments, the PKC inhibitor is a compound represented by formula (128)

    ##STR00158## [0793] or a salt thereof. This compound is also known as K252a.

    [0794] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII)

    ##STR00159## [0795] wherein each R.sub.1 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl; [0796] each R.sub.2 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-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl; [0797] each R.sub.5 is, independently, H, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical with up to 29 carbon atoms in each case, or a heterocyclic or heterocyclic-aliphatic radical with up to 20 carbon atoms in each case, and in each case up to 9 heteroatoms, or acyl with up to 30 carbon atoms; [0798] each R.sub.8 is, independently, acyl with up to 30 carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical with up to 29 carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical with up to 20 carbon atoms in each case, and in each case up to 9 heteroatoms; [0799] each R.sub.9 is, independently, 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, carbonyidioxy, esterified carboxy, carbamoyl, N-mono- or N,N-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl; [0800] each R.sub.10 is, independently, acyl with up to 30 carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical with up to 29 carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical with up to 20 carbon atoms in each case, and in each case up to 9 heteroatoms; [0801] each X is, independently, O, OH and H, or a pair of hydrogen atoms; [0802] each n is, independently, an integer from 0-4; [0803] each m is, independently, an integer from 0-4; [0804] each n is, independently, an integer from 0-4; and [0805] each m is, independently, an integer from 0-4; [0806] or a salt thereof.

    [0807] In some embodiments, the PKC inhibitor is a staurosporine variant described in U.S. Pat. No. 5,624,949, the disclosure of which is incorporated herein by reference in its entirety. Examples of such staurosporine variants are represented by formula (XXVIII)

    ##STR00160## [0808] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0809] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0810] or a salt thereof.

    [0811] In some embodiments, the PKC inhibitor is a compound represented by formula (XXIX)

    ##STR00161## [0812] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0813] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0814] or a salt thereof.

    [0815] In some embodiments, the PKC inhibitor is a compound represented by formula (XXX)

    ##STR00162## [0816] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0817] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0818] or a salt thereof.

    [0819] In some embodiments, the PKC inhibitor is a compound represented by formula (XXXI)

    ##STR00163## [0820] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and [0821] R.sub.2 is optionally substituted C.sub.1-6 alkyl; [0822] or a salt thereof.

    [0823] In some embodiments, the PKC inhibitor is a compound selected from:

    ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171## ##STR00172##

    [0824] In some embodiments, the cell is further contacted with stauprimide, e.g., as described in Caravatti et al. Bioorg. Medic. Chem. Letters 4:199-404, 1994, the disclosure of which is hereby incorporated by reference in its entirety.

    Interfering RNA

    [0825] Exemplary PKC modulating agents that may be used in conjunction with the compositions and methods of the disclosure include interfering RNA molecules, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), and/or micro RNA (miRNA), that diminish PKC gene expression. Methods for producing interfering RNA molecules are known in the art and are described in detail, for example, in WO 2004/044136 and U.S. Pat. No. 9,150,605, the disclosures of each of which are incorporated herein by reference in their entirety.

    HDAC Inhibitors

    [0826] A variety of agents can be used to inhibit histone deacetylases in order to increase the expression of a transgene during viral transduction. Without wishing to be bound by theory, reduced transgene expression from viral vectors may be caused by epigenetic silencing of vector genomes carried out by histone deacetylates. Accordingly, the methods described herein may further include contacting a cell with an HDAC inhibitor, e.g., prior to, concurrently with, or after contacting a cell with a diblock copolymer in order to improve viral transduction and/or increase transgene expression. Hydroxamic acids represent a particularly robust class of HDAC inhibitors that inhibit these enzymes by virtue of hydroxamate functionality that binds cationic zinc within the active sites 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 by reference herein). Other HDAC inhibitors include Panobinostat, described in Drugs of the Future 32(4): 315-322 (2007), the disclosure of which is incorporated herein by reference.

    ##STR00173##

    [0827] Additional examples of hydroxamic acid inhibitors of histone deacetylases include the compounds shown below, described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference:

    ##STR00174## ##STR00175##

    [0828] Other HDAC inhibitors that do not contain a hydroxamate substituent 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 disclosure of each of which is incorporated herein by reference. Other small molecule inhibitors that exploit chemical functionality distinct from a hydroxamate include those described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference:

    ##STR00176##

    [0829] Additional examples of chemical modulators of histone acetylation useful with the compositions and methods of the invention include modulators of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, Sirt1, Sirt2, and/or HAT, such as butyrylhydroxamic acid, M344, LAQ824 (Dacinostat), AR-42, Belinostat (PXD101), CUDC-101, Scriptaid, Sodium Phenylbutyrate, Tasquinimod, Quisinostat (JNJ-26481585), Pracinostat (SB939), CUDC-907, Entinostat (MS-275), Mocetinostat (MGCD0103), Tubastatin A HCl, PCI-34051, Droxinostat, PCI-24781 (Abexinostat), RGFP966, Rocilinostat (ACY-1215), C1994 (Tacedinaline), Tubacin, RG2833 (RGFP109), Resminostat, Tubastatin A, BRD73954, BG45, 4SC-202, CAY10603, LMK-235, Nexturastat A, TMP269, HPOB, Cambinol, and Anacardic Acid.

    [0830] In some particular embodiments, the HDAC inhibitor is Scriptaid.

    [0831] The cell may be contacted with the diblock copolymer and with the HDAC inhibitor simultaneously. Alternatively, the cell may be contacted with the diblock copolymer before being contacted with the HDAC inhibitor. In some embodiments, the cell is contacted with the HDAC inhibitor before being contacted with the diblock copolymer.

    Cyclosporines

    [0832] In some embodiments, the cell is further contacted with a cyclosporine, such as cyclosporine A (CsA) or cyclosporine H (CsH), during viral transduction. The cell may be contacted with the diblock copolymer and with the cyclosporine simultaneously. Alternatively, the cell may be contacted with the diblock copolymer before being contacted with the cyclosporine. In some embodiments, the cell is contacted with the cyclosporine before being contacted with the diblock copolymer.

    [0833] In some embodiments, the cyclosporine is CsH.

    [0834] In some embodiments, the concentration of the cyclosporine, when contacted with the cell, is from about 1 M to about 10 M (e.g., about 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4 M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9 M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, or 10 M). In some embodiments, the cyclosporine is CsA and the concentration of the cyclosporine, when contacted with the cell, is about 6 M. In some embodiments, the cyclosporine is CsH and the concentration of the cyclosporine, when contacted with the cell, is about 8 M.

    Activator of Prostaglandin E Receptor Signaling

    [0835] In some embodiments, the cell is further contacted with an activator of prostaglandin E receptor signaling. The cell may be contacted with the diblock copolymer and with the activator of prostaglandin E receptor signaling simultaneously. Alternatively, the cell may be contacted with the diblock copolymer before being contacted with the activator of prostaglandin E receptor signaling. In some embodiments, the cell is contacted with the activator of prostaglandin E receptor signaling before being contacted with the diblock copolymer.

    [0836] In some embodiments, the activator of prostaglandin E receptor signaling is a small molecule, such as a compound described in WO 2007/112084 or WO 2010/108028, the disclosures of each of which are incorporated herein by reference as they pertain to prostaglandin E receptor signaling activators.

    [0837] In some embodiments, the activator of prostaglandin E receptor signaling is a small molecule, such as a small organic molecule, a prostaglandin, a Wnt pathway agonist, a cAMP/PI3K/AKT pathway agonist, a Ca.sup.2+ 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, Bicuclline, Vesamicol, Peruvoside, Imipramine, Chlorpropamide, 1,5-Pentamethylenetetrazole, 4-Aminopyridine, Diazoxide, Benfotiamine, 12-Methoxydodecenoic acid, N-Formyl-Met-Leu-Phe, Gallamine, IAA 94, Chlorotrianisene, and or a derivative of any of these compounds.

    [0838] In some embodiments, the activator of prostaglandin E receptor signaling is a naturally-occurring or synthetic chemical molecule or polypeptide that binds to and/or interacts with a prostaglandin E receptor, typically to activate or increase one or more of the downstream signaling pathways associated with a prostaglandin E receptor.

    [0839] 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.

    [0840] In some embodiments, the activator of prostaglandin E receptor signaling is PGE2.

    [0841] In some embodiments, the activator of prostaglandin E receptor signaling is 15d-PGJ2, deltal2-PGJ2, 2-hydroxyheptadecatrienoic acid (HHT), Thromboxane (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-deoxy PGE1, Misoprostol, and Butaprost, and Corey alcohol-A ([3aa,4a,5,6aa]-()-[Hexahydro-4-(hydroxymetyl)-2-oxo-2H-cyclopenta/b/furan-5-yl][1,1-biphenyl]-4-carboxylate), Corey alcohol-B (2H-Cyclopenta[b]furan-2-on,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).

    [0842] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin E receptor ligand, such as prostaglandin E2 (PGE2), or an analogs or derivative thereof. Prostaglandins refer generally to hormone-like molecules that are derived from fatty acids containing 20 carbon atoms, including a 5-carbon ring, as described herein and known in the art. Illustrative examples of PGE2 analogs or derivatives include, but are not limited to, 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16, 16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-keto Fluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinol amide, PGE2 methyl ester, 16-phenyl tetranor 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) hydroxyy PGE2.

    [0843] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin analog or derivative having a similar structure to PGE2 that is substituted with halogen at the 9-position (see, e.g., WO 2001/12596, herein incorporated by reference in its entirety), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives, such as those described in US 2006/0247214, herein incorporated by reference in its entirety).

    [0844] In some embodiments, the activator of prostaglandin E receptor signaling is a non-PGE2-based ligand. In some embodiments, the activator of prostaglandin E receptor signaling is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Additional examples of non-PGE2-based EP2 agonists include the carbazoles and fluorenes disclosed in WO 2007/071456, herein incorporated by reference for its disclosure of such agents. Illustrative examples of non-PGE2-based EP.sub.3 agonist include, but are not limited to, AE5-599, MB28767, GR 63799X, ONO-NT012, and ONO-AE-248. Illustrative examples of non-PGE.sub.2-based EP.sub.4 agonist include, but are not limited to, ONO-4819, APS-999 Na, AH23848, and ONO-AE 1-329. Additional examples of non-PGE2-based EP4 agonists can be found in WO 2000/038663; U.S. Pat. Nos. 6,747,037; and 6,610,719, each of which are incorporated by reference for their disclosure of such agonists In some embodiments, the activator of prostaglandin E receptor signaling is a Wnt agonist.

    [0845] Illustrative examples of Wnt agonists include, but are not limited to, Wnt polypeptides and glycogen synthase kinase 3 (GSK3) inhibitors. Illustrative examples of Wnt polypeptides suitable for use as compounds that stimulate the prostaglandin EP receptor signaling pathway include, but are not limited to, Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt1Ob, Wnt11, Wnt14, Wnt15, or biologically active fragments thereof. GSK3 inhibitors suitable for use as agents that stimulate the prostaglandin EP receptor signaling pathway bind to and decrease the activity of GSK3a, or GSK3. Illustrative examples of GSK3 inhibitors include, but are not limited to, BIO (6-bromoindirubin-3-oxime), LiCl, Li.sub.2CO.sub.3, or other GSK-3 inhibitors, as exemplified in U.S. Pat. Nos. 6,057,117 and 6,608,063, as well as US 2004/0092535 and US 2004/0209878, and ATP-competitive, selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also referred to as CT-99021/CHIR-99021 and CT-98023/CHIR-98023, respectively) (Chiron Corporation (Emeryville, CA)). The structure of CHIR-98023 is

    ##STR00177## [0846] or a salt thereof.

    [0847] In some embodiments, method further includes contacting the cell with a GSK3 inhibitor.

    [0848] In some embodiments, the GSK3 inhibitor is CHIR-99021.

    [0849] In some embodiments, the GSK3 inhibitor is Li.sub.2CO.sub.3.

    [0850] 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 ester, forskolin, sclareline, 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.

    [0851] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the Ca.sup.2+ second messenger pathway, such as an agent selected from the group consisting of Bapta-AM, Fendiline, Nicardipine, and derivatives of these agents.

    [0852] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the NO/Angiotensin signaling, such as an agent selected from the group consisting of L-Arg, Sodium Nitroprusside, Sodium Vanadate, Bradykinin, and derivatives thereof.

    Polycationic Polymers

    [0853] In some embodiments of the methods described herein, the cell is further contacted with a polycationic polymer. The cell may be contacted with the diblock copolymer and with the polycationic polymer simultaneously. Alternatively, the cell may be contacted with the diblock copolymer before being contacted with the polycationic polymer. In some embodiments, the cell is contacted with the polycationic polymer before being contacted with the diblock copolymer.

    [0854] In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethylenimine, or a polyethylene glycol/poly-L-lysine block copolymer.

    [0855] In some embodiments, the polycationic polymer is protamine sulfate.

    [0856] In some embodiments, the cell is further contacted with an expansion agent during the transduction procedure. The cell may be, for example, a hematopoietic stem cell and the expansion agent may be a hematopoietic stem cell expansion agent, such as a hematopoietic stem cell expansion agent known in the art or described herein.

    Additional Transduction Enhancers

    [0857] In some embodiments of the methods described herein, during the transduction procedure, the cell is further contacted with an agent that inhibits mTor signaling. The agent that inhibits mTor signaling may be, for example, rapamycin, among other suppressors of mTor signaling.

    [0858] In some embodiments of the methods described herein, during the transduction procedure, the cell is further contacted with an agent that enhances transduction, e.g., in addition to the diblock copolymer. Additional transduction enhancers include, for example, tacrolimus and vectorfusin. In some embodiments, the additional transduction enhancer is tacrolimus. In some embodiments, the additional transduction enhancer is Vectorfusin.

    Spinoculation

    [0859] In some embodiments of the disclosure, a cell targeted for transduction may 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 may occur with a centripetal force of, e.g., from about 200g to about 2,000g. The centripetal force may be, e.g., from about 300g to about 1,200g (e.g., about 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1,000g, 1,100g, or 1,200g, or more). In some embodiments, the cell is spun for from 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 cell is spun at room temperature, such as at a temperature of about 25 C.

    [0860] Exemplary transduction protocols involving a spinoculation step are described, e.g., 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.

    Target Cells

    [0861] Cells that may be used in conjunction with the compositions and methods described herein include cells that are capable of undergoing further differentiation. For example, one type of cell that can be used in conjunction with the compositions and methods described herein is a pluripotent cell. A pluripotent cell is a cell that possesses the ability to develop into more than one differentiated cell type. 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, endoderm, which forms the gastrointestinal and respiratory tracts, endocrine glands, liver, and pancreas, and mesoderm, which forms bone, cartilage, muscles, connective tissue, and most of the circulatory system.

    [0862] Cells that may be used in conjunction with the compositions and methods described herein include hematopoietic stem cells and hematopoietic progenitor cells. Hematopoietic stem cells (HSCs) are immature blood cells that have the capacity to self-renew and to differentiate into mature blood cells including diverse lineages including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), 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 also refer to long term repopulating HSC (LT-HSC) and short-term repopulating HSC (ST-HSC). Any of these HSCs can be used in conjunction with the compositions and methods described herein.

    [0863] HSCs and other pluripotent progenitors can be obtained from blood products. A blood product is a product obtained from the body or an organ of the body containing 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 having HSC or myeloid progenitor cell characteristics in a number of ways. For example, the more mature, differentiated cells can be selected against based on cell surface molecules they express. The blood product may be fractionated by positively selecting for CD34+ cells, which include a subpopulation of hematopoietic stem cells capable of self-renewal, multi-potency, and that can be re-introduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Such selection is accomplished using, for example, 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 a donor or retrieved from cryopreservative storage. HSCs and myeloid progenitor cells can also be obtained from by differentiation of ES cells, PS cells or other reprogrammed mature cells types.

    [0864] Cells that may be used in conjunction with the compositions and methods described herein include allogeneic cells and autologous cells. When allogeneic cells are used, the cells may optionally be HLA-matched to the subject receiving a cell treatment.

    [0865] Cells that may be used in conjunction with the compositions and methods described herein include CD34+/CD90+ cells and CD34+/CD164+ cells. These cells 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 disclosures of each of which are hereby incorporated by reference in their entirety.

    Viral Vectors for Transgene Expression

    [0866] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Pat. No. 5,801,030), the teachings of which are incorporated herein by reference.

    Retroviral Vectors

    [0867] The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that may 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, conferring stable, long-term expression of the transgene. An overview of optimization strategies 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.

    [0868] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, incapsidation, and expression, in which the sequences to be expressed are inserted.

    [0869] A LV used in the methods and compositions described herein may include one or more of a 5-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3-splice site (SA), elongation factor (EF) 1-alpha promoter and 3-self inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in U.S. Pat. No. 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE. The lentiviral vector may further include a pHR backbone, which may include for example as provided below.

    [0870] The Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) may be used to express the DNA molecules and/or transduce cells. A LV used in the methods and compositions described herein may a 5-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3-splice site (SA), elongation factor (EF) 1-alpha promoter and 3-self inactivating L TR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.

    [0871] Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.

    [0872] In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. The vector used in the methods and compositions described herein may include multiple promoters that permit expression more than one polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow 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 pertain to protein cleavage sites that allow expression of more than one polypeptide. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein.

    [0873] The vector used in the methods and compositions described herein may, be a clinical grade vector.

    Methods of Treatment

    Exemplary Diseases that May be Treated Using the Compositions and Methods of the Disclosure

    [0874] Transgenes that may be introduced into a target cell and ultimately delivered to a patient (e.g., by administration of the target cell to a patient) using the compositions and methods of the disclosure include those that encode therapeutic proteins. The recipient of the transgene (e.g., the recipient of a cell transduced to express the transgene) may be suffering from a disease characterized by deficiency in the encoded protein. For example, transgenes that can expressed in a target cell and delivered to a patient in accordance with the compositions and methods of the disclosure include transgenes encoding beta-globin, which are particularly useful for the treatment of patients having beta-thalassemia. Exemplary nucleic acid and amino acid sequences of human beta-globin cDNA and protein are shown below.

    Exemplary Wild-Type Human Beta-Globin cDNA Sequence:

    TABLE-US-00004 (SEQIDNO:1) ATGGTGCATCTGACCCCGGAAGAAAAAAGCGCGGTGACCGCGCTGTGGG GCAAAGTGAACGTGGATGAAGTGGGCGGCGAAGCGCTGGGCCGCCTGCT GGTGGTGTATCCGTGGACCCAGCGCTTTTTTGAAAGCTTTGGCGATCTG AGCACCCCGGATGCGGTGATGGGCAACCCGAAAGTGAAAGCGCATGGCA AAAAAGTGCTGGGCGCGTTTAGCGATGGCCTGGCGCATCTGGATAACCT GAAAGGCACCTTTGCGACCCTGAGCGAACTGCATTGCGATAAACTGCAT GTGGATCCGGAAAACTTTCGCCTGCTGGGCAACGTGCTGGTGTGCGTGC TGGCGCATCATTTTGGCAAAGAATTTACCCCGCCGGTGCAGGCGGCGTA TCAGAAAGTGGTGGCGGGCGTGGCGAACGCGCTGGCGCATAAATATCAT

    Exemplary Wild-Type Human Beta-Globin Amino Acid Sequence:

    TABLE-US-00005 (SEQIDNO:2) MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDL STPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLH VDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

    [0875] Additional examples of transgenes that may be used in conjunction with the compositions and methods of the disclosure include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), calcitonin, growth hormone releasing factor (GRF), thyroid stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), prolactin, melatonin, vasopressin, -endorphin, met-enkephalin, leu-enkephalin, prolactin-releasing factor, prolactin-inhibiting factor, corticotropin-releasing hormone, thyrotropin-releasing hormone (TRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG), vascular endothelial growth factor (VEGF), angiopoietins, 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 (TGF), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor (TGF) superfamily comprising TGF, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1 15, any one of the heregulin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurtuin, persephin, agrin, any one of the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

    [0876] Further examples of transgenes that may be used in conjunction with the compositions and methods of the disclosure include those that encode proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1, IL-1, 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-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), Fas ligand, tumor necrosis factors and (TNF and TNF), interferons (IFN) IFN-, IFN-, and IFN-, stem cell factor, flk-2/flt3 ligand. Transgenes encoding protein products produced by the immune system are also encompassed by the present disclosure. These include, without limitations, 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 engineered MHC molecules including single chain MHC molecules. Useful gene products also include complement regulatory proteins such as membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2 and CD59.

    [0877] Additional examples of suitable transgenes include those that encode any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. Examples of such receptors include flt-1, flk-1, TIE-2; the trk family of receptors such as TrkA, MuSK, Eph, PDGF receptor, EGF receptor, HER2, insulin receptor, IGF-1 receptor, the FGF family of receptors, the TGF receptors, the interleukin receptors, the interferon receptors, serotonin receptors, -adrenergic receptors, -adrenergic receptors, the GDNF receptor, p75 neurotrophin receptor, among others. Further examples are transgenes encoding extracellular matrix proteins, such as integrins, counter-receptors for transmembrane-bound proteins, such as intercellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3 and ICAM-4), vascular cell adhesion molecules (VCAM), and selectins E-selectin, P-selectin and L-selectin. The invention encompasses receptors for cholesterol regulation, including the LDL receptor, HDL receptor, VLDL receptor, and the scavenger receptor. Additional examples are transgenes encoding the apolipoprotein ligands for these receptors, including ApoAI, ApoAIV and ApoE. Additional transgenes include those encoding antimicrobial peptides such as defensins and maginins, transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, NF-B, members of the leucine zipper family, C.sub.2H.sub.4 zinc finger proteins, including Zif268, EGR1, EGR2, C.sub.6 zinc finger proteins, including the glucocorticoid and estrogen receptors, POU domain proteins, exemplified by Pit 1, homeodomain proteins, including HOX-1, basic helix-loop-helix proteins, including myc, 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 regulation factor 1 (IRF-1), Wilms' tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

    [0878] Other useful transgenes include those encoding 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, pro-urokinase, urokinase, plasminogen activator, heparin cofactor II, activated protein C (Factor V Leiden), Protein C, antithrombin, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also referred to as P-protein), H-protein, T-protein, Menkes disease protein, tumor suppressors (e.g., p53), cystic fibrosis transmembrane regulator (CFTR), the product of Wilson's disease gene PWD, Cu/Zn superoxide dismutase, aromatic amino acid decarboxylase, tyrosine hydroxylase, acetylcholine synthetase, prohormone convertases, protease inhibitors, lactase, lipase, trypsin, gastrointestinal enzymes including chymotrypsin, and pepsin, adenosine deaminase, a1 anti-trypsin, tissue inhibitor of metalloproteinases (TIMP), GLUT-1, GLUT-2, trehalose phosphate synthase, hexokinases I, II and III, glucokinase, any one or more of the individual chains or types of collagen, elastin, fibronectin, thrombospondin, vitronectin and tenascin, and suicide genes such as thymidine kinase and cytosine deaminase. Other useful proteins include those involved in lysosomal storage disorders, including acid -glucosidase, -galactosidase a, -1-iduronidase, iduroate sulfatase, lysosomal acid -glucosidase, sphingomyelinase, hexosaminidase A, hexomimidases A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, -fucosidase, -, -mannosidosis, aspartylglucosaminidase, neuramidase, galactosylceramidase, heparan-N-sulfatase, N-acetyl--glucosaminidase, Acetyl-CoA: -glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B, -glucuoronidase and hexosaminidases A and B.

    [0879] Other useful transgenes include those encoding non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides or polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins could be useful in certain immunocompromised patients. Other useful proteins include truncated receptors which lack their transmembrane and cytoplasmic domain. These truncated receptors can be used to antagonize the function of their respective ligands by binding to them without concomitant signaling by the receptor. Other types of non-naturally occurring gene sequences include sense and antisense molecules and catalytic nucleic acids, such as ribozymes, which could be used to modulate expression of a gene.

    [0880] Exemplary transgenes that can be expressed in a target cell, which may then be administered to a patient for the treatment of a disease characterized by a deficiency or dysfunction of the encoded product, include those encoding a protein product listed in Table 3 below.

    TABLE-US-00006 TABLE 3 Exemplary disorders associated with gene deficiency or dysfunction Disease associated with Exemplary amino acid Protein deficiency in protein sequence of protein acid -glucosidase (GAA) Pompe NP _000143.2, NP _001073271.1, NP_001073272.1 Methyl CpG binding protein 2 Rett syndrome NP _001104262.1, (MECP2) NP_004983.1 Aromatic L-amino acid Parkinson's disease NP _000781.1, decarboxylase (AADC) NP _001076440.1, NP _001229815.1, NP_001229816.1, NP _001229817.1, NP _001229818.1, NP_001229819.1 Glial cell-derived neurotrophic Parkinson's disease NP _000505.1, factor (GDNF) NP _001177397.1, NP _001177398.1, NP _001265027.1, NP_954701.1 Glutamate decarboxylase 1 Parkinson's disease NP_ 000808.2, NP_038473.2 (GAD1) Glutamate decarboxylase 2 Parkinson's disease NP _000809.1, (GAD2) NP_001127838.1 Neurturin (NRTN) Parkinson's disease NP_004549.1 neuropeptide Y (NPY) Parkinson's disease, epilepsy NP_000896.1 Cystic fibrosis transmembrane Cystic fibrosis NP_000483.3 conductance regulator (CFTR) Tumor necrosis factor receptor Arthritis, Rheumatoid arthritis SEQ ID NO. 1 of fused to an antibody Fe W02013025079 (TNFR:Fc) Sarcoglycan , , , , , or Muscular dystrophy SGCA (SGCA, SGCB, SGCG, SGCD, NP _000014.1, SGCE, or SGCZ) NP_001129169.1 SGCB NP_000223.1 SGCG NP_000222.1 SGCD NP_ 000328.2, NP _001121681.1, NP_758447.1 SGCE NP _001092870.1, NP _001092871.1, NP_003910.1 SGCZ NP_631906.2 -1-antitrypsin (AAT) Hereditary emphysema or -1- NP_ 000286.3, antitrypsin deficiency NP _001002235.1, NP _001002236.1, NP _001121172.1, NP _001121173.1, NP_ 001121174.1, NP_ 001121175.1, NP_001121176.1, NP _001121177.1, NP _001121178.1, NP_001121179.1 Aspartoacylase (ASPA) Canavan's disease NP _000040.1, NP_001121557.1 Nerve growth factor (NGF) Alzheimer's disease NP_002497.2 Granulocyte-macrophage Prostate cancer NP_000749.2 colonystimulating factory (GM- CSF) Cluster of Differentiation 86 Malignant melanoma NP _001193853.1, (CD86 or B7-2) NP _001193854.1, NP _008820.3, NP _787058.4, NP_795711.1 Interleukin 12 (IL-12) Malignant melanoma NP _000873.2, NP_002178.2 ATPase, Ca.sup.2+ transporting, Chronic heart failure NP _001672.1, cardiac muscle, slow twitch 2 NP_733765.1 (SERCA2) Dystrophin or Minidystrophin Muscular dystrophy NP _000100.2, NP _003997.1, NP _004000.1, NP _004001.1, NP_ 004002.2, NP _004003.1, NP _004004.1, NP _004005.1, NP _004006.1, NP _004007.1, NP _004008.1, NP _004009.1, NP _004010.1, NP_ 004011.2, NP _004012.1, NP _004013.1, NP_004014.1 Ceroid lipofuscinosis neuronal 2 Late infantile neuronal NP_000382.3 (CLN2) ceroidlipofuscinosis or Batten's disease N-acetylglucosaminidase, Sanfilippo syndrome (MPSIIIB) NP_000254.2 (NAGLU) Iduronidase, -1 (IDUA) MPSI-Hurler NP_000194.2 Iduronate 2-sulfatase (IDS) MPSII-Hunter NP _000193.1, NP _001160022.1, NP_006114.1 Glucuronidase, (GUSB) MPSVII-Sly NP_000172.2, NP 001271219.1 Hexosaminidase A, Tay-Sachs NP_000511.2 polypeptide (HEXA) Retinal pigment epithelium- Leber congenital amaurosis NP_000320.1 specific protein 65 kDa (RPE65) Factor IX (FIX) Hemophilia B NP_000124.1 Adenine nucleotide translocator progressive external NP_001142.2 (ANT-I) ophthalmoplegia ApaLI mitochondrial heteroplasmy, YP_007161330.1 myoclonic epilepsy with ragged red fibers (MERRF) or mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (ME LAS) NADH ubiquinone Leber hereditary optic YP_003024035.1 oxidoreductase subunit 4 (ND4) very long-acyl-CoA very long-chain acyl-CoA NP _000009.1, dehydrogenase (VLCAD) dehydrogenase (VLCAD) NP _001029031.1, deficiency NP _001257376.1, NP_001257377.1 short-chain acyl-CoA short-chain acyl-CoA NP_000008.1 dehydrogenase (SCAD) dehydrogenase (SCAD) deficiency medium-chain acyl-CoA medium-chain acyl-CoA NP _000007.1, dehydrogenase (MCAD) dehydrogenase (MCAD) NP _001120800.1, deficiency NP _001272971.1, NP _001272972.1, NP_001272973.1 Myotubularin 1 (MTM1) X-linked myotubular myopathy NP_000243.1 Myophosphorylase (PYGM) McArdle disease (glycogen NP_001158188.1, storage disease type V, NP_005600.1 myophosphorylase deficiency) Lipoprotein lipase (LPL) LPL deficiency NP_000228.1 sFLTOI (VEGF/PIGF (placental Age-related macular SEQ ID NO: 2, 8, 21, 23, or 25 growth factor) binding domain of degeneration of W02009105669 human VEGFRI/FIt-1 (hVEGFRI) fused to the Fe portion of human IgG(I) through a polyglycine linker) Glucocerebrosidase (GC) Gaucher disease NP _000148.2, NP _001005741.1, NP _001005742.1, NP _001165282.1, NP_001165283.1 Calsequestrin 2 (CASQ2) Catecholaminergic polymorphic NP_001223.2 ventricular tachycardia (CPVT) UDP glucuronosyltransferase 1 Crigler-Najjar syndrome NP_000454.1 family member A1 (UGT1A1) Glucose 6-phosphatase GSD-Ia NP _000142.2, (G6Pase) NP_001257326.1 Omithine carbamoyltransferase OTC deficiency NP_000522.3 (OTC) Cystathionine--synthase (CBS) Homocystinuria NP_ 000062.1, NP_ 001171479.1, NP_001171480.1 Factor VIII (F8) Haemophilia A NP _000123.1, NP_063916.1 Hemochromatosis (HFE) Hemochromatosis NP _000401.1, NP _620572.1, NP _620573.1, NP _620575.1, NP _620576.1, NP _620577.1, NP _620578.1, NP _620579.1, NP_620580.1 Low density lipoprotein receptor Phenylketonuria (PKU) NP _000518.1, (LDLR) NP _001182727.1, NP _001182728.1, NP_001182729.1, NP_001182732.1 Galactosidase, (AGA) Fabry disease NP_000160.1 Phenylalanine hydroxylase (P Hypercholesterolaemia or NP_000268.1 AH) Phenylketonuria (PKU) Propionyl CoA carboxylase, Propionic acidaemias NP _000273.2, alpha polypeptide (PCCA) NP_ 001121164.1, NP_001171475.1 Arylsulfatase Metachromatic leukodystrophy NP_000478, (MLD) NP_001078894, NP_001078895, NP_001078896, NP_001078897 Heparan N-sulfatase Sanfilippo type A (MPS-IIIA) NP_000190, NP_001339850, NP_001339851 Adenosine deaminase Adensoine deaminase severe NP_000013, combined immunodeficiency NP_001308979, (ADA-SCID) NP_001308980 Wiscott-Aldrich syndrome Wiskott-Aldrich syndrome NP_000368, protein (WAS) NP_000368.1, NP_033541 NADPH oxidase 2 X-linked chronic granulomatous NP_000388 disease (X-CGD) Progranlin Frontotemporal dementia (FTD) NP_002078 Superoxide dismutase 1 (SOD1) Amytrophic lateral sclerosis NP_000445 (ALS) Apolipoprotein E2 Alzheimer's disease NP_000032, NP_001289617, NP_001289618, NP_001289619, NP_001289620 Palmitoyl-protein thioesterase 1 CLN1 disease NP_000301, (PPT1) NP_001136076, NP_001350624 Tripeptidyl peptidase 1 (TPP1) CLN2 NP_000382 Bruton's tyrosin kinase (BTK) X-linked agammaglobulinemia NP_000052, (XLA) NP_001274273, NP_001274274 Glycine amidinotransferase Crohn's disease NP_001473.1, (GATM) NP_001307944.1

    Selection of Donor Cells

    [0881] In some embodiments, the subject undergoing treatment is the donor that provides cells (e.g., pluripotent cells, such as CD34+ HSCs or HPCs) which are subsequently modified to express one or more therapeutic proteins of the disclosure before being re-administered to the patient. In such cases, withdrawn cells (e.g., CD34+ HSCs or HPCs) may be re-infused into the subject following, for example, incorporation of a transgene encoding one or more therapeutic proteins of the disclosure, and/or disruption of an allelic variant harboring a deleterious mutation), such that the cells may subsequently home to hematopoietic tissue and establish productive hematopoiesis, thereby restoring expression of the transgene in the patient. In cases in which the subject undergoing treatment also serves as the cell donor, the transplanted cells (e.g., HSCs or HPCs) are less likely to undergo graft rejection. This stems from the fact that the infused cells are derived from the patient and express the same HLA class I and class II antigens as expressed by the patient. Alternatively, the subject and the donor may be distinct. In some embodiments, the subject and the donor are related, and may, for example, be HLA-matched. As described herein, HLA-matched donor-recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells within the transplant recipient are less likely to recognize the incoming hematopoietic stem or progenitor cell graft as foreign, and, are thus less likely to mount an immune response against the transplant. Exemplary HLA-matched donor-recipient pairs are donors and recipients that are genetically related, such as familial donor-recipient pairs (e.g., sibling donor-recipient pairs). In some embodiments, the subject and the donor are HLA-mismatched, which occurs when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient. To reduce the likelihood of graft rejection, for example, one haplotype may be matched between the donor and recipient, and the other may be mismatched.

    Pharmaceutical Compositions and Dosing

    [0882] In cases in which a subject is administered a population of cells that together express one or more therapeutic proteins of the disclosure, the number of cells administered may depend, for example, on the expression level of the desired protein(s), the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the disease being treated, and whether or not the patient has been treated with agents to ablate endogenous pluripotent cells (e.g., endogenous CD34+ cells, hematopoietic stem or progenitor cells, or microglia, among others). The number of cells administered may be, for example, from about 110.sup.4 cells/kg to about 110.sup.14 cells/kg, or more. Cells may be administered in an undifferentiated state, or after partial or complete differentiation into a target cell type. The number of pluripotent cells may be administered in any suitable dosage form.

    EXAMPLES

    [0883] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

    Example 1. Synthesis of Diblock Copolymer

    [0884] Poly(ethylene oxide-b-propylene oxide) diblock copolymer was prepared by living anionic polymerization. The scheme of reaction is shown below:

    ##STR00178##

    An aliquot of the anionic block was terminated and analyzed by size exclusion chromatography (SEC) to obtain the molecular weight of the first block. The molecular weight of the second block was calculated from proton NMR spectroscopy by comparing the peak area of the ethylene oxide protons at 3.6 ppm with the propylene oxide protons at 1.08 ppm. The polydispersity of the final diblock copolymer was obtained by SEC. Poly(ethylene oxide-b-propylene oxide) is soluble in chloroform, THF, methanol and ethanol. The polymer precipitates from hexane and ether.

    Example 2. Diblock Copolymer Transduction

    [0885] Peripheral mobilized blood CD34.sup.+ stem cells were transduced with lentiviral vector, (Vector only, multiplicity of infection (MOI) 10-20), in the presence of diblock polymers compounds (DBP1-6). The effect of DBP compounds on cell viability (determined 1 day post-transduction; FIG. 1) and transduction efficiency (expressed as fold change in percentage of transduced cells induced by the addition of DBP, relative to cells treated with vector alone; FIGS. 2A-2F) was determined at 12-14 days post transduction.

    [0886] The effect of DBP on integrated vector copy number per cell (VCN) was determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA harvested from cell cultures 12 days post-transduction (FIG. 3). Percentage of transduced cells was assessed by flow cytometry detection of transgene expression (FIG. 4). The effect of DBP compounds in combination with various transduction enhancer elements on cell viability (FIG. 5) and transduction efficiency (FIG. 6) was determined.

    [0887] Notably, diblock copolymers were found to effectuate increases in transduction efficiency of CD34.sup.+ stem cells across a range of PEO and PPO compositions (FIGS. 7 and 8). Diblock copolymers were also found to be compatible with RetroNectin (FIG. 9). Surprisingly, diblock copolymers were also found to engender improvements in transduction efficiency relative to triblock copolymers, particularly poloxamer 338 (FIG. 10).

    Other Embodiments

    [0888] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.

    [0889] Other embodiments are in the claims.