COMPOSITIONS AND METHODS FOR MODIFYING EUKARYOTIC CELLS

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 poloxamer. 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 poloxamer. Examples of poloxamers that may be used in conjunction with the compositions and methods of the disclosure are those having a molar mass in excess of 10,000 g/mol, as well as those having a molar mass of polyoxypropylene subunits greater than 2,000 g/mol and/or an ethylene oxide content of greater than 40% by mass.

Claims

1. A method of transducing a eukaryotic cell to express a transgene, the method comprising contacting the cell with (i) a viral vector encoding the transgene and (ii) a poloxamer, wherein the concentration of the poloxamer, when contacted with the cell, is less than 10 μg/ml.

2-3. (canceled)

4. The method of claim 1, wherein the cell is: (a) a mammalian cell; (b) a pluripotent cell; (c) a CD34+ cell; (d) an embryonic stem cell; (e) an induced pluripotent stem cell; or (f) a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC).

5. The method of claim 4, wherein the mammalian cell is a human cell.

6-10. (canceled)

11. The method of claim 1, further comprising contacting the cell with a substance that reduces activity and/or expression of protein kinase C (PKC).

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

13-15. (canceled)

16. The method of claim 12, wherein the agent comprises a nucleic acid, such as an interfering RNA or an antisense oligonucleotide.

17-22. (canceled)

23. The method of claim 12, wherein the PKC inhibitor is a compound represented by formula (I) ##STR00138## 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; 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; 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; R.sub.c is O, NR.sub.d, or S; 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; 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; 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; represents a bond that is optionally present; n is an integer from 0-4; and m is an integer from 0-4; or a salt thereof.

24-58. (canceled)

59. The method of claim 1, wherein the concentration of the poloxamer, when contacted with the cell, is from about 10 ng/ml to about 9 μg/ml.

60-64. (canceled)

65. The method of claim 1, wherein the poloxamer: (a) has an average molar mass of polyoxypropylene subunits of greater than 2,050 g/mol, 2,250 g/mol, 2,750 g/mol, 3,250 g/mol, or 3,625 g/mol; (b) has an average molar mass of polyoxypropylene subunits of from about 2,050 g/mol to about 4,000 g/mol; (c) has an average ethylene oxide content of greater than 40%, 50%, 60%, or 70% by mass; (d) has an average molar mass of greater than 10,000 g/mol, 11,000 g/mol, 12,000 g/mol, or 12,500 g/mol; (e) has an average molar mass of from about 10,000 g/mol to about 15,000 g/mol; or (f) is P407, P338, P288, or P188.

66-88. (canceled)

89. The method of claim 1, wherein the viral vector: (a) 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; or (b) is a pseudotyped viral vector.

90. (canceled)

91. The method of claim 89, wherein: (a) the Retroviridae family viral vector is a lentiviral vector; or (b) 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.

92-96. (canceled)

97. The method of claim 1, wherein: (a) the contacting occurs ex vivo; (b) the cell is further contacted with a cyclosporine, such as cyclosporine A or cyclosporine H; (c) the cell is further contacted with an activator of prostaglandin E receptor signaling, such as prostaglandin E2; (d) the cell is further contacted with a polycationic Polymer, such as polybrene, protamine sulfate, polyethylenimine, or a Polyethylene glycol/poly-L-lysine block copolymer; or (e) the cell is spun by centrifugation while being contacted with the viral vector.

98-107. (canceled)

108. A method of expressing a transgene in a subject, delivering a population of genetically modified cells to a subject, or 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.

109-110. (canceled)

111. The method of claim 108, wherein the cells are allogeneic, HLA-matched, or autologous with respect to the subject.

112-113. (canceled)

114. The method of claim 108, wherein: (a) 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; (b) 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; (c) 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; (d) the method further 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; and/or (e) 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, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes.

115. The method of claim 114, wherein the precursor cells are CD34+ HSCs, and wherein the precursor cells are expanded without loss of HSC functional potential, or wherein 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.

116-122. (canceled)

123. The method of claim 108, wherein the subject has been diagnosed as having a deficiency of an endogenous protein encoded by the transgene.

124. The method of claim 123, wherein the subject has been diagnosed as having a disease set forth in Table 2.

125. (canceled)

126. The method of claim 1, wherein the transgene encodes a beta-globin protein.

127-136. (canceled)

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

138-146. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0396] FIG. 1A is a graph showing the effect of poloxamer P407, when incubated with CD34+ hematopoietic stem cells at various concentrations, on lentiviral transduction efficiency, as described in Example One, below. Briefly, CD34+ cells were transduced with a lentiviral vector containing a transgene encoding green fluorescent protein (GFP). Cells were either untreated (“Untr”) or incubated with: the lentivirus alone (“Vector only”), the lentivirus in combination with vehicle (“Vector+Vehicle”), or the lentivirus in combination with particular concentrations of P407, as indicated along the x axis. Values along the y axis represent fold increase in the quantity of transduced cells 12 days after exposure to each of the specified conditions relative to the quantity of transduced cells 12 days after treatment with lentiviral vector alone (“Vector only”). Quantities of transduced cells were determined by measuring expression of the encoded transgene, which was evaluated using flow cytometry to detect GFP-mediated fluorescence. Values along the y axis denote the mean±standard error of the mean (SEM) for n=4 independent experiments.

[0397] FIG. 1B is a graph showing the effect of poloxamer P407, when incubated with CD34+ hematopoietic stem cells at various concentrations, on lentiviral vector copy number, as described in Example One, below. CD34+ cells were transduced with a lentiviral vector containing a transgene encoding GFP. Cells were either untreated (“Untr”) or incubated with the lentivirus alone (“Vector only”), the lentivirus in combination with vehicle (“Vector+Vehicle”), or the lentivirus in combination with particular concentrations of P407, as indicated along the x axis. Values along the y axis represent fold increase in vector copy number of cells 12 days after exposure to each of the specified conditions relative to vector copy number of cells 12 days after exposure to lentiviral vector alone (“Vector only”). Values along the y axis denote the mean±standard error of the mean (SEM) for n=3 independent experiments.

[0398] FIGS. 2A-2C are graphs showing the effect of poloxamers P407 and P338 on lentiviral transduction of human hematopoietic stem cells, as described in Example Two, below. CD34+ hematopoietic stem cells from peripheral mobilized blood were either untreated (“Untr”) or incubated with a lentiviral vector containing a transgene encoding GFP. Among those cells incubated with a lentiviral vector, cells were either transduced with the vector alone (“Vector only,” multiplicity of infection 5-20) or in the presence of various concentrations of poloxamer P407 (final concentration 1 μg/mL-10 mg/mL) or poloxamer P338 (final concentration 10 μg/mL-4 mg/mL). FIG. 2A is a plot summarizing the percentage of viable cells 18-24 hours post-transduction, as determined by flow cytometry detection of 7AAD-AnnexinV-cells. FIG. 2B is a plot summarizing the fold change in percentage of transduced cells induced by the addition of P407 or P338, relative to cells treated with vector alone. The percentage of transduced cells was assessed by flow cytometry detection of transgene expression 12 days post-transduction. FIG. 2C is a plot summarizing the fold change in mean transgene copy number (VCN) relative to cells treated with vector alone. VCN was determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA harvested from cell cultures 12 days post-transduction. The data shown are mean±SEM for n=3-9 (FIGS. 2A and 2B) and n=3-6 (FIG. 2C) independent experiments with healthy human donor cells. Statistical analysis was conducted using a paired T-test of test conditions relative to vector and poloxamer vehicle treated cells, where “ns” indicates no statistical significance was (p<0.05).

[0399] FIGS. 3A and 3B are graphs showing the effect of poloxamer P407 or poloxamer P338 in combination with protamine sulfate on lentiviral transduction of human hematopoietic stem cells, as described in Example Three, below. CD34+ hematopoietic stem cells from peripheral mobilized blood were transduced with a lentiviral vector encoding GFP in the presence of poloxamer P407 (7 μg/mL) or poloxamer P338 (1 mg/mL), in combination with protamine sulfate (“PS,” 3.5 μg/mL). The plots summarize the fold change in the percentage of transduced cells as assessed by flow cytometry (FIG. 3A) and mean transgene copy number (VCN, FIG. 3B) induced by the addition of P407 or P338, in combination with protamine sulfate, relative to cells treated with vector alone. At 12 days post-transduction, percentage of transduced cells was assessed by flow cytometry detection of transgene expression, and VCN was determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA. The data show mean±range of n=5-8 independent experiments with healthy human donor cells. Statistical analysis was conducted using a T-test (p<0.05).

[0400] FIGS. 4A and 4B are graphs showing the effect of poloxamer P407 and protamine sulfate, in combination with either cyclosporine H or lithium carbonate, on lentiviral transduction of human hematopoietic stem cells, as described in Example Three, below. CD34+ stem cells freshly isolated from peripheral mobilized blood were transduced with a clinical therapeutic lentiviral vector in the presence of poloxamer P407 (7 μg/mL) in combination with protamine sulfate (“PS,” 3.5 μg/mL) and either cyclosporine H (“CSH,” 8 μM, dark grey) or lithium carbonate (Li.sub.2CO.sub.3, represented in the figures as “LiCO,” 2 mM, light grey). Plots summarize the percentage of transduced colonies determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA isolated from Colony Forming Units (CFU, FIG. 4A), and mean transgene copy number (VCN) per cell, detected 12 days post transduction, achieved when using a range of vector doses (moi, multiplicity of infection, FIG. 4B). White bars show transduction efficiency and VCN achieved by addition of vector alone. Data show mean±range of n=4 independent experiments with clinical scale isolation of healthy human donor CD34+ cells.

DETAILED DESCRIPTION

[0401] 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.

[0402] 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.

[0403] To augment the extent of transduction and/or the rate at which the target cell is transduced, the cell may be contacted with a poloxamer, such as a poloxamer having a molar mass in excess of 10,000 g/mol, a molar mass of polyoxypropylene subunits greater than 2,000 g/mol, and/or an ethylene oxide content of greater than 40% by mass.

[0404] 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.

Poloxamers

[0405] Poloxamers that may be used in conjunction with the compositions and methods of the disclosure include those having an average molar mass of polyoxypropylene subunits of greater than 2,050 g/mol (e.g., an average molar mass of polyoxypropylene subunits of about 2,055 g/mol, 2,060 g/mol, 2,075 g/mol, 2,080 g/mol, 2,085 g/mol, 2,090 g/mol, 2,095 g/mol, 2,100 g/mol, 2,200 g/mol, 2,300 g/mol, 2,400 g/mol, 2,500 g/mol, 2,600 g/mol, 2,700 g/mol, 2,800 g/mol, 2,900 g/mol, 3,000 g/mol, 3,100 g/mol, 3,200 g/mol, 3,300 g/mol, 3,400 g/mol, 3,500 g/mol, 3,600 g/mol, 3,700 g/mol, 3,800 g/mol, 3,900 g/mol, 4,000 g/mol, 4,100 g/mol, 4,200 g/mol, 4,300 g/mol, 4,400 g/mol, 4,500 g/mol, 4,600 g/mol, 4,700 g/mol, 4,800 g/mol, 4,900 g/mol, or 5,000 g/mol).

[0406] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of greater than 2,250 g/mol (e.g., an average molar mass of polyoxypropylene subunits of about 2,300 g/mol, 2,400 g/mol, 2,500 g/mol, 2,600 g/mol, 2,700 g/mol, 2,800 g/mol, 2,900 g/mol, 3,000 g/mol, 3,100 g/mol, 3,200 g/mol, 3,300 g/mol, 3,400 g/mol, 3,500 g/mol, 3,600 g/mol, 3,700 g/mol, 3,800 g/mol, 3,900 g/mol, 4,000 g/mol, 4,100 g/mol, 4,200 g/mol, 4,300 g/mol, 4,400 g/mol, 4,500 g/mol, 4,600 g/mol, 4,700 g/mol, 4,800 g/mol, 4,900 g/mol, or 5,000 g/mol).

[0407] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of greater than 2,750 g/mol (e.g., an average molar mass of polyoxypropylene subunits of about 2,800 g/mol, 2,900 g/mol, 3,000 g/mol, 3,100 g/mol, 3,200 g/mol, 3,300 g/mol, 3,400 g/mol, 3,500 g/mol, 3,600 g/mol, 3,700 g/mol, 3,800 g/mol, 3,900 g/mol, 4,000 g/mol, 4,100 g/mol, 4,200 g/mol, 4,300 g/mol, 4,400 g/mol, 4,500 g/mol, 4,600 g/mol, 4,700 g/mol, 4,800 g/mol, 4,900 g/mol, or 5,000 g/mol).

[0408] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of greater than 3,250 g/mol (e.g., an average molar mass of polyoxypropylene subunits of about 3,300 g/mol, 3,400 g/mol, 3,500 g/mol, 3,600 g/mol, 3,700 g/mol, 3,800 g/mol, 3,900 g/mol, 4,000 g/mol, 4,100 g/mol, 4,200 g/mol, 4,300 g/mol, 4,400 g/mol, 4,500 g/mol, 4,600 g/mol, 4,700 g/mol, 4,800 g/mol, 4,900 g/mol, or 5,000 g/mol).

[0409] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of greater than 3,625 g/mol (e.g., an average molar mass of polyoxypropylene subunits of about 3,700 g/mol, 3,800 g/mol, 3,900 g/mol, 4,000 g/mol, 4,100 g/mol, 4,200 g/mol, 4,300 g/mol, 4,400 g/mol, 4,500 g/mol, 4,600 g/mol, 4,700 g/mol, 4,800 g/mol, 4,900 g/mol, or 5,000 g/mol).

[0410] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of from about 2,050 g/mol to about 4,000 g/mol (e.g., about 2,050 g/mol, 2,055 g/mol, 2,060 g/mol, 2,065 g/mol, 2,070 g/mol, 2,075 g/mol, 2,080 g/mol, 2,085 g/mol, 2,090 g/mol, 2,095 g/mol, 2,100 g/mol, 2,105 g/mol, 2,110 g/mol, 2,115 g/mol, 2,120 g/mol, 2,125 g/mol, 2,130 g/mol, 2,135 g/mol, 2,140 g/mol, 2,145 g/mol, 2,150 g/mol, 2,155 g/mol, 2,160 g/mol, 2,165 g/mol, 2,170 g/mol, 2,175 g/mol, 2,180 g/mol, 2,185 g/mol, 2,190 g/mol, 2,195 g/mol, 2,200 g/mol, 2,205 g/mol, 2,210 g/mol, 2,215 g/mol, 2,220 g/mol, 2,225 g/mol, 2,230 g/mol, 2,235 g/mol, 2,240 g/mol, 2,245 g/mol, 2,250 g/mol, 2,255 g/mol, 2,260 g/mol, 2,265 g/mol, 2,270 g/mol, 2,275 g/mol, 2,280 g/mol, 2,285 g/mol, 2,290 g/mol, 2,295 g/mol, 2,300 g/mol, 2,305 g/mol, 2,310 g/mol, 2,315 g/mol, 2,320 g/mol, 2,325 g/mol, 2,330 g/mol, 2,335 g/mol, 2,340 g/mol, 2,345 g/mol, 2,350 g/mol, 2,355 g/mol, 2,360 g/mol, 2,365 g/mol, 2,370 g/mol, 2,375 g/mol, 2,380 g/mol, 2,385 g/mol, 2,390 g/mol, 2,395 g/mol, 2,400 g/mol, 2,405 g/mol, 2,410 g/mol, 2,415 g/mol, 2,420 g/mol, 2,425 g/mol, 2,430 g/mol, 2,435 g/mol, 2,440 g/mol, 2,445 g/mol, 2,450 g/mol, 2,455 g/mol, 2,460 g/mol, 2,465 g/mol, 2,470 g/mol, 2,475 g/mol, 2,480 g/mol, 2,485 g/mol, 2,490 g/mol, 2,495 g/mol, 2,500 g/mol, 2,505 g/mol, 2,510 g/mol, 2,515 g/mol, 2,520 g/mol, 2,525 g/mol, 2,530 g/mol, 2,535 g/mol, 2,540 g/mol, 2,545 g/mol, 2,550 g/mol, 2,555 g/mol, 2,560 g/mol, 2,565 g/mol, 2,570 g/mol, 2,575 g/mol, 2,580 g/mol, 2,585 g/mol, 2,590 g/mol, 2,595 g/mol, 2,600 g/mol, 2,605 g/mol, 2,610 g/mol, 2,615 g/mol, 2,620 g/mol, 2,625 g/mol, 2,630 g/mol, 2,635 g/mol, 2,640 g/mol, 2,645 g/mol, 2,650 g/mol, 2,655 g/mol, 2,660 g/mol, 2,665 g/mol, 2,670 g/mol, 2,675 g/mol, 2,680 g/mol, 2,685 g/mol, 2,690 g/mol, 2,695 g/mol, 2,700 g/mol, 2,705 g/mol, 2,710 g/mol, 2,715 g/mol, 2,720 g/mol, 2,725 g/mol, 2,730 g/mol, 2,735 g/mol, 2,740 g/mol, 2,745 g/mol, 2,750 g/mol, 2,755 g/mol, 2,760 g/mol, 2,765 g/mol, 2,770 g/mol, 2,775 g/mol, 2,780 g/mol, 2,785 g/mol, 2,790 g/mol, 2,795 g/mol, 2,800 g/mol, 2,805 g/mol, 2,810 g/mol, 2,815 g/mol, 2,820 g/mol, 2,825 g/mol, 2,830 g/mol, 2,835 g/mol, 2,840 g/mol, 2,845 g/mol, 2,850 g/mol, 2,855 g/mol, 2,860 g/mol, 2,865 g/mol, 2,870 g/mol, 2,875 g/mol, 2,880 g/mol, 2,885 g/mol, 2,890 g/mol, 2,895 g/mol, 2,900 g/mol, 2,905 g/mol, 2,910 g/mol, 2,915 g/mol, 2,920 g/mol, 2,925 g/mol, 2,930 g/mol, 2,935 g/mol, 2,940 g/mol, 2,945 g/mol, 2,950 g/mol, 2,955 g/mol, 2,960 g/mol, 2,965 g/mol, 2,970 g/mol, 2,975 g/mol, 2,980 g/mol, 2,985 g/mol, 2,990 g/mol, 2,995 g/mol, 3,000 g/mol, 3,005 g/mol, 3,010 g/mol, 3,015 g/mol, 3,020 g/mol, 3,025 g/mol, 3,030 g/mol, 3,035 g/mol, 3,040 g/mol, 3,045 g/mol, 3,050 g/mol, 3,055 g/mol, 3,060 g/mol, 3,065 g/mol, 3,070 g/mol, 3,075 g/mol, 3,080 g/mol, 3,085 g/mol, 3,090 g/mol, 3,095 g/mol, 3,100 g/mol, 3,105 g/mol, 3,110 g/mol, 3,115 g/mol, 3,120 g/mol, 3,125 g/mol, 3,130 g/mol, 3,135 g/mol, 3,140 g/mol, 3,145 g/mol, 3,150 g/mol, 3,155 g/mol, 3,160 g/mol, 3,165 g/mol, 3,170 g/mol, 3,175 g/mol, 3,180 g/mol, 3,185 g/mol, 3,190 g/mol, 3,195 g/mol, 3,200 g/mol, 3,205 g/mol, 3,210 g/mol, 3,215 g/mol, 3,220 g/mol, 3,225 g/mol, 3,230 g/mol, 3,235 g/mol, 3,240 g/mol, 3,245 g/mol, 3,250 g/mol, 3,255 g/mol, 3,260 g/mol, 3,265 g/mol, 3,270 g/mol, 3,275 g/mol, 3,280 g/mol, 3,285 g/mol, 3,290 g/mol, 3,295 g/mol, 3,300 g/mol, 3,305 g/mol, 3,310 g/mol, 3,315 g/mol, 3,320 g/mol, 3,325 g/mol, 3,330 g/mol, 3,335 g/mol, 3,340 g/mol, 3,345 g/mol, 3,350 g/mol, 3,355 g/mol, 3,360 g/mol, 3,365 g/mol, 3,370 g/mol, 3,375 g/mol, 3,380 g/mol, 3,385 g/mol, 3,390 g/mol, 3,395 g/mol, 3,400 g/mol, 3,405 g/mol, 3,410 g/mol, 3,415 g/mol, 3,420 g/mol, 3,425 g/mol, 3,430 g/mol, 3,435 g/mol, 3,440 g/mol, 3,445 g/mol, 3,450 g/mol, 3,455 g/mol, 3,460 g/mol, 3,465 g/mol, 3,470 g/mol, 3,475 g/mol, 3,480 g/mol, 3,485 g/mol, 3,490 g/mol, 3,495 g/mol, 3,500 g/mol, 3,505 g/mol, 3,510 g/mol, 3,515 g/mol, 3,520 g/mol, 3,525 g/mol, 3,530 g/mol, 3,535 g/mol, 3,540 g/mol, 3,545 g/mol, 3,550 g/mol, 3,555 g/mol, 3,560 g/mol, 3,565 g/mol, 3,570 g/mol, 3,575 g/mol, 3,580 g/mol, 3,585 g/mol, 3,590 g/mol, 3,595 g/mol, 3,600 g/mol, 3,605 g/mol, 3,610 g/mol, 3,615 g/mol, 3,620 g/mol, 3,625 g/mol, 3,630 g/mol, 3,635 g/mol, 3,640 g/mol, 3,645 g/mol, 3,650 g/mol, 3,655 g/mol, 3,660 g/mol, 3,665 g/mol, 3,670 g/mol, 3,675 g/mol, 3,680 g/mol, 3,685 g/mol, 3,690 g/mol, 3,695 g/mol, 3,700 g/mol, 3,705 g/mol, 3,710 g/mol, 3,715 g/mol, 3,720 g/mol, 3,725 g/mol, 3,730 g/mol, 3,735 g/mol, 3,740 g/mol, 3,745 g/mol, 3,750 g/mol, 3,755 g/mol, 3,760 g/mol, 3,765 g/mol, 3,770 g/mol, 3,775 g/mol, 3,780 g/mol, 3,785 g/mol, 3,790 g/mol, 3,795 g/mol, 3,800 g/mol, 3,805 g/mol, 3,810 g/mol, 3,815 g/mol, 3,820 g/mol, 3,825 g/mol, 3,830 g/mol, 3,835 g/mol, 3,840 g/mol, 3,845 g/mol, 3,850 g/mol, 3,855 g/mol, 3,860 g/mol, 3,865 g/mol, 3,870 g/mol, 3,875 g/mol, 3,880 g/mol, 3,885 g/mol, 3,890 g/mol, 3,895 g/mol, 3,900 g/mol, 3,905 g/mol, 3,910 g/mol, 3,915 g/mol, 3,920 g/mol, 3,925 g/mol, 3,930 g/mol, 3,935 g/mol, 3,940 g/mol, 3,945 g/mol, 3,950 g/mol, 3,955 g/mol, 3,960 g/mol, 3,965 g/mol, 3,970 g/mol, 3,975 g/mol, 3,980 g/mol, 3,985 g/mol, 3,990 g/mol, 3,995 g/mol, or 4,000 g/mol).

[0411] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of from about 2,750 g/mol to about 4,000 g/mol (e.g., about 2,750 g/mol, 2,755 g/mol, 2,760 g/mol, 2,765 g/mol, 2,770 g/mol, 2,775 g/mol, 2,780 g/mol, 2,785 g/mol, 2,790 g/mol, 2,795 g/mol, 2,800 g/mol, 2,805 g/mol, 2,810 g/mol, 2,815 g/mol, 2,820 g/mol, 2,825 g/mol, 2,830 g/mol, 2,835 g/mol, 2,840 g/mol, 2,845 g/mol, 2,850 g/mol, 2,855 g/mol, 2,860 g/mol, 2,865 g/mol, 2,870 g/mol, 2,875 g/mol, 2,880 g/mol, 2,885 g/mol, 2,890 g/mol, 2,895 g/mol, 2,900 g/mol, 2,905 g/mol, 2,910 g/mol, 2,915 g/mol, 2,920 g/mol, 2,925 g/mol, 2,930 g/mol, 2,935 g/mol, 2,940 g/mol, 2,945 g/mol, 2,950 g/mol, 2,955 g/mol, 2,960 g/mol, 2,965 g/mol, 2,970 g/mol, 2,975 g/mol, 2,980 g/mol, 2,985 g/mol, 2,990 g/mol, 2,995 g/mol, 3,000 g/mol, 3,005 g/mol, 3,010 g/mol, 3,015 g/mol, 3,020 g/mol, 3,025 g/mol, 3,030 g/mol, 3,035 g/mol, 3,040 g/mol, 3,045 g/mol, 3,050 g/mol, 3,055 g/mol, 3,060 g/mol, 3,065 g/mol, 3,070 g/mol, 3,075 g/mol, 3,080 g/mol, 3,085 g/mol, 3,090 g/mol, 3,095 g/mol, 3,100 g/mol, 3,105 g/mol, 3,110 g/mol, 3,115 g/mol, 3,120 g/mol, 3,125 g/mol, 3,130 g/mol, 3,135 g/mol, 3,140 g/mol, 3,145 g/mol, 3,150 g/mol, 3,155 g/mol, 3,160 g/mol, 3,165 g/mol, 3,170 g/mol, 3,175 g/mol, 3,180 g/mol, 3,185 g/mol, 3,190 g/mol, 3,195 g/mol, 3,200 g/mol, 3,205 g/mol, 3,210 g/mol, 3,215 g/mol, 3,220 g/mol, 3,225 g/mol, 3,230 g/mol, 3,235 g/mol, 3,240 g/mol, 3,245 g/mol, 3,250 g/mol, 3,255 g/mol, 3,260 g/mol, 3,265 g/mol, 3,270 g/mol, 3,275 g/mol, 3,280 g/mol, 3,285 g/mol, 3,290 g/mol, 3,295 g/mol, 3,300 g/mol, 3,305 g/mol, 3,310 g/mol, 3,315 g/mol, 3,320 g/mol, 3,325 g/mol, 3,330 g/mol, 3,335 g/mol, 3,340 g/mol, 3,345 g/mol, 3,350 g/mol, 3,355 g/mol, 3,360 g/mol, 3,365 g/mol, 3,370 g/mol, 3,375 g/mol, 3,380 g/mol, 3,385 g/mol, 3,390 g/mol, 3,395 g/mol, 3,400 g/mol, 3,405 g/mol, 3,410 g/mol, 3,415 g/mol, 3,420 g/mol, 3,425 g/mol, 3,430 g/mol, 3,435 g/mol, 3,440 g/mol, 3,445 g/mol, 3,450 g/mol, 3,455 g/mol, 3,460 g/mol, 3,465 g/mol, 3,470 g/mol, 3,475 g/mol, 3,480 g/mol, 3,485 g/mol, 3,490 g/mol, 3,495 g/mol, 3,500 g/mol, 3,505 g/mol, 3,510 g/mol, 3,515 g/mol, 3,520 g/mol, 3,525 g/mol, 3,530 g/mol, 3,535 g/mol, 3,540 g/mol, 3,545 g/mol, 3,550 g/mol, 3,555 g/mol, 3,560 g/mol, 3,565 g/mol, 3,570 g/mol, 3,575 g/mol, 3,580 g/mol, 3,585 g/mol, 3,590 g/mol, 3,595 g/mol, 3,600 g/mol, 3,605 g/mol, 3,610 g/mol, 3,615 g/mol, 3,620 g/mol, 3,625 g/mol, 3,630 g/mol, 3,635 g/mol, 3,640 g/mol, 3,645 g/mol, 3,650 g/mol, 3,655 g/mol, 3,660 g/mol, 3,665 g/mol, 3,670 g/mol, 3,675 g/mol, 3,680 g/mol, 3,685 g/mol, 3,690 g/mol, 3,695 g/mol, 3,700 g/mol, 3,705 g/mol, 3,710 g/mol, 3,715 g/mol, 3,720 g/mol, 3,725 g/mol, 3,730 g/mol, 3,735 g/mol, 3,740 g/mol, 3,745 g/mol, 3,750 g/mol, 3,755 g/mol, 3,760 g/mol, 3,765 g/mol, 3,770 g/mol, 3,775 g/mol, 3,780 g/mol, 3,785 g/mol, 3,790 g/mol, 3,795 g/mol, 3,800 g/mol, 3,805 g/mol, 3,810 g/mol, 3,815 g/mol, 3,820 g/mol, 3,825 g/mol, 3,830 g/mol, 3,835 g/mol, 3,840 g/mol, 3,845 g/mol, 3,850 g/mol, 3,855 g/mol, 3,860 g/mol, 3,865 g/mol, 3,870 g/mol, 3,875 g/mol, 3,880 g/mol, 3,885 g/mol, 3,890 g/mol, 3,895 g/mol, 3,900 g/mol, 3,905 g/mol, 3,910 g/mol, 3,915 g/mol, 3,920 g/mol, 3,925 g/mol, 3,930 g/mol, 3,935 g/mol, 3,940 g/mol, 3,945 g/mol, 3,950 g/mol, 3,955 g/mol, 3,960 g/mol, 3,965 g/mol, 3,970 g/mol, 3,975 g/mol, 3,980 g/mol, 3,985 g/mol, 3,990 g/mol, 3,995 g/mol, or 4,000 g/mol).

[0412] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of from about 3,250 g/mol to about 4,000 g/mol (e.g., about 3,250 g/mol, 3,255 g/mol, 3,260 g/mol, 3,265 g/mol, 3,270 g/mol, 3,275 g/mol, 3,280 g/mol, 3,285 g/mol, 3,290 g/mol, 3,295 g/mol, 3,300 g/mol, 3,305 g/mol, 3,310 g/mol, 3,315 g/mol, 3,320 g/mol, 3,325 g/mol, 3,330 g/mol, 3,335 g/mol, 3,340 g/mol, 3,345 g/mol, 3,350 g/mol, 3,355 g/mol, 3,360 g/mol, 3,365 g/mol, 3,370 g/mol, 3,375 g/mol, 3,380 g/mol, 3,385 g/mol, 3,390 g/mol, 3,395 g/mol, 3,400 g/mol, 3,405 g/mol, 3,410 g/mol, 3,415 g/mol, 3,420 g/mol, 3,425 g/mol, 3,430 g/mol, 3,435 g/mol, 3,440 g/mol, 3,445 g/mol, 3,450 g/mol, 3,455 g/mol, 3,460 g/mol, 3,465 g/mol, 3,470 g/mol, 3,475 g/mol, 3,480 g/mol, 3,485 g/mol, 3,490 g/mol, 3,495 g/mol, 3,500 g/mol, 3,505 g/mol, 3,510 g/mol, 3,515 g/mol, 3,520 g/mol, 3,525 g/mol, 3,530 g/mol, 3,535 g/mol, 3,540 g/mol, 3,545 g/mol, 3,550 g/mol, 3,555 g/mol, 3,560 g/mol, 3,565 g/mol, 3,570 g/mol, 3,575 g/mol, 3,580 g/mol, 3,585 g/mol, 3,590 g/mol, 3,595 g/mol, 3,600 g/mol, 3,605 g/mol, 3,610 g/mol, 3,615 g/mol, 3,620 g/mol, 3,625 g/mol, 3,630 g/mol, 3,635 g/mol, 3,640 g/mol, 3,645 g/mol, 3,650 g/mol, 3,655 g/mol, 3,660 g/mol, 3,665 g/mol, 3,670 g/mol, 3,675 g/mol, 3,680 g/mol, 3,685 g/mol, 3,690 g/mol, 3,695 g/mol, 3,700 g/mol, 3,705 g/mol, 3,710 g/mol, 3,715 g/mol, 3,720 g/mol, 3,725 g/mol, 3,730 g/mol, 3,735 g/mol, 3,740 g/mol, 3,745 g/mol, 3,750 g/mol, 3,755 g/mol, 3,760 g/mol, 3,765 g/mol, 3,770 g/mol, 3,775 g/mol, 3,780 g/mol, 3,785 g/mol, 3,790 g/mol, 3,795 g/mol, 3,800 g/mol, 3,805 g/mol, 3,810 g/mol, 3,815 g/mol, 3,820 g/mol, 3,825 g/mol, 3,830 g/mol, 3,835 g/mol, 3,840 g/mol, 3,845 g/mol, 3,850 g/mol, 3,855 g/mol, 3,860 g/mol, 3,865 g/mol, 3,870 g/mol, 3,875 g/mol, 3,880 g/mol, 3,885 g/mol, 3,890 g/mol, 3,895 g/mol, 3,900 g/mol, 3,905 g/mol, 3,910 g/mol, 3,915 g/mol, 3,920 g/mol, 3,925 g/mol, 3,930 g/mol, 3,935 g/mol, 3,940 g/mol, 3,945 g/mol, 3,950 g/mol, 3,955 g/mol, 3,960 g/mol, 3,965 g/mol, 3,970 g/mol, 3,975 g/mol, 3,980 g/mol, 3,985 g/mol, 3,990 g/mol, 3,995 g/mol, or 4,000 g/mol).

[0413] In some embodiments, the poloxamer has an average molar mass of polyoxypropylene subunits of from about 3,625 g/mol to about 4,000 g/mol (e.g., about 3,625 g/mol, 3,630 g/mol, 3,635 g/mol, 3,640 g/mol, 3,645 g/mol, 3,650 g/mol, 3,655 g/mol, 3,660 g/mol, 3,665 g/mol, 3,670 g/mol, 3,675 g/mol, 3,680 g/mol, 3,685 g/mol, 3,690 g/mol, 3,695 g/mol, 3,700 g/mol, 3,705 g/mol, 3,710 g/mol, 3,715 g/mol, 3,720 g/mol, 3,725 g/mol, 3,730 g/mol, 3,735 g/mol, 3,740 g/mol, 3,745 g/mol, 3,750 g/mol, 3,755 g/mol, 3,760 g/mol, 3,765 g/mol, 3,770 g/mol, 3,775 g/mol, 3,780 g/mol, 3,785 g/mol, 3,790 g/mol, 3,795 g/mol, 3,800 g/mol, 3,805 g/mol, 3,810 g/mol, 3,815 g/mol, 3,820 g/mol, 3,825 g/mol, 3,830 g/mol, 3,835 g/mol, 3,840 g/mol, 3,845 g/mol, 3,850 g/mol, 3,855 g/mol, 3,860 g/mol, 3,865 g/mol, 3,870 g/mol, 3,875 g/mol, 3,880 g/mol, 3,885 g/mol, 3,890 g/mol, 3,895 g/mol, 3,900 g/mol, 3,905 g/mol, 3,910 g/mol, 3,915 g/mol, 3,920 g/mol, 3,925 g/mol, 3,930 g/mol, 3,935 g/mol, 3,940 g/mol, 3,945 g/mol, 3,950 g/mol, 3,955 g/mol, 3,960 g/mol, 3,965 g/mol, 3,970 g/mol, 3,975 g/mol, 3,980 g/mol, 3,985 g/mol, 3,990 g/mol, 3,995 g/mol, or 4,000 g/mol).

[0414] In some embodiments, the poloxamer 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).

[0415] In some embodiments, the poloxamer 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).

[0416] In some embodiments, the poloxamer 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).

[0417] In some embodiments, the poloxamer 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).

[0418] In some embodiments, the poloxamer 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%).

[0419] In some embodiments, the poloxamer 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%).

[0420] In some embodiments, the poloxamer 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%).

[0421] In some embodiments, the poloxamer has an average molar mass of greater than 10,000 g/mol (e.g., about 10,100 g/mol, 10,200 g/mol, 10,300 g/mol, 10,400 g/mol, 10,500 g/mol, 10,600 g/mol, 10,700 g/mol, 10,800 g/mol, 10,900 g/mol, 11,000 g/mol, 11,100 g/mol, 11,200 g/mol, 11,300 g/mol, 11,400 g/mol, 11,500 g/mol, 11,600 g/mol, 11,700 g/mol, 11,800 g/mol, 11,900 g/mol, 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0422] In some embodiments, the poloxamer has an average molar mass of greater than 11,000 g/mol (e.g., about 11,100 g/mol, 11,200 g/mol, 11,300 g/mol, 11,400 g/mol, 11,500 g/mol, 11,600 g/mol, 11,700 g/mol, 11,800 g/mol, 11,900 g/mol, 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0423] In some embodiments, the poloxamer has an average molar mass of greater than 12,000 g/mol (e.g., about 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0424] In some embodiments, the poloxamer has an average molar mass of greater than 12,500 g/mol (e.g., about 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0425] In some embodiments, the poloxamer has an average molar mass of from about 10,000 g/mol to about 15,000 g/mol (e.g., about 10,000 g/mol, 10,100 g/mol, 10,200 g/mol, 10,300 g/mol, 10,400 g/mol, 10,500 g/mol, 10,600 g/mol, 10,700 g/mol, 10,800 g/mol, 10,900 g/mol, 11,000 g/mol, 11,100 g/mol, 11,200 g/mol, 11,300 g/mol, 11,400 g/mol, 11,500 g/mol, 11,600 g/mol, 11,700 g/mol, 11,800 g/mol, 11,900 g/mol, 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0426] In some embodiments, the poloxamer has an average molar mass of from about 11,000 g/mol to about 15,000 g/mol (e.g., about 11,000 g/mol, 11,100 g/mol, 11,200 g/mol, 11,300 g/mol, 11,400 g/mol, 11,500 g/mol, 11,600 g/mol, 11,700 g/mol, 11,800 g/mol, 11,900 g/mol, 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0427] In some embodiments, the poloxamer has an average molar mass of from about 11,500 g/mol to about 15,000 g/mol (e.g., about 11,500 g/mol, 11,600 g/mol, 11,700 g/mol, 11,800 g/mol, 11,900 g/mol, 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0428] In some embodiments, the poloxamer has an average molar mass of from about 12,000 g/mol to about 15,000 g/mol (e.g., about 12,000 g/mol, 12,100 g/mol, 12,200 g/mol, 12,300 g/mol, 12,400 g/mol, 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0429] In some embodiments, the poloxamer has an average molar mass of from about 12,500 g/mol to about 15,000 g/mol (e.g., about 12,500 g/mol, 12,600 g/mol, 12,700 g/mol, 12,800 g/mol, 12,900 g/mol, 13,000 g/mol, 13,100 g/mol, 13,200 g/mol, 13,300 g/mol, 13,400 g/mol, 13,500 g/mol, 13,600 g/mol, 13,700 g/mol, 13,800 g/mol, 13,900 g/mol, 14,000 g/mol, 14,100 g/mol, 14,200 g/mol, 14,300 g/mol, 14,400 g/mol, 14,500 g/mol, 14,600 g/mol, 14,700 g/mol, 14,800 g/mol, 14,900 g/mol, or 15,000 g/mol).

[0430] Poloxamers P288, P335, P338, and P407

[0431] Poloxamers that may be used in conjunction with the compositions and methods of the disclosure include “poloxamer 288” (also referred to in the art as “P 288” and poloxamer “F98”) having the approximate chemical formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is about 236.36, and z is about 44.83. The average molecular weight of P288 is about 13,000 g/mol.

[0432] In some embodiments, the poloxamer is a variant of P288, such as a variant of the formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is from about 220 to about 250, and z is from about 40 to about 50. In some embodiments, the average molecular weight of the poloxamer is from about 12,000 g/mol to about 14,000 g/mol.

[0433] Poloxamers that may be used in conjunction with the compositions and methods of the disclosure further include “poloxamer 335” (also referred to in the art as “P 335” and poloxamer “P105”), having the approximate chemical formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is about 73.86, and z is about 56.03. The average molecular weight of P335 is about 6,500 g/mol.

[0434] In some embodiments, the poloxamer is a variant of P335, such as a variant of the formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is from about 60 to about 80, and z is from about 50 to about 60. In some embodiments, the average molecular weight of the poloxamer is from about 6,000 g/mol to about 7,000 g/mol.

[0435] Poloxamers that may be used in conjunction with the compositions and methods of the disclosure further include “poloxamer 338” (also referred to in the art as “P 338” and poloxamer “F108”), having the approximate chemical formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is about 265.45, and z is about 50.34. The average molecular weight of P335 is about 14,600 g/mol.

[0436] In some embodiments, the poloxamer is a variant of P338, such as a variant of the formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is from about 260 to about 270, and z is from about 45 to about 55. In some embodiments, the average molecular weight of the poloxamer is from about 14,000 g/mol to about 15,000 g/mol.

[0437] Poloxamers that may be used in conjunction with the compositions and methods of the disclosure further include “poloxamer 407” (also referred to in the art as “P 407” and poloxamer “F127”), having the approximate chemical formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is about 200.45, and z is about 65.17. The average molecular weight of P335 is about 12,600 g/mol.

[0438] In some embodiments, the poloxamer is a variant of P407, such as a variant of the formula HO(C.sub.2H.sub.4O).sub.x(C.sub.3H.sub.6O).sub.y(C.sub.2H.sub.4O).sub.zH, wherein the sum of x and y is from about 190 to about 210, and z is from about 60 to about 70. In some embodiments, the average molecular weight of the poloxamer is from about 12,000 g/mol to about 13,000 g/mol.

[0439] For clarity, the terms “average molar mass” and “average molecular weight” are used interchangeable herein to refer to the same quantity. The average molar mass, ethylene oxide content, and propylene oxide content of a poloxamer, 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.

PKC Modulating Agents

[0440] 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

[0441] 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 (I)

##STR00071##

[0442] 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;

[0443] 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;

[0444] 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;

[0445] R.sub.c is O, NR.sub.d, or S;

[0446] 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;

[0447] 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;

[0448] 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;

[0449] represents a bond that is optionally present;

[0450] n is an integer from 0-4; and

[0451] m is an integer from 0-4;

[0452] or a salt thereof.

[0453] 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)

##STR00072##

[0454] 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;

[0455] 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;

[0456] 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;

[0457] R.sub.c is O or S;

[0458] 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;

[0459] 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;

[0460] n is an integer from 0-4; and

[0461] m is an integer from 0-4;

[0462] or a salt thereof.

[0463] Additional examples of such staurosporine variants are represented by formula (III)

##STR00073##

[0464] wherein R.sub.1 is H, OH, oxo, or thiocarbonyl;

[0465] 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;

[0466] Ring A is an optionally substituted and optionally fused heterocycloalkyl ring;

[0467] R.sub.c is O or S;

[0468] 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;

[0469] 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;

[0470] n is an integer from 0-4; and

[0471] m is an integer from 0-4;

[0472] or a salt thereof.

[0473] Further examples of such staurosporine variants are represented by formula (IV)

##STR00074##

[0474] wherein R.sub.1 is H, OH, or oxo;

[0475] Ring B is an optionally substituted heteroaryl or heterocycloalkyl ring;

[0476] R.sub.c is O or S;

[0477] W is O, NH, or S;

[0478] 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;

[0479] 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;

[0480] n is an integer from 0-4; and

[0481] m is an integer from 0-4;

[0482] or a salt thereof.

[0483] Additional examples of such staurosporine variants are represented by formula (V)

##STR00075##

[0484] wherein R.sub.1 is H, OH, or oxo;

[0485] R.sub.c is O or S;

[0486] W is O, NH, or S;

[0487] 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

[0488] p is 0 or 1;

[0489] or a salt thereof.

[0490] Additional examples of such staurosporine variants are represented by formula (VI)

##STR00076##

[0491] wherein R.sub.1 is H, OH, or oxo;

[0492] 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

[0493] s is an integer from 0-8;

[0494] or a salt thereof.

[0495] Further examples of such staurosporine variants are represented by formula (VII)

##STR00077##

[0496] wherein R.sub.1 is H, OH, or oxo;

[0497] R.sub.2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; and

[0498] R.sub.3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido

[0499] or a salt thereof.

[0500] Additional examples of such staurosporine variants are represented by formula (VIII)

##STR00078##

[0501] wherein R.sub.1 is H, OH, or oxo;

[0502] R.sub.2 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy; and

[0503] R.sub.3 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, or optionally substituted amido

[0504] or a salt thereof.

[0505] Further examples of such staurosporine variants are represented by formula (IX)

##STR00079##

[0506] 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;

[0507] 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;

[0508] n is an integer from 0-4; and

[0509] m is an integer from 0-4;

[0510] or a salt thereof.

[0511] Additional examples of such staurosporine variants are represented by formula (1)

##STR00080##

[0512] or a salt thereof.

[0513] 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)

##STR00081##

[0514] or a salt thereof.

[0515] Further examples of such staurosporine variants are represented by formula (X)

##STR00082##

[0516] wherein R.sub.1 is H, OH, or oxo;

[0517] 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

[0518] t is an integer from 0-6;

[0519] or a salt thereof.

[0520] Additional examples of such staurosporine variants are represented by formula (XI)

##STR00083##

[0521] wherein R.sub.1 is H, OH, or oxo; and

[0522] R.sub.4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy;

[0523] or a salt thereof.

[0524] Further examples of such staurosporine variants are represented by formula (XII)

##STR00084##

[0525] wherein R.sub.1 is H, OH, or oxo; and

[0526] R.sub.4 is H, OH, optionally substituted alkoxy, or optionally substituted acyloxy;

[0527] or a salt thereof.

[0528] Additional examples of such staurosporine variants are represented by formula (XIII)

##STR00085##

[0529] 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;

[0530] 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;

[0531] n is an integer from 0-4; and

[0532] m is an integer from 0-4;

[0533] or a salt thereof.

[0534] Additional examples of such staurosporine variants are represented by formula (3)

##STR00086##

[0535] or a salt thereof.

[0536] Additional examples of such staurosporine variants are represented by formula (4)

##STR00087##

[0537] or a salt thereof.

[0538] Additional examples of such staurosporine variants are:

##STR00088## ##STR00089## ##STR00090##

or a salt thereof.

[0539] 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)

##STR00091##

[0540] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and

[0541] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0542] or a salt thereof.

[0543] In some embodiments, the PKC inhibitor is a compound represented by formula (XV)

##STR00092##

[0544] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and

[0545] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0546] or a salt thereof.

[0547] In some embodiments, the PKC inhibitor is a compound selected from:

##STR00093##

or a salt thereof.

[0548] 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)

##STR00094##

[0549] 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;

[0550] or a salt or quaternized variant thereof.

[0551] In some embodiments, the PKC inhibitor is a compound represented by formula (XVII)

##STR00095##

[0552] 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;

[0553] or a salt or quaternized variant thereof.

[0554] In some embodiments the PKC inhibitor is a compound selected from:

##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114##

or a salt thereof.

[0555] 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)

##STR00115##

[0556] wherein R is H, OH, C.sub.1-6 alkoxy, or oxo; and

[0557] R.sub.2 is

##STR00116##

optionally wherein the configuration of the sugar moiety is derived from D-glucose, D-galactose, or D-mannose;

[0558] 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;

[0559] 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;

[0560] R.sub.5 is H or C.sub.1-6 alkyl;

[0561] 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

[0562] 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;

[0563] or a salt thereof.

[0564] In some embodiments, the PKC inhibitor is a compound represented by formula (XIX)

##STR00117##

[0565] wherein R is H, OH, C.sub.1-6 alkoxy, or oxo; and

[0566] R.sub.2 is

##STR00118##

[0567] 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;

[0568] 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;

[0569] R.sub.5 is H or C.sub.1-6 alkyl;

[0570] 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

[0571] 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;

[0572] or a salt thereof.

[0573] In some embodiments, the PKC inhibitor is a compound selected from N-(1-α-O-Benzyl-2-N-acetylmuramyl)staurosporine, N-(2-N-Acetyl-muramyl)staurosporine, N-(6-0-Mesyl-1-α-O-benzyl-2-N-acetylmuramyl)staurosporine, N-(6-Azido-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-Amino-1-α-O-benzyl-2-N-acetyl-6-deoxymuramyl)staurosporine, N-(6-Amino-6-deoxy-2-N-acetylmuramyl)staurosporine, N-(6-O-Mesyl-2-N-acetylmuramyl)staurosporine, N-(2-N-Acetyl-demethylmuramyl)staurosporine, N-(1-α-O-Benzyl-2-N-acetylhomomuramyl)staurosporine, N-(1-α-O-Benzyl-2—N-acetyl-L-homomuramyl)staurosporine, the 1-α-anomer of N-(2-N-acetyl-L-homomuramyl)staurosporine, N-(1-α-O-Benzyl-4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α-O-Benzyl-4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporin, N-(1-Deoxy-2-N-acetylmuramyl)staurosporine, the 1-α-anomer of N-(4-O-acetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, the 1-α-anomer of N-(4,6-O-diacetyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4-O-diacetyl-6-O-stearoyl-2-N-acetylmuramyl)staurosporine, N-(1-α,4,6-O-Triacetyl-2-N-acetylmuramyl)staurosporine, 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-toluylsulfonyl-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.

[0574] 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)

##STR00119##

[0575] wherein Z.sub.1 is H or OH;

[0576] Z.sub.2 is H or OH;

[0577] R.sub.1 is H, halogen, or optionally substituted alkyl;

[0578] R.sub.2 is H or halogen;

[0579] R is OH or optionally substituted alkoxy; and

[0580] X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH.sub.2—NH-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;

[0581] or a salt thereof.

[0582] 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)

##STR00120##

[0583] wherein Z.sub.1 is H or OH;

[0584] Z.sub.2 is H or OH;

[0585] R.sub.1 is H, halogen, or optionally substituted alkyl;

[0586] R.sub.2 is H or halogen;

[0587] R is OH or optionally substituted alkoxy; and

[0588] X is optionally substituted alkyl or optionally substituted acyl, optionally wherein X is CH.sub.2—NH-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;

[0589] or a salt thereof.

[0590] 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)

##STR00121##

[0591] 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;

[0592] 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;

[0593] 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

[0594] each X is, independently, O, OH and H, or a pair of hydrogen atoms;

[0595] 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;

[0596] 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;

[0597] each n is, independently, an integer from 0-4; and

[0598] each m is, independently, an integer from 0-4;

[0599] or a salt thereof.

[0600] In some embodiments, the PKC inhibitor is a compound represented by formula (XXVI) or (XXVII)

##STR00122##

[0601] 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;

[0602] 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;

[0603] 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;

[0604] 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;

[0605] 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, carbonyldioxy, esterified carboxy, carbamoyl, N-mono- or N,N-di-substituted carbamoyl, sulfo, substituted sulfonyl, aminosulfonyl or N-mono- or N,N-di-substituted aminosulfonyl;

[0606] 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;

[0607] each X is, independently, O, OH and H, or a pair of hydrogen atoms;

[0608] each n is, independently, an integer from 0-4;

[0609] each m is, independently, an integer from 0-4;

[0610] each n′ is, independently, an integer from 0-4; and

[0611] each m′ is, independently, an integer from 0-4;

[0612] or a salt thereof.

[0613] 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)

##STR00123##

[0614] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and

[0615] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0616] or a salt thereof.

[0617] In some embodiments, the PKC inhibitor is a compound represented by formula (XXIX)

##STR00124##

[0618] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and

[0619] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0620] or a salt thereof.

[0621] In some embodiments, the PKC inhibitor is a compound represented by formula (XXX)

##STR00125##

[0622] wherein R.sub.1 is H or optionally substituted 01-6 alkyl; and

[0623] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0624] or a salt thereof.

[0625] In some embodiments, the PKC inhibitor is a compound represented by formula (XXXI)

##STR00126##

[0626] wherein R.sub.1 is H or optionally substituted C.sub.1-6 alkyl; and

[0627] R.sub.2 is optionally substituted C.sub.1-6 alkyl;

[0628] or a salt thereof.

[0629] In some embodiments, the PKC inhibitor is a compound selected from:

##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137##

Interfering RNA

[0630] 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.

Spinoculation

[0631] 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 200×g to about 2,000×g. The centripetal force may be, e.g., from about 300×g to about 1,200×g (e.g., about 300×g, 400×g, 500×g, 600×g, 700×g, 800×g, 900×g, 1,000×g, 1,100×g, or 1,200×g, 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.

[0632] 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

[0633] 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.

[0634] 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.

[0635] 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, N.Y.). 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, iPS cells or other reprogrammed mature cells types.

[0636] 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.

Viral Vectors for Transgene Expression

[0637] 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

[0638] 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.

[0639] 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, encapsidation, and expression, in which the sequences to be expressed are inserted.

[0640] 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.

[0641] 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.

[0642] 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.

[0643] 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.

[0644] The vector used in the methods and compositions described herein may, be a clinical grade vector.

Methods of Treatment

[0645] Exemplary diseases that may be treated using the compositions and methods of the disclosure 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-00002 (SEQ ID NO: 1) ATGGTGCATCTGACCCCGGAAGAAAAAAGCGCGGTG ACCGCGCTGTGGGGCAAAGTGAACGTGGATGAAGT GGGCGGCGAAGCGCTGGGCCGCCTGCTGGTGGTGT ATCCGTGGACCCAGCGCTTTTTTGAAAGCTTTGGC GATCTGAGCACCCCGGATGCGGTGATGGGCAACCC GAAAGTGAAAGCGCATGGCAAAAAAGTGCTGGGCG CGTTTAGCGATGGCCTGGCGCATCTGGATAACCTG AAAGGCACCTTTGCGACCCTGAGCGAACTGCATTG CGATAAACTGCATGTGGATCCGGAAAACTTTCGCC TGCTGGGCAACGTGCTGGTGTGCGTGCTGGCGCAT CATTTTGGCAAAGAATTTACCCCGCCGGTGCAGGC GGCGTATCAGAAAGTGGTGGCGGGCGTGGCGAACG CGCTGGCGCATAAATATCAT

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

[0646]

TABLE-US-00003 (SEQ ID NO: 2) MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPW TQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGL AHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCV LAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0647] 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, β-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 α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor β (TGFβ) superfamily comprising TGFβ, 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), neurturin, 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.

[0648] 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-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, and IL-17, monocyte chemoattractant protein (MCP-1), leukemia inhibitory factor (LIF), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), Fas ligand, tumor necrosis factors α and β (TNFα and TNFβ), interferons (IFN) IFN-α, IFN-β, and IFN-γ, stem cell factor, 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.

[0649] 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 TGFβ receptors, the interleukin receptors, the interferon receptors, serotonin receptors, α-adrenergic receptors, β-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.

[0650] 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-κB, members of the leucine zipper family, C.sub.2H.sub.4 zinc finger proteins, including Zif268, EGR1, EGR2, C6 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.

[0651] 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, α1 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 β-glucosidase, α-galactosidase a, α-1-iduronidase, iduronate sulfatase, lysosomal acid α-glucosidase, sphingomyelinase, hexosaminidase A, hexomimidases A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, α-fucosidase, α-, β-mannosidosis, aspartylglucosaminidase, neuramidase, galactosylceramidase, heparan-N-sulfatase, N-acetyl-α-glucosaminidase, Acetyl-CoA: α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B, β-glucuoronidase and hexosaminidases A and B.

[0652] 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.

[0653] 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 2, below.

TABLE-US-00004 TABLE 2 Exemplary disorders associated with gene deficiency or dysfunction Disease associated with Exemplary amino acid Protein deficiency in protein sequence of protein acid α-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 α, β, γ, Δ, ε, or ζ 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 α-1-antitrypsin (AAT) Hereditary emphysema or α-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, α Sanfilippo syndrome (MPSIIIB) NP_000254.2 (NAGLU) Iduronidase, α -1 (IDUA) MPSI-Hurler NP_000194.2 Iduronate 2-sulfatase (IDS) MPSII-Hunter NP_000193.1, NP_001160022.1, NP_006114.1 Glucuronidase, β (GUSB) MPSVII-Sly NP_000172.2, NP_001271219.1 Hexosaminidase A, α 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/Flt-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-β-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, α (AGA) Fabry disease NP_000160.1 Phenylalanine hydroxylase Hypercholesterolaemia or NP_000268.1 (PAH) Phenylketonuria (PKU) Propionyl CoA carboxylase, Propionic acidaemias NP_000273.2, alpha polypeptide (PCCA) NP_001121164.1, NP_001171475.1

Selection of Donor Cells

[0654] 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

[0655] 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 1×10.sup.4 cells/kg to 1×10.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

[0656] 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 One. Poloxamers, when Used at Low Concentrations, Serve to Increase Viral Transduction Efficiency

[0657] This example describes a series of experiments designed to evaluate the effect of poloxamers, particularly poloxamer P407, on lentiviral transduction efficiency. Human CD34+ hematopoietic stem cells were mobilized into peripheral circulation and isolated from donor subjects. Cells were cultured in the presence of a lentiviral vector at a multiplicity of infection of 10. The lentiviral vector used in these experiments contained a transgene encoding green fluorescent protein (GFP), enabling the detection of transgene-expressing cells by monitoring fluorescence at about 510 nm.

[0658] During the transduction procedure, cells were exposed to the GFP-encoding lentiviral vector either alone or in combination with poloxamer P407 at a concentration of 7 μg/ml, 10 μg/ml, or 100 μg/ml. For cells transduced in the presence of poloxamer, the cells were exposed to the lentivirus and P407 simultaneously. After 18 hours of incubation, cells were washed to remove the lentivirus (and the poloxamer, for co-cultured cells). The proportion of transgene-expressing cells and the vector copy number of each population of cells was then assessed 12 days after the transduction procedure. The results of these studies are reported in FIGS. 1A and 1B, respectively.

[0659] To assess the quantity of transgene-expressing cells, each population of cells was monitored using flow cytometry to detect GFP+ cells. As shown in FIG. 1A, surprisingly, lower concentrations of P407 appear to promote a general increase in the proportion of transgene-expressing cells. Vector copy number was evaluated using polymerase chain reaction techniques designed to measure the number of lentiviral genomes present in each cell. As shown in FIG. 1B, cells transduced with 7 μg/ml of P407 appeared to exhibit elevated vector copy numbers relative to cells transduced with 10 μg/ml.

Example Two. Effects of Poloxamers P407 and P338 on Lentiviral Transduction of Hematopoietic Stem Cells

[0660] This example describes a series of experiments designed to compare the effects of poloxamers P407 and P338 on lentiviral transduction efficiency. Human CD34+ hematopoietic stem cells were mobilized into peripheral circulation and isolated from donor subjects. Cells were cultured in the presence of a lentiviral vector at a multiplicity of infection of from 5 to 20. The lentiviral vector used in these experiments contained a transgene encoding GFP, enabling the detection of transgene-expressing cells by monitoring fluorescence at about 510 nm.

[0661] During the transduction procedure, cells were exposed to the GFP-encoding lentiviral vector either alone or in combination with poloxamer P407 or poloxamer 338 at various concentrations. The proportion of transgene-expressing cells and the vector copy number of each population of cells was then assessed 12 days after the transduction procedure. The results of these studies are reported in FIGS. 2A-2C.

[0662] As shown in FIG. 2A, both CD34+ stem cells were tolerant of both poloxamer P407 and poloxamer P338, as neither poloxamer exerted a detrimental effect on cell viability at the concentrations tested. Surprisingly, lower concentrations of P407, such as concentrations less than 10 μg/ml, were found to enhance lentiviral transduction to a greater extent than the same or higher concentrations of poloxamer P338, as shown in FIGS. 2B and 2C.

Example Three. Poloxamers can be Used in Combination with Protamine Sulfate, Cyclosporine H, and/or Lithium Carbonate to Enhance Lentiviral Transduction

[0663] This example describes a series of experiments designed to evaluate the effects of poloxamers, such as poloxamer P407, in combination with protamine sulfate on lentiviral transduction efficiency. Briefly, human CD34+ hematopoietic stem cells were mobilized into peripheral circulation and isolated from donor subjects in accordance with the method described in Examples One and Two, above. Cells were then transduced with a lentiviral vector encoding GFP in the presence of poloxamer P407 (7 μg/mL) or poloxamer P338 (1 mg/mL), each in combination with protamine sulfate (3.5 μg/mL). At 12 days post-transduction, the percentage of transduced cells was assessed by flow cytometry detection of transgene expression, and VCN was determined by droplet digital PCR detection of integrated transgene sequences in genomic DNA. The results of these experiments are shown in FIGS. 3A and 3B.

[0664] As FIGS. 3A and 3B show, poloxamer P407, in the presence of protamine sulfate, exhibited a surprisingly greater improvement in lentiviral transduction efficiency relative to poloxamer P338 in the presence of protamine sulfate.

[0665] In addition to analyzing the effects of poloxamers in combination with protamine sulfate, a series of experiments was conducted in which poloxamer P407 was combined with protamine sulfate and either cyclosporine H or lithium carbonate (Li.sub.2CO.sub.3) in order to evaluate the effects of three-component combinations of agents on lentiviral transduction efficiency. In these experiments, CD34+ stem cells freshly isolated from peripheral mobilized blood were transduced with a clinical therapeutic lentiviral vector encoding a beta-globin transgene in the presence of poloxamer P407 (7 μg/mL) in combination with protamine sulfate (3.5 μg/mL) and either cyclosporine H (8 μM) or Li.sub.2CO.sub.3, (2 mM). The results of these experiments are shown in FIGS. 4A and 4B, as well as in Table 3, below.

TABLE-US-00005 TABLE 3 Effects of poloxamer P407, protamine sulfate, and either cyclosporine H or Li.sub.2CO.sub.3 on lentiviral transduction efficiency Range Range Mean Mean VCN per Range % VCN Mean VCN VCN (range) % cell in trans- per cell (range) in (range) in Trans- total CFU/ duction in CFU Transduction eryth. LC myel. LC duction only BFU (MOI (MOI Conditions (MOI 100).sup.1 (MOI 100).sup.2 (MOI 100).sup.3 (MOI 100).sup.4 10-20) 10-20) n Vector only 0.37 0.24 28.3 0.4-3.9/ 13-28 0.15-0.36 5 (0.18-0.82) (0.18-0.34) (22-31) 0.8-6.7  P407 + PS + 3.4  1.81 76.9 2.1-3.4/ 35-65 0.76-1.44 4 CSH (3.02-4.05) (1.38-2.32) (65-94) 0.8-4.1  P407 + PS + 1.74 1.37 63   0.9-3.1/ 33-64 0.56-0.90 4 Li.sub.2CO.sub.3 (0.73-2.9)  (0.93-2.33) (50-78) 1.2-2.6  .sup.1, 2Mean VCN (and VCN range) detected in bulk liquid cultures for erythroid and myeloid differentiation .sup.3Range shows percentage of LV+ colonies detected of total colonies screened in CFU assays .sup.4Shows the lower and upper range of VCN/cell detected within all LV+ colonies detected within a CFU assay, followed by the range of VCN detected within LV+ BFU-E colonies within each CFU assay (n = 4-5 independent healthy donors)

[0666] As the results in FIGS. 4A, 4B, and Table 3 show, poloxamers, such as poloxamer P407, can be used to enhance lentiviral transduction efficiency in the presence of additional agents, such as protamine sulfate, cyclosporine H, and Li.sub.2CO.sub.3.

OTHER EMBODIMENTS

[0667] 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.

[0668] Other embodiments are in the claims.