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Title:
ENHANCED NON-VIRAL NANOPARTICLE DELIVERY TO PLURIPOTENT STEM CELLS
Document Type and Number:
WIPO Patent Application WO/2016/018924
Kind Code:
A1
Abstract:
The invention provides methods to enhance the efficiency of nanoparticle delivery to pluripotent stem cells, particularly human embryonic stem cells. The method includes contacting a pluripotent stem cell with an effective amount of a Rho-associated kinase (ROCK) inhibitor such as Y-27632 for an effective period of time and contacting the pluripotent stem cell with a nanoparticle composition, thereby delivering one or more nanoparticles to the cytoplasm of the pluripotent stem cell. The delivery efficiency of the nanoparticles was found to be significantly enhanced compared to delivery efficiency in the absence of the ROCK inhibitor.

Inventors:
CHENG JIANJUN (US)
YEN JONATHAN (US)
Application Number:
US2015/042495
Publication Date:
February 04, 2016
Filing Date:
July 28, 2015
Export Citation:
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Assignee:
UNIV ILLINOIS (US)
International Classes:
A61K31/44; C12N5/0735; C12N5/0775; C12N5/16
Foreign References:
US20090318485A12009-12-24
US4548716A1985-10-22
Other References:
YEN: "Materials And Biological Approach To Gene Delivery In Human Embryonic Stem Cells", THESIS, SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOENGINEERING IN THE GRADUATE COLLEGE OF THE UNIVERSITY OF ILLINOIS AT URBANA CHAMPAIGN, 24 April 2013 (2013-04-24)
Attorney, Agent or Firm:
HAUKAAS, Michael H. (5100 Eden Ave. Suite 30, Edina Minnesota, US)
Download PDF:
Claims:
What is claimed is:

1. A method to enhance the efficiency of nanoparticle delivery to a pluripotent stem cell comprising contacting a pluripotent stem cell with an effective amount of Y-27632 for an effective period of time and contacting the pluripotent stem cell with a nanoparticle composition comprising one or more nanoparticles, thereby delivering one or more nanoparticles to the cytoplasm of the pluripotent stem cell; wherein the delivery efficiency of the one or more nanoparticles is enhanced compared to the corresponding delivery efficiency in the absence of Y-27632.

2. The method of claim 1 wherein the nanoparticle composition comprises a nanocomplex comprising a) a non-viral delivery vector, and b) genetic material, a protein, a drug, or a combination thereof.

3. A method to enhance the efficiency of non-viral gene delivery to a pluripotent stem cell comprising contacting a pluripotent stem cell with an effective amount of Y-27632 for an effective period of time and contacting the pluripotent stem cell with a nanoparticle composition comprising a nanocomplex that includes a non-viral delivery vector and genetic material, thereby delivering the genetic material to the cytoplasm or nucleus of the pluripotent stem cell; wherein the delivery efficiency of the genetic material is enhanced compared to the corresponding delivery efficiency in the absence of Y-27632.

4. The method of claim 3 wherein the delivery efficiency of the genetic material is enhanced by at least 50%.

5. The method of claim 3 wherein the delivery efficiency of the genetic material is enhanced by at least 85%.

6. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 prior to contacting the pluripotent stem cell with the nanoparticle composition.

7. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 concurrently with contacting the pluripotent stem cell with the nanoparticle composition.

8. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 both prior to and concurrently with contacting the pluripotent stem cell with the nanoparticle composition.

9. The method of claim 3 wherein the non-viral delivery vector comprises lipofectamine 2000, Fugene HD, poly-L-lysine, poly-L-arginine, polyethyleneimine, or a combination thereof.

10. The method of claim 3 wherein the genetic material comprises plasmid DNA or siRNA.

1 1. The method of claim 1 or 3 wherein the pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

12. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 at a concentration of about 5 μΜ to about 100 μΜ.

13. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 at a concentration of about 25 μΜ to about 60 μΜ.

14. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 for at least 30 minutes prior to contacting the pluripotent stem cell with the nanoparticle composition.

15. The method of claim 1 or 3 wherein the pluripotent stem cell is contacted with Y- 27632 for a period of about 1 hour to about 6 hours, prior to contacting the pluripotent stem cell with the nanoparticle composition.

16. The method of any one of claims 3-15 wherein delivery of the genetic material to the cytoplasm of the pluripotent stem cell results in delivery to the nucleus, thereby resulting in transfection of the cell.

17. A method to enhance the transfection of a human pluripotent stem cell or human induced pluripotent stem cell comprising contacting a human pluripotent stem cell or a human induced pluripotent stem cell with an effective amount of Y-27632 for an effective period of time and contacting the stem cell with a nanocomplex composition comprising a non-viral delivery vector and genetic material, resulting in the delivery of the genetic material to the cytoplasm of the pluripotent stem cell and transfection of the cell; wherein the transfection of the stem cell is enhanced compared to the corresponding transfection in the absence of Y-27632.

18. A kit comprising the compound Y-27632 and one or more transfection reagents for the condensation and delivery of genetic material, such as a plasmid.

19. The kit of claim 18 wherein the Y-27632 is lyophilized and sterile.

20. The kit of claim 18 or 19 wherein the kit further includes ultrapure water, optionally in containers containing pre-measured amounts of the ultrapure water.

Description:
ENHANCED NON-VIRAL NANOP ARTICLE DELIVERY

TO PLURIPOTENT STEM CELLS

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/029,964, filed July 28, 2014, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Award Nos.

1DP2OD007246 and 1R21EB013379, awarded by the National Institutes of Health, and Grant No. 0965918 IGERT, awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Human embryonic stem cells (hESC) hold tremendous potential in the field of regenerative medicine largely due to their pluripotency. These cells have the ability to differentiate into endoderm, mesoderm, and ectoderm lineages, which compose virtually any cell type in the body. Therefore, the genetic manipulation of embryonic stem cells is an important tool in regenerative medicine. The control and expression of specific genes afforded by gene delivery is valuable not only in efforts to control stem cell fate, but also to study cell behavior in differentiation and gene targeting studies.

Lentiviral transduction has been established as an effective method for gene delivery to hESCs because of their consistently high transfection efficiency and capability to maintain stable transgene expression. However, virus-based delivery systems pose risks of immunogenicity, insertional mutagenesis, and viral integration into the host system. In this regard, non- viral gene delivery, often characterized by its desired biocompatibility and minimal immunogenicity, provides an ideal alternative to viral gene delivery. Nevertheless, non-viral systems applied to human embryonic stem cell colonies are hampered by low transfection efficiency, which limits their applications.

The low efficiency is, to some extent, attributed to the distinct physiology of hESCs. hESCs are mildly intrinsically stiff in structure due to the fact that they grow in tight colonies and in rounded up shapes. Because of such tight two dimensional colonies, cells in the center are often compressed by the surrounding cells and exposure of the centered cells to exogenous materials is greatly limited. This limited exposure prevents effective

internalization of gene delivery materials and thus leads to low transfection efficiency. Such cases have been widely noted in previous gene delivery studies (see, e.g., Yen et al, Biomaterials Science 2013, 1, (7), 719-727) showing that the outer edge of the hESCs have notably higher uptake efficiency. These physical properties of the hESC colony growth pose a large limitation in gene delivery that may not be able to be solved through the material design of the delivery vector. Accordingly, alternative strategies are needed to increase the gene delivery efficiency to hESCs.

Considering the colony-forming properties of hESCs that limit non- viral gene delivery, it might be possible that increasing cell spreading would increase total surface area for interaction with transfection reagents and thus increase cellular uptake and the gene transfection efficiency. Growing hESCs on stiffer substrates has shown to disperse cells and promote cell spreading, and it has also been demonstrated in other cell types that an increase in the substrate stiffness can lead to higher transfection efficiency (Kong et al, Nature Materials 2005, 4, (6), 460-464). However, such an approach is infeasible for hESCs cells, mainly because of the sensitive nature of hESCs to their external environment; it has been reported that hESCs grown on stiff substrates will start differentiating (Zoldan et al, Biomaterials 2011, 32, (36), 9612-9621).

Accordingly, there is a need for new non-viral gene delivery compositions and methods that can avoid the risks associated with viral delivery systems. There is also a need for compositions and methods that enhance the delivery efficiency of current delivery vectors to hESCs.

SUMMARY

Non-viral gene delivery into human embryonic stem cells (hESCs) is an important tool for controlling cell fate. However, the delivery efficiency of current non-viral methods remains low due in part to the tight colony structure of hESCs, which prevents effective exposure of all of the cells to delivery vectors. The invention provides a novel approach to enhance non-viral nanoparticle delivery to hESCs by transiently altering the cell and colony structure.

The small molecule (R)-(+)-£raws-4-(l-aminoethyl)-N-(4-pyridyl)cyclohexane- carboxamide (Y-27632) inhibits the rho-associated protein kinase pathway and can be used to induce transient colony spreading. Inducing pluripotent stem cell spreading was found to increase transfection efficiency by 1.5 to 2 fold for a wide variety of non-viral transfection reagents. After removal of Y-27632 post-transfection, cells can revert back to their normal state and do not show alteration of pluripotency. This approach provides a simple, effective tool to enhance non-viral nanoparticle and/or gene delivery into adherent hESCs for genetic manipulation.

Accordingly, the invention provides methods to enhance the efficiency of nanoparticle delivery to pluripotent stem cells, particularly human embryonic stem cells and human induced pluripotent stem cells. The method includes contacting a pluripotent stem cell with an effective amount of a Rho-associated kinase (ROCK) inhibitor and contacting the pluripotent stem cell with a nanoparticle composition, thereby delivering one or more nanoparticles via endocytosis to the cytoplasm of the pluripotent stem cell. The delivery efficiency of the nanoparticles was found to be significantly enhanced compared to delivery efficiency in the absence of the ROCK inhibitor. In one embodiment, the ROCK inhibitor is a small molecule compound such as Y-27632, blebbistatin, or fasudil. Additional examples of ROCK inhibitors are described herein below.

The invention also provides a method to enhance the efficiency of nanoparticle delivery to a pluripotent stem cell comprising contacting a pluripotent stem cell with an effective amount of Y-27632 for an effective period of time and contacting the pluripotent stem cell with a nanoparticle composition comprising one or more nanoparticles, thereby delivering one or more nanoparticles to the cytoplasm of the pluripotent stem cell; wherein the delivery efficiency of the one or more nanoparticles is enhanced compared to the corresponding delivery efficiency in the absence of Y-27632. The nanoparticle composition can be a nanoparticle or nanocomplex that includes a non-viral delivery vector and one or more of genetic material, a protein, and a drug.

The invention further provides a method to enhance the efficiency of non-viral gene delivery to a pluripotent stem cell comprising contacting a pluripotent stem cell with an effective amount of Y-27632 for an effective period of time and contacting the pluripotent stem cell with a nanoparticle composition comprising a nanocomplex that includes a non- viral delivery vector and genetic material, thereby delivering the genetic material to the cytoplasm or nucleus of the pluripotent stem cell; wherein the delivery efficiency of the genetic material is enhanced compared to the corresponding delivery efficiency in the absence of Y-27632.

In one embodiment, the delivery efficiency of the nanoparticles, such as a nanocomplex containing genetic material, is enhanced by at least 50% (e.g., compared to the corresponding delivery efficiency in the absence of same ROCK inhibitor). In another embodiment, the delivery efficiency of the genetic material is enhanced by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, or by at least 90%.

In some embodiments, the pluripotent stem cell is contacted with the ROCK inhibitor prior to contacting the pluripotent stem cell with the nanoparticle composition. As used herein, the term contacting includes exposing to and/or incubating with the ROCK inhibitor.

In various embodiments, the pluripotent stem cell is contacted with the ROCK inhibitor concurrently with contacting the pluripotent stem cell with the nanoparticle composition.

In additional embodiments, the pluripotent stem cell is contacted with the ROCK inhibitor both prior to and concurrently with contacting the pluripotent stem cell with the nanoparticle composition.

In one embodiment, the nanoparticle is a nanocomplex as described herein, or a non- viral nanoparticle as described in U.S. Patent No. 9,028,880 (Cheng et al), U.S. Patent Publication Nos. 2013/0236510 (Cheng et al.) or 2013/0274173 (Cheng et al), or

International Publication No. WO 2014/169264 (Cheng et al.). In various embodiments, the nanoparticle is a nanocomplex wherein the nanocomplex includes a non-viral delivery vector such as poly-L-lysine, poly-L-arginine, polyethyleneimine, lipofectamine 2000, L-dioleoyl phosphatidylethanolamine (DOPE), cationic lipids, Fugene HD (a nonliposomal transfection reagent), or a combination thereof.

In one embodiment, the genetic material comprises plasmid DNA or siRNA. Further examples of suitable genetic material are described herein below.

In any embodiment described herein, the pluripotent stem cell can be an embryonic stem cell, and specifically, can specifically be a human embryonic stem cell or a human induced pluripotent stem cell.

In some embodiments, the pluripotent stem cell is contacted with the ROCK inhibitor at a concentration of about 5 μΜ to about 100 μΜ. In one embodiment, the pluripotent stem cell is contacted with Y-27632 at a concentration of about 5 μΜ to about 100 μΜ. In other embodiments, the pluripotent stem cell is contacted with the ROCK inhibitor at a

concentration of about 20 μΜ to about 75 μΜ, about 25 μΜ to about 60 μΜ, about 30 μΜ to about 50 μΜ, or a concentration about 30 μΜ, about 40 μΜ, or about 50 μΜ. In one embodiment, the pluripotent stem cell is contacted with Y-27632 at a concentration of about 20 μΜ to about 75 μΜ, about 25 μΜ to about 60 μΜ, about 30 μΜ to about 50 μΜ, or a concentration about 30 μΜ, about 40 μΜ, or about 50 μΜ. In some embodiments, the pluripotent stem cell is contacted with a ROCK inhibitor for at least 30 minutes prior to contacting the pluripotent stem cell with the nanoparticle composition. In various embodiments, the pluripotent stem cell is contacted with a ROCK inhibitor for a period of about 1 hour to about 6 hours, prior to contacting the pluripotent stem cell with the nanoparticle composition. Typically, the pluripotent stem cell is contacted with a ROCK inhibitor such as Y-27632 for about 2-5 hours prior to contacting the pluripotent stem cell with the nanoparticle composition.

The delivery of the genetic material to the cytoplasm of the pluripotent stem cell can efficiently deliver the genetic material to the nucleus, thereby resulting in transfection of the cell.

The invention yet further provides a method to enhance the transfection of a human embryonic stem cell comprising contacting a human embryonic stem cell with an effective amount of a ROCK inhibitor such as Y-27632 for an effective period of time and contacting the human embryonic stem cell with a nanocomplex composition comprising of a non- viral delivery vector and genetic material, resulting in the delivery of the genetic material to the cytoplasm of the embryonic stem cell and transfection of the cell; wherein the transfection of the human embryonic stem cell is enhanced compared to the corresponding transfection in the absence of the ROCK inhibitor.

The invention additionally provides a kit comprising a ROCK inhibitor such as the compound Y-27632 and one or more transfection reagents for the condensation and delivery of genetic material, such as a plasmid. The ROCK inhibitor can be provided in containers containing pre- measured amounts of the ROCK inhibitor. The ROCK inhibitor, for example, Y-27632, can be lyophilized and sterile. Additionally, the kit can further include ultrapure water, optionally in containers containing pre-measured amounts of the ultrapure water.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention. Figure 1. Y-27632 enhances the transfection efficiencies of various polyplexes or lipoplexes in hESCs via increased membrane exposure through transient spreading of the cells.

Figure 2A-B. Y-27632 enhances the transfection efficiencies of various

nanocomplexes in hESCs. a) Size and zeta potential of various nanocomplexes

(DNA:polymer, w/w): PLR (1 : 10), PLL (1 : 10), PEI (1 :5), LPF (1 :2), and FHD (1 :3.5). b) Transfection efficiencies of various reagents in hESCs in the absence or presence (50 μΜ) of Y-27632, as measured by flow cytometry. Cells were pre-treated with Y-27632 for 4 h before removal of Y-27632 and addition of various nanocomplexes (n=3) (* p <0.05, ** /?<0.01).

Figure 3A-D. Y-27632 promotes transfection in hESCs cells by extending the cell surface area, a) Transfection efficiencies of FHD/DNA nanocomplexes in HI hESCs in the presence of Y-27632 or blebbistatin at various concentrations, b) Fluorescence images of HI hESCs 48 h post transfection with FHD/pEGFP nanocomplexes. Cells were pre-treated with 0 μΜ, 10 μΜ, 30 μΜ, or 50 μΜ Y-27632 for 4 h before transfection and during the 4-h transfection process (scale bar = 250 μιη). c) Bright- field imaging showing the

morphological change (spreading) of hESCs after 4-h treatment with Y-27632 at various concentrations (scale bar = 250 μιη). d) Alteration of the cytoplasm area (μιη 2 ) per nucleus of hESCs following treatment with Y-27632 of various concentrations. Images were taken and analyzed with the GE InCell Analyzer (n=5) (* p <0.05, ** <0.01).

Figure 4. Representative images of the alteration of the hESC cytoplasmic area following treatment with Y-27632 of various concentrations (0, 10, 30 and 50 μΜ). The top row represents the Hoechst channel and the middle row represents CMTPX cell tracking channel. The bottom row represents the software generated segmentation used for analysis. The blue circles (solid circles within the lighter area of the images) represent the identified nuclei and the green outline (lighter area of the images outside the nuclei) is the identified CMTPX stain of the cellular cytoplasm from which the cell area was calculated.

Figure 5. Representative images of the transfection of hESCs after alteration of the cytoplasm area with the treatment of Y-27632 at various concentrations (0, 10, 30 and 50 μΜ). The top row represents the Hoechst channel and the middle row represents the GFP channel. The bottom row represent the software generated segmentation used for analysis. The blue circles (solid circles within the lighter area of the images) represent the identified nuclei and green outline (lighter area of the images outside the nuclei) is the determined cell cytoplasm as extrapolated by the nuclei and GFP channel. Figure 6. Y-27632 promotes cellular internalization of the gene cargo as a result of increased surface area. Cell uptake level of FHD/Y OYO-l-DNA nanocomplexes in hESCs in the presence of various concentrations of Y-27632 (n =3) (* p <0.05, ** p<0.0l).

Figure 7A-B. Y-27632 does not compromise the pluripotency of HI hESCs. a)

DAPI and SSEA-4 staining patterns of HI hESCs without Y-27632 treatment (control group) or transfected with FHD in the presence of 50 μΜ Y-27632 (FHD group, scale bar = 250 μιη). b) Western blot analysis on the OCT4 expression in hESCs 4 days post FHD

transfection and treatment of Y-27632 at various concentrations. ("-" demonstrates the cells without transfection and "+" indicates cells with FHD transfection).

Figure 8. Cytotoxicity of HI hESCs 48 h after transfection with FHD at various concentrations: MTT cell viability assay of the FHD transfection with varying concentrations (0-50 μΜ) of Y-27632 treatment in HI hESCs after 48 h.

DETAILED DESCRIPTION

Dissociation of hESCs into single cells or small colonies can potentially facilitate cell spreading. However, since hESCs are considered to be in the primed state, they are intolerant to single cell passaging and usually exhibit <1% clonal efficiency due to apoptosis upon cellular detachment and dissociation. Rho-associated kinase (ROCK) inhibitors can diminish dissociation induced apoptosis, resulting in increased survival of individual hESCs. The compound Y-27632 [(R)-(+)-?ra«s-4-(l-aminoethyl)-N-(4-pyridyl)cyclohexanecar boxamide] (Figure 1), a ROCK inhibitor, can also decrease cell-generated tension. Experimentation with Y-27632 led to the discovery that Y-27632 facilitates the spreading and flattening of hESC colonies on plates due to decreased membrane tension. The decreased membrane tension results in increased surface area exposure of the cells to allow for a more efficient uptake of nanoparticles, such as gene delivery vector-containing nanoparticles. The more efficient uptake directly results in enhanced gene transfection.

A broad spectrum of non- viral gene delivery vectors, including commercially available reagents (Lipofectamine 2000 (LPF), Fugene HD (FHD), poly-L-lysine (PLL), poly- L-arginine (PLR), and polyethyleneimine (PEI)), were evaluated in terms of their transfection efficiencies in the presence or absence of Y-27632. Mechanistic analysis into the effect of Y- 27632 on cell spreading, cell uptake property, and pluripotency of hESCs was also performed. The treatment of hESCs with Y-27632 (Figure 1) demonstrated significantly increase transfection efficiency of all the tested materials by 1.5 to 2 folds in hESCs, demonstrating an alternative use of Y-27632 as a tool for enhancing non- viral gene delivery. Inhibition of rho-associated kinase facilitates non-viral gene transfection. Prior to the transfection assessment, pEGFP- l plasmid nanocomplexes with gene transfection vectors were evaluated by dynamic light scattering and zetasizer to determine their complexation capacities. We confirmed that all tested materials were able to form nanocomplexes with DNA. Upon complexation with DNA, the nanocomplexes of DNA with PLR, PLL and PEI formed particles having diameters of about 60-70 nm, while DNA complexes with LPF and FHD following the standard protocol afforded particle of a much larger size (above 300 nm, Figure 2a). PLR, PLL, and PEI are cationic polymers with sufficiently long backbone so that they can well condense the anionic DNA into compact nanocomplexes via electrostatic interaction as well as intermolecular entanglement. In comparison, LPF and FHD are cationic lipid based materials, and their short molecular length appear to prevent significant entanglement with the DNA molecules, thus leading to relatively larger complexes.

The transfection efficiency of each material was then evaluated in the presence (50 μΜ) or absence of Y-27632, by monitoring the EGFP expression, to probe the effect of Y- 27632 on gene transfection. As shown in Figure 2b, PLR was unable to mediate transfection in HI hESCs (transfection efficiency < 1%), which is consistent with previous findings in various cell lines (Yin et al, Biomaterials 2013, 34, (9), 2340-2349; Tang et al., Chemical Science 2013, 4, (10), 3839-3844). Upon the treatment of Y-27632, all other materials significantly increased their transfection efficiency of hESCs as measured by flow cytometry.

Specifically, the PLL transfection efficiency with Y-27632 increased from 3.0 ± 0.8% to 5.8 ± 0.8%. For PEI mediated transfection, the transfection efficiency increased from 7.7 ± 0.8% to 13.7 ± 1.3% in the presence of Y-27632. Similar enhancement of gene transfection efficiencies were observed with the use of Y-27632 when LPF and FHD were used as transfection agents. Their transfection efficiencies were improved from 8.8 ± 0.8% to 15.3 ± 1.2% and 21.5 ± 0.9% to 37.0 ± 1.0%, respectively. These studies clearly demonstrate that Y-27632 treatment can universally increase the transfection efficiency by approximately 1.7 to 1.9 fold in non- viral gene delivery to hESCs.

Increased cell spreading by Y-27632 treatment. To further investigate the treatment effect of Y-27632, transfection studies were performed on HI hESC colonies that were treated with varying concentration of Y-27632 for 4 h prior to and during transfection. DNA/FHD complexes, which demonstrated the highest transfection efficiency among the systems tested, were selected for further studies. With no treatment, the EGFP transfection efficiency was 14.4 ± 0.9%. With 10 μΜ treatment of Y-27632, the transfection efficiency was increased to 17.6 ± 1.4%. When the concentration of Y-27632 was increased to 30 μΜ and 50 μΜ, the respective efficiency was increased to 22.7 ± 0.8% and 26.4 ± 1.1% (Figure 3a-b). To confirm that the mechanism of increased transfection efficiency was due to the spreading and actin-myosin interactions, we treated the cells with blebbistatin, a small molecule that acts downstream of Y-27632, inhibits non-museie myosin HA, reduces actin-myosin interactions, and alters the intracellular structure and morphology. Treatment of blebbistatin at 10 μΜ also resulted in increased transfection efficiency of DNA/FHD complex up to 17.8 ± 0.4%, indicating a strong correlation between cell structure and transfection efficiency (Figure 3a).

When hESC colonies were treated with Y-27632 for 4 h at varying concentrations (10, 30, and 50 μΜ), significant morphological changes of the cells were observed. In the absence of Y-27632, the cells maintained a two dimensional cobble stone like colony morphology, and the cells were tight and rounded up (Figure 3c). At 10 μΜ, the colonies started to spread and lose their rounded-up structure. At 30 and 50 μΜ, the cells were even more spread out and elongated, indicating that they had relaxed their original structure. The treatments thus increased the surface area and decreased the surface membrane tension.

To further verify the Y-27632-mediated cell spreading, the cytoplasm and nuclei of the Y-27632-treated cells were respectively labelled before cells were imaged and analyzed using the GE InCell Analyzer (Figure 4). Without Y-27632 treatment, the cells were calculated to have a cytoplasmic area of 445 ± 29.8 μιη 2 per cell. When treated with Y- 27632 at 10, 30, and 50 μΜ, the cytoplasmic area per cell increased to 516 ± 8.0, 563 ± 32.7 and 589 ± 18.6 μιη 2 , respectively (Figure 3d). The increased cytoplasmic area thus substantiated the promotion of cell spreading upon Y-27632 treatment. Through further analysis of the cell spreading effect on transfection efficiency, the GFP efficiency was also measured using the GE InCell Analyzer and InCell Workstation (Figure 5). With the increased spreading, the GFP transfection efficiency increases from about 6.3 ± 3.4% at 0 μΜ to 10.9 ± 4.4%, 22.8 ± 0.6% and 30.6 ± 1.7% at 10, 30, and 50 μΜ of Y-27632 treatment, respectively (Figure 3d). A correlation between the cell spreading and GFP transfection upon the treatment of Y-27632 was thus demonstrated through the GE InCell imaging analysis of the hESCs cytoplasmic area and GFP expression.

Sequential treatment of Y-27632 and uptake inhibition study. To study the importance of morphological changes, hESCs were treated with Y-27632 in three different stages, with FHD as the model vector. When the cells were treated with Y-27632 for 4 h and removed before transfection, the transfection efficiency was 28.9 ± 1.8%. When the cells were treated with Y-27632 only during the transfection for 4 h, the transfection efficiency decreased to 25.4 ± 1.9%. Finally, when the cells were treated for 4 h before and during the transfection with Y-27632, the transfection efficiency increased to 31.3 ± 3.6% (Table 1). Y- 27632 therefore promotes cellular internalization of the gene cargo as a result of increased surface area. The increase in transfection when the hESCs were pretreated with Y-27632 indicates the importance of altering the cell morphology before the transfection.

Table 1. EGFP Transfection Efficiency Dependence on the Presence of Y-27632 either before and/or during the Transfection Step. Y-27632 was applied to the cells for 4 h before transfection, during the 48-h transfection period, or a combination thereof.

Pre-incubation (4 h) Transfection (4 h) % GFP Positive

19.6 ± 0.8

50 μΜ 25.4 ± 1 .9

50 μΜ — 28.9 ± 1 .8

50 μΜ 50 μΜ 31 .3 ± 3.6

Uptake study of the YOYO-l -DNA was also conducted at in the presence of various concentrations of Y-27632. In the absence of Y-27632, the fluorescence unit of the

FHD/YOYO- 1 -DNA nanocomplexes in the cells was found to be 56.9 ± 2.1. When the concentration of Y-27632 was increased from 10 to 50 μΜ, the uptake level continued to increase up to 84.4 ± 0.7 fluorescence unit in the case of 50 μΜ Y-27632 treatment, which was consistent with the trend of transfection efficiency enhancement (Figure 6).

Treatment of Y-27632 maintains pluripotency of hESCs. Treatment of hESCs with Y-27632 at the concentration of 50 μΜ significantly increases non- viral transfection efficiency. The treated cells can also revert back to their normal hESC morphology 48 h post-treatment (Figure 3b). This study demonstrates that Y-27632 only transiently alters the hESC morphology to facilitate improved gene delivery.

To further confirm whether the hESCs were only transiently altered by the treatment of Y-27632 and FHD transfection, extracellular stage specific embryonic antigen 4 (SSEA-4) was stained with PE conjugated antibodies and imaged 72 h post transfection with 50 μΜ Y- 27632 treatment (Figure 7a). The cells were stained uniformly positive for SSEA-4, indicating well maintained pluripotency. A western blot assay of the hESCs conducted 4 days post FHD/DNA transfection with Y-27632 also demonstrated unaltered expression of OCT4, a gene indicator of hESC pluripotency. With the treatment of Y-27632 at all concentrations, there was no change in the OCT4 expression. However, when treated with 10, 30, and 50 μΜ of Y-27632 in the presence of FHD, slight reduction in the OCT4 expression level was observed, indicating that Y-27632 itself did not affect pluripotency and that the minor change of OCT4 expression could be induced by the transfection reagent. These experiments substantiated that gene transfection and Y-27632 treatment were transient and that cells were able to revert back to their natural pluripotent state after removal of Y-27632 (Figure 7b). The treatment of hESCs with Y- 27632 also showed no cytotoxicity to the cells at any treatment concentration (Figure 8). The Y-27632 treatment at any of the concentrations for transfection thus demonstrated only transient alteration of the cells with well-retained pluripotency and low cytotoxicity.

Accordingly, we demonstrated that with the treatment of Y-27632, transfection efficiencies of a variety of non-viral gene delivery materials in hESC, including PLR, PLL, PEI, LPF, and FHD, are markedly augmented. Y-27632 allowed the hESC colonies to effectively transform their internal mechanical structure by inhibiting the actin-myosin contractility and cell-to-cell adhesion, thus facilitating spreading of the cells. As such, the exposed surface area was increased and the membrane tension was decreased, ultimately leading to an increase in the internalization level of exogenous genetic materials.

Using one of the most efficient commercial transfection reagents, FHD, we demonstrated a dose dependent effect of Y-27632 small molecule treatment in hESCs. An increase in the Y-27632 concentration from 0 to 50 μΜ correlated to a 1-fold increment in the transfection efficiency. To ensure that the increased transfection efficiency was due to the actin-myosin inhibition effect on the spreading or morphology change of the cells and not the other pathways associated with the rho-associated protein kinase pathway, the cells were also treated with 10 μΜ blebbistatin. Blebbistatin acts downstream of the rho-associated protein kinase and directly inhibits non-muscle myosin IIA. This inhibition directly decreases the affinity of myosin with actin, indicating that the Y-27632's role in the cell morphological change is responsible for the increased gene transfection.

From the treatment of varying concentrations of Y-27632, the morphology of cells changed from a rounded up structure to an elongated flat morphology, and disassociation from the colony was observed. This flattening and spreading of the cells increased the cell surface area that was exposed to exogenous nanocomplexes, and thus the nanocomplexes were more readily taken up by the cells. A higher Y-27632 concentration resulted in enhanced cell spreading and ultimately increased transfection efficiency. The important role of Y-27632 in increasing cell adhesion and survival also helped the cells to maintain physiological functions, thus contributing to the transfection process. In addition, through the spreading and elongation of the cells, the cell membrane likely showed decreased membrane tension, allowing for a higher rate of endocytosis of the nanocomplexes.

It is therefore important that the cells be transiently altered before transfection, such that spreading of the cells and decrease of the membrane tension allow efficient nanocomplex uptake. As described herein for the adding sequence of Y-27632, pre-treated cells showed significantly higher transfection efficiency than post-treated cells, indicating the importance of morphological change prior to the transfection process. The altered morphology primes the cells by increasing the cytoplasmic area and facilitating the uptake of the nanocomplexes during transfection. In consistence with our findings that cells in smaller colonies afforded higher transfection efficiencies (data not shown), it was further demonstrated that Y-27632- mediated cell spreading and larger cell surface area is attributed to the decreased surface tension and declumping of the cell colonies. Furthermore, the use of Y-27632 does not permanently affect the pluripotency and hESC cell state, and the cells recovered to their natural, tight, two-dimensional colony morphology 72 h after removal of Y-27632.

Expression of SSEA-4 and OCT4 were also observed, indicating reversible alteration of cell physiology and maintenance of cell pluripotency.

In conclusion, we studied and adapted a new approach to increase the non-viral gene delivery to hESCs by transiently altering the colony structure to increase their susceptibility for uptake of nanocomplexes. Treatment of hESCs with Y-27632 prior to and during transfections effectively increases cell spreading and decreases cell membrane tension, which increases cell uptake and thus potentiates the gene transfection. The hESCs colonies were able to return to their original morphology and maintain their pluripotency within hours after removal of Y-27632. While most current studies in non-viral gene delivery focus on vector material design, the disclosure herein opens a new window to control gene transfection in hESCs on the cellular side. The invention therefore provides a promising approach to manipulate pluripotent stem cells through transient gene therapy, overcoming a significant hurdle against controlling and studying pluripotent stem cell differentiation and development toward various biomedical applications. With the increase in the gene transfection efficiency, an increase in differentiation efficiency of hESCs can be found, which reduces the need for enrichment and sorting of desired cells.

Definitions

The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries reference volumes such as

The Encyclopedia of ' Molecular Biology ; by Kendrew et al. (eds.), published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, by Robert A. Meyers fed.), published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Hawley 's Condensed Chemical Dictionary 14 th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and/or the recitation of claim elements or use of "negative" limitations.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the end-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio.

Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo. The term contacting therefore includes exposing to and incubating, as described herein.

The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein that provides a desired effect. As used herein, an "effective amount of a ROCK inhibitor" is an amount of a ROCK inhibitor required to inhibit expression of ROCK or inhibit activity of ROCK. For example, when the ROCK inhibitor is a small molecule, an effective amount is the concentration required to partially or completely eliminate ROCK activity, such as its kinase activity. In some embodiments, the ROCK inhibitor is Y-27632. In one embodiment, an effective amount of Y-27632 is at least 1 μΜ. In another embodiment, an effective amount of Y-27632 is at least 5 μΜ. In another embodiment, an effective amount of Y-27632 is at least 10 μΜ. In some embodiments, the effective amount of ROCK inhibitor is about 1 to about 100 μΜ, about 25 to about 60 μΜ, or about 50 μΜ. In another embodiment, the ROCK inhibitor is an antisense compound. An effective amount of an antisense compound specific for ROCK is an amount required to inhibit ROCK mRNA level by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%. An effective amount of a ROCK inhibitor can also refer to the amount required to achieve a particular effect, such as inducement of transient colony spreading.

The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells or activity of an enzyme. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth, progression, or activity that occurs in the absence of the treatment or contacting.

The terms "transfection" and "transformation" refer to the insertion of an exogenous molecule (e.g., a nucleic acid sequence encoding a peptide or protein, or fragment, homolog, or variant thereof), into a host cell, irrespective of the method used for the insertion, the molecular form of the molecule that is inserted, or the nature of the cell. Accordingly, transfection can refer to the introduction of foreign DNA into cells, or the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

Rho -associated kinase mhibitors. Mammalian cells encode two Rho kinases, known as ROCK1 and ROCK2. These kinases are activated by binding to an active GTP-bound Rho GTPase. ROCK phosphorylates substrates on serine or threonine residues. These substrates are involved in a wide range of cell behavior. Rho-associated kinase inhibitors ("ROCK inhibitors") inhibit the activity of these kinases, for example, by binding to the kinase domain to inhibit ROCK enzymatic activity.

In one embodiment, the ROCK inhibitor used in the methods described herein is a small molecule such as Y-27632, blebbistatin, or fasudil. In another embodiment, the ROCK inhibitor is a derivative of Y-27632, blebbistatin, or fasudil. Suitable derivatives of Y-27632 are described in U.S. Patent No. 4,997,834 (Muro et al.) and International Publication No. WO 1998/006433 (Uehata et al). Fasudil, also known as HA 1077, is described by Asano et al. (J. Pharmacol. Exp. Ther. 241 : 1033-1040, 1987). Other small molecules that can inhibit ROCK and that can be used in the methods described herein include H-l 152 ((5)-(+)-2- methyl-l-[(4-methyl-5-isoquinolinyl)sijlfony1]homopiperazine , ikenoya et al., J. Neurochem, 81 :9, 2002; Sasaki et al., Pharmacol. Ther. 93:225, 2002); N-(4-pyridyl)--V-(2,4,6- trichlorophenyl)urea (Takami et al., Bioorg. Med. Chem. 12:2115, 2004); and 3-(4-pyridyl)- lH-indole (Yarrow et al., Chem. Biol. 12:385, 2005). Additional small molecule Rho kinase inhibitors that can be used in the methods described herein include those described in U.S. Patent Nos. 7,199,147 (Imazaki et al.) and 7,217,722 (Tamaki et al.); U.S. Patent Application Publication Nos. 2003/0220357 (Blankston et al), 2005/0182040 (Imazaki et al.)

2005/0197328 (Bailey et al), and 2006/0241127 (Feurer et al); and International Publication Nos. WO 03/059913, WO 03/062225, WO 03/062227, WO 03/064397, WO 04/112719, and WO 05/003101.

Nanoparticles. As used herein, nanoparticles are particles small enough to be taken up by a cell via endocytosis. Typically the nanoparticle has a diameter of less than about one micron, often less than about 500 nm. Suitable nanoparticles can be about 50 nm to about 400 nm, about 50 nm to about 100 nm, about 100 nm to about 500 nm, about 200 nm to about 400 nm, or about 300 nm to about 400 nm. A nanoparticle can be a nanocomplex (a non- viral delivery vector in combination with genetic material or other cargo), e.g., a nanoparticle formed by non-covalent interactions between a polymeric material or delivery vector and genetic material, often by self-assembly. Examples of nanocomplexes include lipoplexes (e.g., DNA or RNA complexed with a lipid-based material such as lipofectamine) and polyplexes (e.g., DNA or RNA complexed with polypeptides and/or other complex forming materials).

Nanoparticles can be formed from delivery vectors such as polypeptide-based delivery systems including polyarginine (PLR), polylysine (PLL), PVBLG-8, or cell penetrating peptides (e.g., HIV-TAT). Lipid-based delivery vectors / systems include lipofectamine systems (2000, 3000, LTX RNAI max), DOTAP/DOTMA/DOPE lipids, Fugene HD/Fugene 9, or X-tremeGene. Other useful polymeric systems include PEI (e.g., JETPEI), polyethylene glycol and PEG-based systems, PLGA-based systems, and poly(P- amino esters) (see J. Green and coworkers, Nano Lett. 2008, 8, (10), 3126-3130; Gene Ther. 2009, 16, (4), 533-546; Proc. Natl. Acad. Sci. USA 2010, 107, (8), 3317-3322). Additional useful nanoparticles-based systems include silica nanoparticles, gold nanoparticles, and quantum dots.

Nanoparticle Cargo. Nanoparticles can carry various types of cargo for delivery to the pluripotent stem cells. Nanoparticle cargo can be genetic material, polypeptides, proteins, drugs, or a combination thereof. Examples of protein cargo include transcription factors, enzymes, nucleases, growth factors, and the like. When the cargo is genetic material, the nanoparticle is typically a nanocomplex of the genetic material and a delivery vector.

Examples of the genetic material include DNA, which encompasses plasmid DNA, DNA minicircles, and oligoDNA, and RNA, which encompasses oligoRNA, mRNA, sgRNA, siRNA, and shRNA. For example, the genetic material can be a ribonucleic acid protein complex such as a Cas9-sgRNA complex. Other examples include protein complexes with various other types of RNA and DNA. In various embodiments, the cargo can include any combination of the cargo described herein, such as the Crispr-Cas9/sgRNA ribonucleic protein complex.

Genetic Material. The enhanced delivery of nanoparticles to pluripotent stem cells as described herein can include the delivery of nanoparticles that include various amounts of genetic material. The phrases "genetic information" and "genetic material" refer to materials found in the nucleus, mitochondria and /or cytoplasm of a cell, which play a fundamental role in determining the structure and nature of cell substances. Genetic material can be a gene, a part of a gene, a group of genes, DNA, RNA, nucleic acid, a nucleic acid fragment, a nucleotide sequence, a polynucleotide, a DNA sequence, a group of DNA molecules, double- stranded RNA (dsRNA), small interfering RNA or small inhibitory RNA (siRNA), microRNA (miRNA), or the genome of an organism. The genetic material can be, for example, any nucleic acid molecule suitable to provide desired coding information to a cell. The term "genetic material" is intended to encompass any DNA or RNA molecule that has basic research or therapeutic use.

The genetic material used in the nanoparticles described herein may be of eukaryotic, prokaryotic, fungal, archaeal or viral origin. The genetic material can be naturally occurring, mutant, or synthetic. The genetic material can include isolated or substantially purified nucleic acids. Naturally occurring nucleotide sequences can be amplified, for example, by polymerase chain reaction (PCR), to obtain suitable quantities for use in the nanoparticles described herein.

The degree of incorporation of genetic material in the nanoparticles can be determined by techniques known in the art including, for example, fluorescence studies, DNA mobility studies, etc., and will vary depending upon desired use. See for example, the techniques described by Hwang and coworkers (Bioconjugate Chem. 2001; 12(2):280-90) and by Liu and coworkers (J. Am. Chem. Soc. 2004; 126(24):7422-23).

The following paragraphs provide further definitions of genetic material that can be incorporated into the nanoparticles described herein (e.g., to form a nanocomplex).

The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al, Nucl. Acids Res., 19:508 (1991); Ohtsuka et al, J. Biol. Chem., 260:2605 (1985); Rossolini et al, Mol. Cell. Probes, 8:91 (1994).

Deoxyribonucleic acid (DNA) in the majority of organisms defines the genetic information while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid", "nucleic acid molecule", "nucleic acid fragment", "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA or RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA. As would be recognized by one of skill in the art, a "nucleic acid fragment" is a portion of a given nucleic acid molecule.

"Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA. The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complimentary copy of the DNA sequence, it is referred to as the primary transcript or it may be an RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated.

The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, a gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

The genetic material can be chimeric DNA. The term "chimeric" refers to any gene or DNA that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, DNA that is either heterologous or homologous to the DNA of a particular cell to be transformed.

Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer. The particles described herein can be used to deliver transgenes to a cell.

A "recombinant DNA" molecule is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook and Russell, Molecular Cloning: A Lab Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (3 rd Ed., 2001).

A "vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self- transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

"Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.

An "expression cassette" refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest, which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. The term "siRNA" (short interfering RNA) has the same meaning as typically in the art, i.e., the term refers to short double stranded RNA complex, typically 19-28 base pairs in length. In other words, siRNA is a is double-stranded nucleic acid molecule comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The complex often includes a 3'-overhang. SiRNA can be made using techniques known to one skilled in the art and a wide variety of siRNA is commercially available from suppliers such as Integrated DNA Technologies, Inc.

(Coralville, IA).

"Operably-linked" refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

A "coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an

"uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES

Example 1. Enhanced Non- Viral Gene Delivery to Human Embryonic Stem Cells

Non-viral cargo delivery into human embryonic stem cells (hESCs) is an important tool for controlling cell fate. However, the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. Described herein are methods to enhance non-viral nanoparticle delivery to hESCs by transiently altering the cell and colony structure. The compound (R)-(+)-trans-A-(\- aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632), a rho-associated protein kinase inhibitor, is utilized to induce transient colony spreading, which leads to increased transfection efficiency by 1.5 to 2 folds in conjunction with a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y- 27632 post-transfection, cells can revert back to their normal state and do not show alteration of pluripotency. This approach provides a simple, effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation, as discussed above. Specific techniques of the methods and corresponding analyses are described below.

MATERIALS AND METHODS

General. Human embryonic stem cell line HI (hESC-Hl) was cultured in mTeSR 1 medium from Stem Cell Technologies (Vancouver, Canada). The commercial non-viral transfection reagent Fugene HD (FHD) was purchased from Promega (Madison, WI, USA). Y-27632 was purchased from Stemgent (Cambridge, MA, USA). Opti-MEM, Lipofectamine 2000 (LPF), and YOYO- 1 were purchased from Invitrogen (Carlsbad, CA, USA). pEGFP- Nl was obtained from Elim Biopharmaceuticals (Hayward, CA, USA). Milli-Mark™ Anti- SSEA-4-PE was purchased from EMD Millipore (Billerica, MA, USA). PLL, PLR, and PEI were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Instrumentation. Flow cytometry analysis was conducted on a BD LSRII color flow cytometer analyzer (Becton Dickinson, Franklin Lakes, NJ). Cells were visualized with a Zeiss Axiovert 40 CFL fluorescence microscope equipped with a lOx and 20x objective (Thornwood, NY). Fluorescence imaging was performed using the GE InCell Analyser 2000 from GE Healthcare Sciences (Piscataway, NJ, USA). Zeta potential and size analysis were conducted on a Malvern Zetasizer (Worcestershire, UK).

Preparation and characterization of nanocomplexes. Various non-viral vectors were used for the transfection analyses. Plasmid DNA (1 μί, 1 mg/mL) was diluted with water (50 μΚ). The transfection reagent, polyarginine (10 μί, 1 mg/mL), polylysine (10 μΐ,, 1 mg/mL), or polyethylenimine (5 μΐ ^ , 1 mg/mL) was diluted with water (50 μί). The two solutions were then vortexed gently and allowed to incubate for 20 min at rt (~22 °C), after which they were combined and allowed to incubate for another 20 min at rt.

Plasmid DNA (1 μΐ ^ , 1 mg/mL) was diluted with mTeSR (50 μΚ). The transfection reagent, LPF (2 μΐ,, 1 mg/mL) was diluted with mTeSR (50 μί). The two solutions were then vortexed gently and allowed to incubate for 20 min at rt, after which they were combined and allowed to incubate for another 20 min at rt. Various non- viral vectors were used for the transfection analyses. Plasmid DNA (1 μΐ,, 1 mg/mL) was diluted with mTeSR (50 μΚ). The transfection reagent, FHD (3.5 μί, 1 mg/mL) was added to the DNA solution. The solution was then vortexed gently and allowed to incubate for 20 min at rt, after which the solutions were combined and allowed to incubate for another 20 min at rt.

Solutions of plasmid DNA (5-25 μg/mL) were prepared in water or medium.

Separately, a solution of 50 μg of the vectors was prepared in 400 μΐ, water. A solution of 50 μϊ ^ LPF and FHD in 400 μϊ ^ media was also prepared as a control. The DNA solution was then mixed with the vectors, LPF, or FHD solution and allowed to incubate at room temperature for 20 min. Dynamic light scattering (DLS) and zeta potential analysis were conducted on the samples with a Malvern Zetasizer.

hESC transfection with Y-27632. hESCs were seeded on Matrigel-coated 24-well plates and cultured in mTeSR medium for 24 h. For the full treatment group, Y-27632 was added into the culture medium at a final concentration of 0, 10, 30 or 50 μΜ for 4 h prior to the transfection. Nanocomplexes prepared from pEGFP-Nl plasmid, different transfection vectors were added dropwise into the media, and cells were cultured at 37°C for 4 h. For the pre-treatment group, cells were treated with Y-27632 (50 μΜ) for 4 h at 37 °C prior to transfection. The cells were then washed with PBS and fresh media was added prior to the addition of the nanocomplex and further incubation at 37 °C for 4 h. For the post-treatment group, cells were treated with Y-27632-free medium for 4 h prior to transfection. Directly prior to transfection, the medium was replaced with Y-27632 (50 μΜ) containing medium, and cells were transfected with various vectors at 37 °C for 4 h. In all three cases, 4 h after treatment with the nanocomplexes, the media was replaced with fresh mTeSR and cultured for 48 h before measurement of the transfection efficiency by flow cytometry. Alternatively, 10 μΜ of blebbistatin, a non-muscle myosin IIA inhibitor, downstream of the rho-associated protein kinase, was added to the cells prior to and during transfection.

Intracellular uptake studies. DNA (1 mg/mL) was labeled with YOYO-1 (20 μΜ) at one dye molecule per 50 bp of DNA (see Gabrielson et al, Biomaterials 2010, 31, (34), 9117-9127), and was allowed to form nanocomplexes with various transfection reagents as described above. hESCs were plated on Matrigel-coated 24-well plate and cultured until they reached medium (50 cells) sized colonies. Nanocomplexes were added at 1 μg YOYO-1- DNA/well and cells were incubated at 37 °C for 4 h. Cells were then harvested, re-suspended in PBS, and subjected to flow cytometry analysis to quantify the cellular uptake level of YOYO-1 -DNA. To elucidate the mechanisms underlying the cellular internalization of FHD/DNA nanocomplexes, we performed the uptake study at 4 °C or in the presence of various endocytic inhibitors. Cells were pre-treated with chlorpromazine (10 μg/mL), genistein (200 μg/mL), methyl- -cyclodextrin (mpCD, 50 μΜ), dynasore (80 μΜ), or wortmannin (50 nM) for 30 min before addition of the nanocomplexes and throughout the uptake study at 37 °C for 2 h. Results were expressed as percentage uptake level of control cells that were treated with the nanocomplexes at 37 °C for 2 h in the presence of Y-27632 (50 μΜ) while in the absence of endocytic inhibitors.

hESC spreading and transfection analysis. hESC HI were plated on 96-well plates as medium sized colonies coated with Matrigel and cultured for 24 h. Y-27632 was added at various concentrations (0, 10, 20, and 50 μΜ) and after 4 h incubation, the cell nuclei were stained with Hoechst 33258 and the cytoplasm was stained with CellTracker Red CMTPX per manufacturer's protocols. Forty five fields were imaged using the GE InCell Analyzer 2000 in the Hoechst and Texas Red channel (Figure 4). The cytoplasmic area of the cells in each field was quantified using the GE analysis software. The cell spreading level was expressed as the total calculated cytoplasmic area normalized by the number of nucleus counted in cytoplasmic area, as further described below.

To prepare the images of Figure 4, hESCs were seeded on Matrigel-coated 96-well plates and cultured in mTeSR medium for 24 h. Y-27632 was added into the culture medium at a final concentration of 0, 10, 30 or 50 μΜ, and incubated at 37°C for 4 h. After 4 h of treatment, the cells were stained with CMTPX Cell Tracker dye and Hoechst according to the manufacturer's instructions. Five wells were imaged with 9 fields each using the GE InCell Analyzer 2000. The images were then analyzed using the GE InCell Analyzer workstation. The nuclei were identified using top-hat segmentation in the Hoechst channel, and the cell area was analyzed and calculated through the analysis in the CMPTX channel using multiscale top-hat. The cytoplasmic area was then normalized to the number of nucleus counted.

Images of the transfection of hESCs after alteration of the cytoplasm area with the treatment of Y-27632. hESC HI were seeded on Matrigel-coated 24-well plates and cultured in mTeSR medium for 24 h. Before transfection, Y-27632 was added into the culture medium at the final concentration of 0, 10, 30 or 50 μΜ, and incubated at 37 °C for 4 h. Nanoparticles from pEGFP- l and FHD were added dropwise into the culture medium and cells were further incubated at 37 °C for 4 h. The medium was then replaced with fresh mTeSR medium, and cells were further cultured for 48 h. After 48 h, the cell nuclei were stained with Hoechst 33258. Forty five fields were imaged using the GE InCell Analyser 2000 in the Hoechst and GFP channel (Figure 5). The transfection efficiency was then analyzed using the GE InCell Workstation by determining the ratio of GFP positive cells to the number of Hoechst stained nuclei, as further described below.

To prepare the images of Figure 5, hESCs were seeded on Matrigel-coated 24-well plates and cultured in mTeSR medium for 24 h. Before transfection, Y-27632 was added into the culture medium at a final concentration of 0, 10, 30 or 50 μΜ, and incubated at 37 °C for 4 h. Nanoparticles from pEGFP- l and FHD were added dropwise into the culture medium and cells were further incubated at 37°C for 4 h. The medium was then replaced with fresh mTeSR medium, and cells were further cultured for 48 h. The hESCs were then stained with Hoechst and imaged using the GE InCell Analyzer. Using the InCell Analyzer Workstation, the nuclei were identified using top-hat segmentation in the Hoechst channel and the numbers of cells were then calculated. The identified nuclei were then further segmented by multiscale top-hat in the GFP signal to determine the number of cells with positive GFP signals. The transfection efficiency was then determined by dividing the number of GFP positive cells by the total number of cells.

Sample preparation and flow cytometry analysis. Prior to analysis by flow cytometry, transfected cells on the 24-well plate were washed with PBS (3 x 500 μί) to remove any residual serum, dead cells, and debris. Accutase (100 μί) was added to detach the cells from the plate, and PBS (100 μί) was then added to re-suspend the cells. An aqueous solution of paraformaldehyde (4%, 100 μί) was added to fix the cells, which were then subjected to flow cytometry analysis.

Western blot analysis and SSEA-4 staining. After 72 h, the cells were stained with DAPI (250 μΐ, 3 nM) and SSEA-4-PE (250 μΐ, 0.02 mg/mL), a pluripotency cell marker, for 30 min at 37 °C. The cells were images using the GE InCell Analyser 2000. After 4 d treatment of 50 μΜ Y-27632 and FHD transfection, the cells were lysed with the RIPA buffer, mixed with Laemmli buffer supplemented with 2-mercaptoethanol, and heated at 100 °C for 5 min to denature the proteins. After being cooled in ice, the samples were subjected to electrophoresis on a 10% SDS PAGE Gel at 120 V for 1.5 h, and wet transferred to the nitrocellulose membrane using the AMRESCO Rapid Western Blot Kit per manufacturer's instructions. The membrane was stained with OCT4 and a-Tubulin primary antibodies and then with HRP -tagged secondary.

Cell Cytotoxicity of Y-27632 Treatment with FHD Transfection. For the MTT assays cytotoxicity assessment, 1 confluent 6-well well of hESCs was plated onto a Matrigel- coated 48-well plate one day before transfection. The cells were then pre-treated for 4 h with 0, 10, 30, and 50 μΜ of Y-27632 for 4 h and transfected with FHD as previously described above. The cells were further incubated for 4 h at 37 °C in the transfection mix before removal of the transfection reagent and being returned to fresh growth media. After 48 h, the cell viability was monitored by the MTT assay. Cell viability was represented as percentage viability of control cells that did not receive any transfection treatment (Figure 8). For the MTT assay, the cells were washed with PBS and MTT solution was added. Following 4-h incubation at 37 °C, MTT solubilization solution (10% Triton X-100 in acidic (0.1M HC1) isopropanol) was added to the cells and the absorbance of 570 nm light was quantified on a Perkins Elmer plate reader (Waltham, MA, USA).

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.