Histone-mediated Transduction as an Efficient Means for Gene Delivery

original article

& The American Society of Gene Therapy

Histone-mediated Transduction as an Efficient Means for Gene Delivery Kylie M Wagstaff1, Dominic J Glover1, David J Tremethick2 and David A Jans1,3 1 Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; 2Chromatin Transcription and Regulation Laboratory, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia; 3ARC Centre of Excellence for Biotechnology and Development, Clayton, Victoria, Australia

Gene delivery into the nucleus of eukaryotic cells is inefficient, largely because of the significant barriers within the target cell of the plasma membrane and nuclear envelope. Recently, a group of basic proteins, including the HIV-1 Tat protein and the four core histones, have been shown to enter cells through a novel energy- and receptor-independent manner. Here, we show that engineered histone H2B proteins are able to mediate the efficient delivery of either green fluorescent protein or DNA into HeLa cells through the process of ‘‘Histone-Mediated Transduction’’ (HMT), with further enhancement achieved by utilizing a dimer of histones H2B and H2A. Subsequent nuclear delivery was accelerated approximately two-fold by the addition of an optimized nuclear localization signal to histone H2B, thereby increasing the affinity of interaction with components of the cellular nuclear import machinery, resulting in increased expression of a reporter gene. Further, we demonstrate that the domains responsible for this histone transduction are located in the N-terminal tail and globular regions of histone H2B. HMT represents a new, efficient, and technically non-demanding means to deliver DNA to the nucleus of intact cells, including embryonic stem cells, which has important applications in gene therapy and cancer therapeutics. Received 16 August 2006; accepted 29 November 2006; advance online publication 27 February 2007. doi:10.1038/mt.sj.6300093

INTRODUCTION Efficient delivery of therapeutic DNA through the plasma membrane and into the double-membrane-bound nucleus of eukaryotic cells represents some of the most important hurdles to gene therapy. Although viral vectors have evolved to include mechanisms to help overcome these, safety concerns, especially in recent years have hampered their use.1 Non-viral vectors, on the other hand, are relatively safe to use, but are much more inefficient, as they must be engineered to include mechanisms/moieties to bypass the natural barriers of the cell.2 In the last decade, a novel class of protein has been identified that is capable of penetrating the cellular membrane of intact

cells in an apparently energy- and receptor-independent manner,3–5 with obvious potential to enhance gene delivery. The cellular transduction of these proteins is attributable to distinct, usually basic or amphipathic a-helical regions, 20–30 amino acids in length known as protein transduction domains (PTDs).3,4 The first PTD containing protein initially reported was the trans-activating transcriptional activator (Tat) protein from human immunodeficiency virus 1 (HIV-1),6,7 although current understanding of the mechanism of cell entry of this protein tends to favor endocytosis (for reviews see refs. 3, 4). Despite this, the PTD containing protein group has rapidly expanded to include several classes of both cellular and viral proteins.3 Importantly, although the PTDs responsible have not been defined, all four core histone proteins have been shown to possess protein transduction ability.8,9 Transport into the nucleus of molecules 445 kDa generally requires specific targeting signals, called nuclear localization signals (NLSs), typically mono- or bipartite clusters of basic amino acids (see refs. 10, 11). The NLS is recognized by members of the importin (Imp) superfamily of proteins,12 and, in particular, either by the Imp a component of the Imp a/b heterodimer, as in the case of the classical NLS from the simian virus 40 large tumor antigen (T-ag),13 or by Imp b1 or homologs thereof directly, as is the case for the core histones.14,15 In this study, we examine the ability of core histone H2B derivatives to mediate the delivery of green fluorescent protein (GFP) or DNA into intact cells in tissue culture, either as a monomeric protein or in heterodimeric form with histone H2A. We demonstrate that the engineered H2B proteins used in this study efficiently deliver DNA to the nuclei of intact cells, a process that is significantly enhanced by the addition of the optimized T-ag NLS to the C-terminus. The significant level of transfection achieved using this Histone-Mediated Transduction (HMT) highlights the potential of engineered histones as vehicles for efficient, safe, and cost-effective non-viral gene delivery.

RESULTS Engineered histone H2B proteins Owing to their essential role in DNA binding and compaction, core histone proteins contain specific NLSs that mediate their active transport into the nucleus.16 Although some debate exists

Correspondence: Professor David A Jans, Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: [email protected]

Molecular Therapy vol. 15 no. 4, 721–731 april 2007

721

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

over the precise details of the import pathway,15,17–19 the general consensus is that Imp b family members play a central role.14,15,17 For this study, we generated several engineered histone H2B fusion proteins, all of which contain a GFP moiety fused to the N-terminus of H2B. An additional optimized NLS from the SV40 large tumor antigen (T-ag NLS), recognized by Imp a/b with high affinity,20–22 was also fused to the C-terminus of H2B to generate the H2B-NLS construct. For purification purposes, protein constructs also contained a (His)6 tag at the N-terminus. A schematic representation of the engineered histones is provided in Figure 1a.

had not altered the ability of H2B to localize to the nucleus of mammalian cells (Figure 1b). The GFP control protein exhibited diffuse localization in both the cytoplasm and nucleus, consistent with its lack of an NLS and size (B27 kDa), which is below the exclusion limit for the nuclear pore complex (NPC). In contrast, each of the H2B proteins as well as the T-ag NLS containing control protein (GFP-NLS) localized predominantly to the nucleus with little to no cytoplasmic localization observed. Similar results were obtained using HTC or HeLa cells, indicating that there are no apparent cell/species-specific effects on the localization. Image analysis of the digitized confocal images was performed to determine the ratio of nuclear to cytoplasmic fluorescence (Figure 1c), revealing that the addition of the T-ag NLS to the H2B protein resulted in approximately two-fold enhanced (Po0.02) localization of the protein in the nucleus (Fn/c of 110–150) compared to that in its absence

Nuclear localization of engineered histone H2B proteins is enhanced by the addition of the T-ag NLS Cos-7 cells were transfected with plasmids encoding each of the engineered histone proteins to confirm that the modifications

a

1 His

GFP

c

125

P < 0.02

180

H2B

160 1 His

GFP

125 H2B

Cos-7 cells HTC cells HeLa cells

140

NLS 111 135

120

b

GFP

Fn/c

100

His

NLS 111 135

80 60

GFP-H2B GFP-H2B-NLS GFP-NLS

GFP

40 20 0 GFP

d

GFP-H2B

− − −

Imp  Imp  Imp /

e

GFP-H2B-NLS

− + −

+ − −

− − −

− − +

15,000 10,000

+ Imp / Kd = 3.0 nM

5000

+ Imp  Kd : ND

5

10 15 20 Imp (nM)

25

30

AlphaScreen counts

AlphaScreen counts

+ Imp  Kd = 6.2 nM

20,000

0

− + −

+ Imp / + Imp  + Imp 

+ − −

− − +

GFP-H2B-NLS

GFP-H2B 30,000 25,000

GFP-H2B GFP-H2B-NLS GFP-NLS

20,000

+ Imp / Kd = 3.8 nM

15,000

+ Imp  Kd = 6.0 nM

10,000

+ Imp  Kd = 2.6 nM

5000 0

5

10 15 20 Imp (nM)

25

30

Figure 1 Addition of an optimized NLS enhances the nuclear localization of histone H2B through binding to Importin a/b. (a) Schematic representation of the engineered histone H2B proteins designed for this study. Constructs for bacterial expression and purification contain an N-terminal (His)6 tag to allow for affinity chromatography purification, whereas the mammalian cell expression constructs (not shown) are identical but lack this tag. Residue numbers are as indicated. NLS, optimized T-ag NLS; His, (His)6 tag. (b) Typical CLSM images of live Cos-7 cells 24 h posttransfection to express the indicated GFP-fusion proteins. (c) Results of quantitative image analysis of images such as those in b, used to determine the ratio of nuclear to cytoplasmic fluorescence (Fn/c). Results shown are mean7SEM (n469) from a single typical experiment from a total of two separate experiments. P-values (Student’s t-test, two-tailed) indicate significant differences between constructs; no significant differences between cell lines were observed. (d) Native PAGE gel mobility shift assay. Bacterially expressed and purifed engineered histone GFP-H2B and GFP-H2B-NLS were incubated in the presence of the indicated Imp proteins (10 mM) and then subjected to native PAGE and the fluorescent proteins visualized by fluorimaging. (e) ALPHAScreen performed in triplicate as described in Materials and Methods (see also ref. 42). Thirty nanomolar of GFP-H2B (left panel) or GFP-H2B-NLS (right panel) was incubated with increasing amounts of the indicated Imps to determine the binding affinities. Values in the ‘‘hooking zone’’ (see Materials and Methods) were excluded before standard three-parameter sigmoidal curves were fitted. ND, not able to be determined. Results are from a single typical experiment, from a series of at least two separate experiments.

722

www.moleculartherapy.org vol. 15 no. 4, april 2007

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

(Fn/c of 50–70). This enhancement is most probably due to a ‘‘switching’’ of the nuclear import pathway of the engineered histone H2B from Imp b to Imp a/b mediated, as has previously been documented for GAL4 fusion proteins.23 This was confirmed using a native PAGE gel mobility shift assay (Figure 1d), whereby the GFP-tagged histones were preincubated with different Imps. The electrophoretic mobility of GFP-H2B was impeded in the presence of both Imp b and Imp a/b, but not Imp a alone, implying that the latter effect is most likely through binding to Imp b within the Imp a/b heterodimer. In contrast, GFP-H2B-NLS showed altered mobility in the presence of both Imp a and Imp a/b, indicating that the nuclear import of this engineered histone is likely to be mediated by the Imp a/b heterodimer rather than Imp b alone. Utilizing our recently established ALPHAScreen assay, binding constants (Kd) for the affinity of the interaction between the engineered histones and the various Imps could be estimated. Results indicated that whereas GFP-H2B is recognized by Imp b with high affinity (Kd of 6.2 nM, Bmax of 24040; Figure 1e), GFP-H2B-NLS is recognized with high affinity by Imp a/b (Kd of 3.8 nM, Bmax of 17792), consistent with the results obtained using gel mobility shift assays (Figure 1d). Binding of Imp b to

d

*

Anisotropy

0.345

AlphaScreen counts

0.350

**

**

0.340 0.335 0.330 0.325

GFP-H2B/H2A + Imp  Kd = 4.9 nM

30,000 25,000 20,000 15,000 10,000 5000

+ Imp / Kd = 0.4 nM

+ Imp  Kd : ND

0

5

10 15 20 Imp (nM)

25

− − −

+ − +

AlphaScreen counts

AlphaScreen counts

+ − −

+ − −

+ + −

15,000

+ Imp  Kd= 3.4 nM

10,000

+ Imp  Kd: ND

5000

35,000

GFP-H2B-NLS/H2A

60,000

+ Imp / Kd= 4.8 nM

50,000

+ Imp  Kd= 4.4 nM

40,000 30,000 20,000

+ Imp  Kd= 6.5 nM

10,000

30

0

5

10 15 20 Imp (nM)

25

30

AlphaScreen counts

H2A − Imp / − Imp  −

b 35,000

20,000

+ Imp / Kd: ND

5

GFP-H2B-NLS

GFP-H2B

20,000

25,000

0

0.320

GFP-H2B + DNA

30,000

AlphaScreen counts

**

0.355

Engineered histone H2B containing dimers can bind to both Imps and DNA with high affinity Histone H2B naturally dimerizes with H2A; that this activity has not been oblated by the addition of either the GFP or the T-ag NLS moieties was investigated by performing fluorescence polarization (Figure 2a) measurements. Fluorescence polarization is a biophysical method that detects changes in anisotropy upon binding of proteins or other molecules to a fluorophore in solution. Extremely significant changes in anisotropy were observed for both engineered histone proteins in the presence of H2A as compared with those in its absence (Po0.001; Figure 2a), indicative of the fact that the fluorescent protein complex is much larger than the monomeric GFP-H2B proteins alone, signifying dimerization of the H2B proteins with H2A. A further significant (Po0.05) change in anisotropy was observed when Imps were added to the dimer (Imp b to GFP-H2B and Imp a/b to GFP-H2B-NLS), indicating that the engineered histonecontaining dimers retain the ability to be recognized by Imps.

10

15 20 Imp (nM)

25

30

GFP-H2B/H2A + DNA

25,000

+ Imp  Kd= 4.1 nM

20,000 15,000 10,000

+ Imp / Kd = 1.7 nM

5000

+ Imp  Kd : ND

0

5

10 15 20 Imp (nM)

c

25

30

DNA

H2A

−

+ GFP + GFP-H2B + GFP-H2B-NLS

−

+

−

+

−

AlphaScreen counts

Unbound DNA

+ Imp  Kd = 5.2 nM + Imp  Kd = 5.4 nM

5000

5

10

15 20 Imp (nM)

25

30

GFP-H2B-NLS/H2A + DNA + Imp / Kd = 5.1 nM

50,000 40,000 30,000

+ Imp  Kd = 1.3 nM

20,000

+ Imp  Kd = 3.4 nM

10,000 0

5

10

15 20 Imp (nM)

25

30

GFP-NLS

120,000

Histone/DNA complex

+ Imp / Kd = 4.5 nM

10,000

60,000

30,000

GFP-H2B-NLS + DNA

15,000

0

AlphaScreen counts

a

GFP-H2B-NLS is apparent in this more sensitive assay (Kd of 6.0 nM, Bmax of 16651), but reduced and of lower affinity as compared with that of Imp a/b.

+ DNA Kd = 10.0 nM

100,000 80,000

–DNA Kd = 9.0 nM

60,000 40,000 20,000 0

10

20 Imp / (nM)

30

Figure 2 Engineered histone H2B monomers and dimers can bind to both Imps and DNA with high affinity. (a) Engineered histone H2B proteins were analyzed by FP as monomers or dimers to determine the average anisotropy measurement (n ¼ 5) in the presence or absence of Imps as indicated. Results are from a single typical experiment from a total of two separate experiments. P-values, determined using the two-tailed Student’s t-test, are indicated; *Po0.05; **Po0.001. (b) ALPHAScreen assay performed as in the legend to Figure 1e to determine the binding affinity of the indicated engineered histone-containing dimers to Imps. (c) Two micrograms of linearized DNA (pUC18) was incubated with the indicated GFP fusion proteins and subjected to agarose gel electrophoresis. The gel was stained with EtBr and imaged using a UV transilluminator. Shifted bands are indicated by the arrows. (d) ALPHAScreen assay performed as in the legend to Figure 1e to determine the binding affinity of Imps to engineered histone containing monomers (upper panels) or dimers (middle panels), when prebound to DNA. Binding of the non-DNA binding GFP-NLS control to Imp a/b in either the presence or absence of DNA is presented as a negative control (lower panel).

Molecular Therapy vol. 15 no. 4, april 2007

723

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

engineered H2B proteins readily transduced 100% of the HeLa cells, as indicated by GFP fluorescence in both the cytoplasm and nucleus (Figure 3a, left panels). Neither GFP nor the GFP-NLS control protein was able to translocate into the HeLa cells either in the presence or absence of H2A, implying that the transduction ability of the engineered H2B proteins is specific to the H2B moiety. Image analysis was performed on the digitized CLSM images to determine the specific cytoplasmic fluorescence, results confirming that GFP-H2B transduced efficiently into the cells (Figure 3b), with translocation significantly enhanced by the addition of H2A (Po0.0001). Similar results were also seen at 41C (Figure 3d and e). Addition of H2A to GFP-H2B-NLS resulted in a decrease in the specific cytoplasmic fluorescence, but determination of the ratio of nuclear to cytoplasmic fluorescence (Fn/c) (Figure 3c) indicated that significantly more of the protein was localized in the nucleus compared to the cytoplasm (Po0.0001), accounting for the apparently lower specific cytoplasmic fluorescence. The Fn/c values also indicate that the NLS-containing protein is significantly more nuclear than GFP-H2B either in the presence or absence of H2A (Po0.0001), confirming functionality of the NLS. Similar results were observed when the transduction experiments were carried out at 41C (Figure 3d and e), indicating that the proteins were not internalized via endocytosis. The only distinguishable difference was that at 41C, nuclear transport was inhibited, resulting in a mainly cytoplasmic distribution of the internalized proteins and a slightly higher specific cytoplasmic fluorescence value, especially in the case of GFP-H2B-NLS þ H2A, which was artifactually low at 371C as already mentioned. The time course of uptake was examined for GFP-H2B and GFP-H2B-NLS in the presence and absence of H2A. Intact HeLa cells were surrounded by GFP-protein containing media and monitored over time by CLSM (Figure 3f). Using this method, fluorescent protein can be seen to move from the surrounding medium into the cells over a period of several minutes (Figure 3f, upper panels show GFP-H2B-NLS as an example). Image analysis of the digitized images (Figure 3f, lower left and middle panels) revealed that addition of H2A to GFP-H2B resulted in an increase in the transduction kinetics (from t1/2 ¼ 28.6 to 4.4 min). A smaller increase was seen for GFP-H2B-NLS in the

ALPHAScreen assays were used to quantitate the binding affinity, with results indicating that the GFP-H2B/H2A dimer binds to Imp b with high affinity (Kd of 4.9 nM, Bmax of 29122; Figure 2b), whereas the GFP-H2B-NLS/H2A dimer, as expected, binds to both Imp a/b and Imp b with high affinity (Kd of 4.8 and 6.5 nM, Bmax of 54696 and 30325, respectively). These complexes showed a higher Bmax than their monomeric counterparts (compare Figure 2b with Figure 1e; Table 1), which indicates that, most likely, there is more than one Imp binding site available in the histone dimer, consistent with each of the histones within the heterodimer retaining a functional NLS. The significant Imp binding and nuclear targeting activity of histones, coupled with their DNA binding ability, makes them interesting prospects for use as gene delivery vehicles, provided, of course, that these abilities are not mutually exclusive. To test this, a gel mobility shift assay was performed. The electrophoretic mobility of the DNA (linearized pUC18 plasmid) was altered in the presence of either engineered histone H2B protein, in the presence or absence of H2A, albeit to a lesser extent in the case of the monomers (Figure 2c). No change was seen for the control GFP protein, indicating that the observed DNA mobility shift was due to binding by the histone proteins and not merely an artifact of coelectrophoresed protein in the wells. The ALPHAScreen assay was used to investigate the ability of the histones to bind both Imps and DNA simultaneously. Results (Figure 2d; Table 1) indicated that in the presence of DNA, the engineered H2B proteins still bound to Imps with high affinity in both monomeric and heterodimeric forms as indicated (Kdo6 nM in all cases). The H2B proteins bind to Imps with slightly lower affinity when in the presence of DNA than in its absence (compare Figure 2d with Figures 2b and Figure 1e; Table 1), which is further indication of DNA binding on the part of the histone, but this remains a high-affinity interaction.

Engineered histones can transduce intact HeLa cells To determine if the engineered histone proteins maintain their ability to translocate into intact cells, HeLa cells were incubated in medium containing bacterially expressed and purified proteins. After removal of excess protein by washing, confocal laser scanning microscopy (CLSM) revealed that each of the

Table 1 Summary of binding parameters for engineered histone monomers and dimers from ALPHAScreen based assay DNA Imp a

+DNA

Imp b

Imp a/b

Imp a

Imp b

Imp a/b

Kd

Bmax

Kd

Bmax

Kd

Bmax

Kd

Bmax

Kd

Bmax

Kd

Bmax

ND

23417144

6.370.1

2705674265

2.970.2

826171785

ND

21967239

3.9270.72

119707419

ND

207071457

GFP-H2B-NLS

2.070.9

104887188

5.371.1

168017212

3.071.2

1925072061

6.371.6

74277383

6.070.8

4.470.2

1317272898

GFP-H2B/H2A

ND

2439711

6.772.5

3045471883

0.570.2

922272257

ND

2050774

4.971.1

200527153

1.270.7

3656783

5.872.0

3705471679

6.270.5

3432275653

4.171.0

5389271138

0.970.6

73937553

2.970.8

46507274

7.273.0

4389272714

9.670.9

915607342

10.170.1

9371077560

GFP-H2B

GFP-H2BNLS/H2A GFP-NLS

521371323

Results are for the mean7SD (n=2) for ALPHAScreen analysis as in Figure 1e. Kd, binding coefficient; Bmax, maximal binding; ND, not able to be determined due to low binding.

724

www.moleculartherapy.org vol. 15 no. 4, april 2007

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

increased nuclear uptake kinetic compared with GFP-H2B (t1/2 ¼ 23.7 and 36.9 min respectively; Figure 3f, lower right panel), demonstrating that the addition of the T-ag NLS has also significantly enhanced nuclear uptake of the protein. We tested several cell lines including human breast cancer MCF-7 cells and rat hepatoma HTC cells (data not shown) with similar results as those seen in HeLa cells. Importantly, we also

presence of H2A, compared with that in its absence, but again significantly more protein was seen in the nuclei of these cells. Interestingly, GFP-H2B-NLS on its own had a kinetic profile similar to that of GFP-H2B in the presence of H2A, in direct accord with the previous transduction results (Figure 3b), indicating that the addition of the T-ag NLS increases the transduction ability of H2B. GFP-H2B-NLS also had an

a

GFP

GFP

GFP-H2B

GFP-H2B

GFP-H2B-NLS

GFP-H2B-NLS

GFP-NLS

GFP-NLS

c P < 0.0001

18

e

P < 0.0001

1.6

Specific cytoplasmic fluorescence

P < 0.0001

20

1.4

16

1.2

14

P < 0.0001

1.0

12 Fn/c

Specific cytoplasmic fluorescence

b

+H2A

d

+H2A

10 8

0.8 0.6

6

0.4

4 0.2

2

0

0 GFP-H2B GFP-H2B-NLS GFP-NLS

GFP

H2A −

+

−

+

−

+

−

+

25

20

15

10

5

0 GFP-H2B GFP-H2B-NLS GFP-NLS

GFP

H2 A −

+

−

+

−

+

−

+

GFP-H2B GFP-H2B-NLS GFP-NLS

GFP

H2 A −

+

−

+

−

+

−

+

Figure 3 Engineered histone H2B monomers and dimers can enter intact HeLa cells by transduction. (a) Intact HeLa cells were incubated for 1 h in the presence of the indicated bacterially expressed and purified engineered GFP-fusion proteins or GFP alone, in the presence or absence of unlabelled histone H2A as indicated. Cells were then washed thoroughly to remove excess protein and examined by CLSM. Arrow heads indicate cell nuclei. (b) Results (mean7SEM) of quantitative analysis of images such as those in a, used to determine the specific cytoplasmic fluorescence (n4110), whereby a value greater than one is indicative of transduction. Results are from a single typical experiment from a total of four separate experiments. P-values (Student’s t-test) indicating significant differences are indicated. (c) Results of quantitative analysis of images, such as those in a, used to determine the ratio of nuclear to cytoplasmic fluorescence (mean7SEM; n4110). P-values (Student’s t-test) indicating significant differences are indicated. (d) Intact HeLa cells were incubated for 1 h at 41C in the presence of the indicated bacterially expressed and purified engineered GFPfusion proteins or GFP alone, in the presence or absence of unlabelled histone H2A as indicated. Cells were then washed thoroughly to remove excess protein and examined by CLSM. (e) Results (mean7SEM) of quantitative analysis of images, such as those in (d), used to determine the specific cytoplasmic fluorescence (n4137). Results are from a single typical experiment from a total of two separate experiments. (f) Intact HeLa cells were incubated with GFP-fusion proteins in the absence or presence of unlabelled histone H2A and examined over time by CLSM. Upper panels show HeLa cells in the presence of GFP-H2B-NLS at the indicated time points as an example. Quantitative analysis of the digitized images to determine the kinetics of the increase of the cytoplasmic to surrounding medium fluorescence ratio (Fc/s) over time is given in the lower panels for GFP-H2B (lower left panel) and GFP-H2B-NLS (lower middle panel) in the presence or absence of H2A as indicated. Quantitative analysis of the increase in kinetics of the nuclear to cytoplasmic fluorescence ratio (Fn/c) over time is given in the lower right panel. (g) Intact murine embryonic stem cells were incubated for 90 min in the presence of the indicated bacterially expressed and purified engineered GFP-fusion proteins or GFP alone, in the presence or absence of unlabelled histone H2A as indicated. Cells were then washed thoroughly to remove excess protein and examined by CLSM.

Molecular Therapy vol. 15 no. 4, april 2007

725

Gene Delivery by Histone Transduction

f

& The American Society of Gene Therapy

GFP-H2B-NLS

g

+ H2A

GFP-H2B

0

+ H2A t1/2 = 4.4 min

– H2A t1/2 = 28.6 min

20

40 60 Time (min)

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

0

9.5 min

51.5 min

GFP-H2B-NLS +H2A t1/2 = 5.04 min –H2A t1/2 = 5.7 min

20

40 60 Time (min)

Fn/c

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

5 min

GFP-H2B

Fc/s

Fc/s

2.5 min

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

0

GFP-H2B-NLS

GFP-H2B-NLS t1/2 = 23.7 min

GFP-H2B t1/2 = 36.9 min

20

GFP

40 60 Time (min)

Figure 3 (continued)

showed that the engineered histone H2B proteins can effectively transduce murine embryonic stem cells (Figure 3g), with significant enhancement by the addition of H2A.

Transduction of H2B is dependent on the N-terminal tail and the globular domain Although transduction of core histone proteins has been observed previously,24 the PTD responsible has not been described. As an initial step toward determining the domain responsible, three H2B fragments were generated as GFP-fusion proteins: the N-terminal tail (GFP-H2B 1–35), which contains a conventional basic NLS;16 the globular domain (GFP-H2B 36–104), which is responsible for octamer formation and DNA binding, and is thought to contain a non-conventional, structurally dependent NLS;16 and the C-terminal tail (GFPH2B 105–125), which plays a role in maintaining nucleosome structure.25,26 Attempts to transduce these fragments into intact HeLa cells resulted in no uptake of any fragment after 1 h incubation (Figure 4a, left panels); GFP-H2B observed significant uptake under these conditions, as previously observed. Extended incubation (100 min) of the cells with the H2B fragments resulted in uptake of both GFP-H2B 1–35 and GFP-H2B 36–104, although not to the extent of the full-length GFP-H2B (Figure 4a, right panels). GFP-H2B 105–125 showed no significant uptake under either condition. Image analysis confirmed that, after 100-min incubation, the specific cytoplasmic fluorescence of both the N-terminal and globular domains of H2B increased above background levels, indicating transduction (Figure 4b). However, both fragments had levels of specific cytoplasmic fluorescence approximately half that of the fulllength protein (4.8 and 5.4 versus 11.1, respectively), suggesting that both domains may be required for efficient protein transduction. It is important to note at this point that, once again, owing to import of the protein into the nucleus, as indicated by an increased Fn/c (Figure 4c), the specific cytoplasmic fluorescence of the full-length protein after 100 min is low. Under the time periods measured, nuclear 726

accumulation was not observed for any of the H2B fragments. This appears to indicate that the full-length protein is required for complete or efficient transport into the nucleus. Minimal uptake of the C-terminal domain was detected, comparable to that of the other two domains after 60-min incubation (Figure 4b). A liposomal disruption assay performed on the H2B fragments confirmed that the N-terminal and globular domains were responsible for the majority of the membrane disruption (Figure 4d). This property was pH dependent, in that minimal disruption was observed at neutral pH, with increasing disruption as the pH was lowered toward endosomal pH (5.0 –5.5). Minimal liposomal disruption activity was observed for the C-terminal fragment of H2B, corresponding well with the lack of transduction in HeLa cells.

HMT is an efficient means for gene delivery into intact cells Having established that the engineered H2B proteins bind to both DNA and Imps with high efficiency and undergo efficient transduction into HeLa cells in culture, their usefulness for gene delivery was investigated. DNA (10 mg) encoding a DsRed2fusion protein that localizes specifically to the nucleus was delivered to intact cells, using the engineered histone proteins as either monomers or dimers with H2A, a process termed HMT. In all cases, expression of the reporter gene was observed in a percentage of the cell population (Figure 5a). Image analysis revealed no significant differences in the Fn/c between proteins expressed from DNA delivered using any of the engineered histone combinations (data not shown). Cells were scored for the expression of the DsRed2 reporter protein (Figure 5b), revealing that, using HMT, engineered histone H2B containing dimers resulted in approximately twice as many cells being transfected as with the monomers (30.9 versus 13.8% for GFPH2B). GFP-H2B-NLS resulted in 1.7-fold more DsRed2 expressing cells than GFP-H2B, indicating that the T-ag NLS significantly enhances the gene delivery process. As expected, www.moleculartherapy.org vol. 15 no. 4, april 2007

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

a

b 18

GFP-H2B (1–35)

3

16 2.5 14

60 min 100 min

60 min 100 min

12

2

10

Fn/c

GFP-H2B

100 min Specific cytoplasmic fluorescence

60 min

c

8 6

1.5

1

4 0.5 2 0

FL

1–35

0

36 –104 105 –125 GFP

d

50

% Total liposome disruption

GFP-H2B (105–125)

1–35 36 –104 105 –125 GFP

GFP-H2B (36–104)

45 GFP-H2B (36–104)

FL

GFP-H2B (1–35)

40 35 30 25 20 15

GFP-H2B (105 –125)

10 5 GFP

0 7.5

7.0

6.5

6.0

5.5

5.0

pH

Figure 4 Histone H2B transduction can be mediated by either the N-terminal tail or the globular domain. (a) Intact HeLa cells were incubated for the times indicated in the presence of either full-length or truncated versions of GFP-H2B or GFP alone, expressed in bacteria. Cells were then washed thoroughly and examined by CLSM. (b) Results of quantitative analysis of images, such as those in a, used to determine the specific cytoplasmic fluorescence, whereby a value greater than one is indicative of transduction. Results (mean7SEM; n478) are from a single typical experiment from a total of three separate experiments. (c) Results of quantitative analysis of images, such as those in a, used to determine the nuclear to cytoplasmic ratio (mean7SEM; n478). (d) Auto-quenched, calcein loaded liposomes were incubated in the presence of the indicated bacterially expressed GFP-fusion proteins at decreasing pH levels. Disruption of the liposomes was indicated by release of the dye and subsequent emitted fluorescence at 520 nm. Results are presented as the percentage of liposome disruption effected by Triton X-100 (n ¼ 3). Results are from a single typical experiment from three separate experiments.

HMT using the GFP-H2B-NLS/H2A dimer resulted in the greatest level of transfection, with 40% of cells positive for expression of the reporter gene, a level approximately twice that of the corresponding monomer. Interestingly, DNA could be delivered to cells using a mixture of GFP and H2A, resulting in a level of transfection comparable to that mediated by GFP-H2B. As GFP alone offered no gene delivery propensity, it can be concluded that this delivery is mediated by the H2A protein, which has been previously shown to have transduction capability.24 Importantly, when compared to DNA (10 mg) delivered using a commercially available transfection reagent (LipofectAMINE 2000), HMT using histone monomers resulted in an equivalent or greater level of transfection. HMT utilizing the engineered histone dimers outperformed the liposomal delivery by approximately 2.5-fold, indicating that HMT is indeed a viable alternative for DNA delivery to intact cells. Importantly, no significant cytotoxicity was observed when utilizing HMT, as determined by cell viability count (data not shown). To establish that the histone-DNA complexes enter cells via transduction rather than through a conventional endocytotic mechanism, a number of inhibitors of endocytosis were tested, with FITC-labelled-poly-L-lysine-Transferrin (Transferrin) used Molecular Therapy vol. 15 no. 4, april 2007

as a positive control, as it is known to enter cells via endocytosis.27 After Five hour incubation, FITC-Transferrin was found to localize to distinct cytoplasmic speckles within MCF-7 cells, indicative of endocytotic vesicles, as well as diffusely in the cytoplasm (Figure 5c, left panel). Due to the ‘‘sticky’’ nature of the positively charged poly-L-lysine moiety in this construct, there was also plasma membrane staining, probably representative of protein both undergoing endocytosis as well as being bound to the external surface of the cell. Upon treatment with the various endocytotic inhibitors (chloroquine, brefeldin A, wortmannin, 0.5 M sucrose (not shown) or incubation at 41C), endocytotic uptake of Transferrin was severely impaired (Figure 5c). In each case, the cytoplasmic speckles were significantly reduced in number compared to Transferrin with no treatment, or absent altogether. A slight staining of the plasma membrane was still seen with all treatments, but as the intensity was very weak compared to no treatment, this could be concluded to represent protein bound to the external surface of the membrane. Consistent with this idea was the fact that cells that are touching did not show staining of this portion of membrane (Figure 5c, arrow head). Having established that the treatments were effective in inhibiting Transferrin internalization, DNA was delivered to 727

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

a

c

+ H2A

GFP No addition

d GFP-H2B

+ Brefeldin

+ Chloroquine + Wortmannin

4°C

50 45 40

No addition Chloroquine Brefeldin Wortmannin Sucrose 4°C

35

b

% Cells transfected

GFP-H2B-NLS

45

% cells transfected

40 35 30

30 25 20 15

25 10

20 15

5

10 5

0

0 DNA

H2A −

GFP + DNA

−

+

GFP-H2B + DNA GFP-H2B-NLS + DNA DNA L

−

+

−

+

−

DNA L H2A −

GFP-H2B + DNA − +

GFP-H2B-NLS + DNA − +

Figure 5 Engineered histone H2B monomers and dimers can deliver DNA to intact cells via transduction, resulting in expression of a reporter gene. (a) Typical CLSM images of intact HeLa cells, showing expression of a DsRed2-fusion protein reporter gene, 48 h after transduction using the indicated engineered histone H2B monomers/dimers bound to DNA (10 mg pGGDsRed2H2A). (b) Cells treated as in a were scored for the expression of the DsRed2-fusion protein reporter gene (mean7SEM; n4235). Results are from a single typical experiment from four separate experiments. DNA L represents cells where an equal amount of DNA (10 mg) was delivered using LipofectAMINE 2000 (Invitrogen). (c) Typical CLSM images of MCF-7 cells 5 h after incubation with FITC-poly-L-lysine-Transferrin without or with the indicated endocytosis treatments. Arrow head represents portion of membrane not showing FITC staining owing to close proximity of neighboring cell. (d) MCF-7 cells treated as in a, without or with the indicated endocytosis treatments, were scored for the expression of the DsRed2-fusion protein reporter gene (mean7SEM; n4300). Results are from a single typical experiment from two separate experiments. DNA L represents cells where an equal amount of DNA (10 mg) was delivered using LipofectAMINE 2000 (Invitrogen).

MCF-7 cells via HMT with the various GFP-H2B constructs, in the presence of each of the endocytosis inhibitors. Five hours after treatment, cells were washed thoroughly to remove excess DNA and incubated in fresh media for 24 h, after which they were scored for expression of the reporter gene. DNA delivered using LipofectAMINE 2000 showed a significant reduction in transfection efficiency in the presence of each of the treatments (Figure 5d), indicating, as expected, that the uptake is mediated by endocytosis. Each of the engineered histone H2B monomers and dimers, however, showed no significant difference with or without any of the treatments tested (Figure 5d). This demonstrates that DNA is delivered to cells by the histones in a non-endocytotic manner, consistent with protein transduction.

DISCUSSION In this study we show that histone H2B can mediate delivery of DNA to intact HeLa cells, resulting in expression of an encoded reporter gene. This HMT is both efficient, resulting in levels of transfection greater than 40%, approximately 2.5-fold that 728

mediated by a commercial liposomal reagent, and applicable to numerous cell types and tissues. Significant enhancement (a twofold increase in the rate of nuclear uptake) was afforded by the addition of an optimized T-ag NLS that binds to Imp a/b with 100-fold greater affinity and has previously been used to target photosensitizer-containing proteins to the nucleus of tumor cells.28 These are significant findings as gene delivery to eukaryotic cells is traditionally inefficient, due in large part to the membranes surrounding the cell itself and the nucleus. Also significant was the demonstration that histone H2B mediates delivery of DNA in a non-endocytotic manner, consistent with protein transduction. This is exciting as most reports examining the delivery of DNA by protein transduction have shown that although the protein can undergo transduction, the delivery of DNA is mediated by conventional endocytosis.3,29,30 We hypothesize that the ability of histone H2B to transduce DNA is probably linked to its intrinsic DNA condensation ability, consistent with the findings of Demirhan et al.,31 who showed that DNA delivery by histone H3 or H4 is dependent on the ability to condense plasmid DNA. www.moleculartherapy.org vol. 15 no. 4, april 2007

& The American Society of Gene Therapy

For this study we engineered several histone proteins based around the core histone H2B. Additional to the optimized T-ag NLS, these novel histones included an N-terminal GFP moiety to enable the proteins to be visualized inside living cells. Our results show that the nuclear localization, histone H2B/H2A dimerization and DNA binding abilities of the engineered histones are unaffected by the modifications, with the additional NLS conferring binding by Imp a/b. That the DNA binding and Imp interacting functions of H2B are not mutually exclusive was demonstrated by the ALPHAScreen assay, highlighting the potential of these proteins as DNA carriers, which is obviously testified to by the results here (Figure 5). Although all the core histones have been previously shown to contain PTDs,8,9,24 H2B had been reported to have relatively low transduction potential.24 Our study, however, shows that the engineered histone H2B constructs readily transduce into intact HeLa cells in culture and indeed into other cell types, including ES cells. This ability to transduce a wide variety of cell types and tissues with 100% efficiency (t1/2 of cellular and nuclear uptake of approximately 5 and 20 min in the presence of the T-ag NLS), typical of PTD-containing proteins,3 emphasizes the usefulness of histones as a gene delivery vehicle. By dimerizing the engineered histone proteins with wild-type H2A, a further 47-fold increase in the rate of cellular uptake was seen (Figure 3f). As both H2A and H2B have been shown to undergo transduction24 and H2A has previously been utilized for DNA delivery,32–34 this enhancement is hardly surprising as the dimeric form possesses two PTDs and should, therefore, enter cells more efficiently, as has been seen for other PTD containing proteins such as Tat.3,35,36 Histone H2A,32–34 H3,31 H4,31 and H137–39 have all previously been used to mediate/enhance DNA delivery, but this is the first report of any two histones being used in conjunction with one another as dimers. Most PTDs that have been defined are short stretches (o30 amino acids), typically having a high arginine or lysine content and often being amphipathic a-helices (for review see refs. 3,35). Our results indicate that both the N-terminal tail and the globular domain of H2B are required for optimal translocation. The NLS-containing N-terminal tail of H2B16,40 has a relatively high lysine/arginine content (B42%) and may therefore contain a typical PTD sequence, whereas the globular domain of H2B has a lysine/arginine content of only 11% and no single defined basic region. This domain has been shown previously to contain a structurally mediated NLS, dependent upon correct folding of the entire globular domain.16 It is intriguing that both NLS containing regions of H2B also appear to have transduction ability, but the T-ag NLS control demonstrates that not all NLS sequences can function as PTDs. Further definition of the PTD in H2B would be useful to enable it to be used efficiently in other applications. In summary, we demonstrate, that HMT utilizing histone H2B is an efficient means of gene delivery into eukaryotic cells. Future work will focus on expanding this work to incorporate the use of reconstituted chromatin as the transfecting DNA. This system will have all the advantages of the current HMT as well as additional DNA compaction and protection properties. HMT is a concept that has applications not only in gene therapy, but also in cancer therapeutics and gene knockdown technologies. Molecular Therapy vol. 15 no. 4, april 2007

Gene Delivery by Histone Transduction

MATERIALS AND METHODS Generation of GFP-fusion protein bacterial and mammalian expression plasmids. Bacterial or mammalian cell expression vectors encoding

GFP-H2B or GFP-H2B-NLS, containing the full-length histone H2B (Xenopus laevis; xH2B) with or without the optimized protein kinase CK2 site-enhanced T-ag NLS (SSDDEATADAQHAAPPKKKRKV; single letter code) fused to the C-terminus or the T-ag NLS alone (GFP-NLS), as well as the GFP-H2B truncation derivatives GFP-H2B (1–35), GFPH2B (36–104), and GFP-H2B (105–125), were generated using the Gateway cloning technology (Invitrogen, Carlsbad, VA). Briefly, DNA fragments flanked by attB recombination sites encoding H2B, H2B-NLS, NLS alone or the H2B fragments 1–35, 36–104, or 105–125, were generated using standard PCR techniques from the template pET3axH2B15 or pET3a-xH2B-NLS, which contains the T-ag NLS inserted Cterminal to H2B. These PCR products were then recombined into either the pDONR201 or pDONR207 vectors (Invitrogen) via the ‘‘BP’’ recombination reaction, and then the DONR vectors subsequently recombined using the ‘‘LR’’ recombination reaction into vector pGFPattC,41,42 used to express GFP-fusion proteins in bacteria, and pDest53 (Invitrogen), for GFP-fusion protein expression in mammalian cells. The integrity of all expression constructs was verified by DNA sequencing. The reporter plasmid pGGDsRed2-H2A used in the HMT and DNA binding assays was generated in a similar manner; attB flanked PCR products encoding histone H2A (X. laevis) were generated from plasmid pET3a-xH2A15 and recombined into pDONR207 via the BP reaction. The resultant pDONR207-H2A plasmid was recombined into vector pBk-CMV-DsRed2-C (used to express DsRed2-fusion proteins in mammalian cells), which we had previously rendered Gateway compatible, to generate vector pDsRed2-H2A.43 To generate plasmid pGGDsRed2-H2A, the DsRed2-H2A coding region was amplified from plasmid pDsRed2-H2A, with primers designed to introduce a BglII restriction endonuclease site 50 to the sequence and a NotI site at the 30 end. The PCR product was then digested with the requisite enzymes (NEB) and ligated into pGeneGrip (Genlantis), predigested with BclI and NotI. Positive clones were identified by PCR and confirmed by DNA sequencing. Protein purification and Imp a/b dimerization. Fusion proteins GFP-

H2B, GFP-H2B-NLS, GFP-NLS, and GFP alone (expressed from plasmid pTRCA-EGFP)44 were expressed and purified from bacteria as (His)6tagged proteins using nickel affinity chromatography under denaturing conditions (8 M area) essentially as previously,41,44 except that KCl was substituted for NaCl in the lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris, 0.1 M KCl; pH 8.0), and proteins were eluted in dimerization buffer (450 mM KCl, 50 mM N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES); pH 7.5). Histone H2A was purified from plasmid pET3a in Escherichia coli strain BL21DE3lysS from inclusion bodies under native conditions as previously described.15 The Imp proteins were purified from bacteria as GST fusion proteins under native conditions as previously described.21,42,45,46 Where required, Imp a and Imp b were predimerized at 13.6 mM for 15 min at room temperature to generate the Imp a/b heterodimer used in binding studies.41 Cell culture. Cos-7 (African green monkey kidney), HeLa (human

cervical adenocarcinoma), HTC (rat hepatoma), and MCF-7 (human breast cancer) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute Media (RPMI) (MCF-7 cells), supplemented with 10% fetal calf serum, 1 mM Lglutamate, 1 mM penicillin/streptomycin, and 20 mM HEPES at 371C in 5% CO2. At 24 or 48 h before transfection or HMT respectively, cells were seeded onto glass coverslips (15  15 mm2). W9.5 murine embryonic stem cells were cultured in DMEM/20% ES cell fetal calf

729

Gene Delivery by Histone Transduction

serum (Gibco), 0.1% non-essential amino acids (Gibco), 2 mM L-glutamate, 2 mM penicillin/streptomycin, 0.1 mM b-mercaptoethanol, and 1000 U/ml ESGRO (Invitrogen) on a mouse embryonic fibroblast (MEF) feeder layer. Before transduction, murine embryonic stem cells were MEF depleted and seeded onto 0.1% gelatin-coated glass coverslips (15  15 mm). Mammalian cell transfection. LipofectAMINE 2000 (Invitrogen) was

used according to the manufacturer’s instructions to transfect DNA into mammalian cells. Cells were imaged live 24 h post-transfection by CLSM (BioRad MRC-500) using a  40 water immersion lens. Digitized images were analyzed using the ImageJ v1.37 public domain software (NIH) to determine the ratio of nuclear (Fn) to cytoplasmic (Fc) fluorescence (Fn/c) according to the formula: Fn/c ¼ (FnFb)/(FcFb), where Fb is background autofluorescence. Native PAGE/fluorimaging. Native PAGE/fluorimaging was performed

as previously to examine protein–protein interactions.46 Fluorescently labelled protein (2 mM) was incubated with 10 mM GST-Imp in a final volume of 25 ml, followed by electrophoresis and fluorimaging. DNA electrophoretic mobility shift assay. Before the assay, GFP-H2B

and GFP-H2B-NLS were predimerized with H2A at 251C for 30 min, followed by centrifugation through a Centricon 50 to remove unbound monomers. DNA (250 ng) (plasmid pUC18, linearized using restriction endonuclease BamH1) was then incubated with 20 mM histone monomer, or dimer, or GFP alone. Samples were subjected to electrophoresis in a 0.75% ethidium-free agarose gel at 40 V for 6 h at 41C in TAE (40 mM Tris, 0.114% glacial acetic acid, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5). The gel was then stained with ethidium bromide and visualized under UV illumination. ALPHAScreen assay. An ALPHAScreen assay was performed in

triplicate as previously described, to quantitate the interaction between the engineered histones and Imp proteins.42 Thirty nanomolar of (His)6 binding partner and increasing concentrations of the biotinylated GSTImps were used. Where DNA was added, 100 ng pGGDsRed2-H2A was added for 30 min before the addition of the Imps. All additions and incubations were performed in subdued lighting conditions because of the photosensitivity of the beads. The assay was measured on a PerkinElmer Fusion-a plate reader, the triplicate values averaged, and the titration curves (standard three-parameter sigmoidal fit) plotted using the SigmaPlot graphing program. As standard practice, values in the ‘‘hooking zone’’ (see ref. 42 for explanation), where quenching had occurred, were excluded from the analysis. Fluorescence polarization. Fluorescence polarization assays were carried out in quintuplicate, essentially as described previously,47,48 using a Cary (Varian) fluorimeter fitted with manual polarization filters, with excitation at 485 nm and emission at 520 nm. GFP fusion protein monomers or dimers were added at a final concentration of 0.2 mM, with Imps added to 0.7 mM. Anisotropy changes were calculated according to A ¼ (I8I>)/(I8 þ 2I>), where A is anisotropy, and I8 and I> are parallel and perpendicular emitted light intensity, respectively. Protein transduction. HeLa cells were incubated in a 6.8 mM solution of GFP-fusion protein in DMEM at 371C. Where appropriate, a molar equivalent amount of H2A was added at the same time. Cells were then washed five times with 1 ml DMEM to remove free protein followed by washing once with DMEM/lacking phenol red. Cells were imaged live by CLSM as per mammalian cell transfection (above). Image analysis was performed on digitized images, using the Image J software as previously

730

& The American Society of Gene Therapy

described, to determine both the Fn/c ratio and the specific cytoplasmic fluorescence (cytoplasmic fluorescence/cellular auto fluorescence). To determine the kinetics of protein transduction, HeLa cells were surrounded by a 6.8 mM solution of GFP-fusion protein in PBS, with or without equimolar amounts of H2A as appropriate. Cells were then imaged live over a period of B70 min by CLSM. Image analysis was performed, as previously described, to determine the ratio of cytoplasmic to surrounding media fluorescence (Fc/s). Liposomal disruption assay. Calcein-loaded liposomes were created by

first evaporating 25 mg of egg yolk phosphatidylcholine (Sigma) in chloroform at 401C in a rotary evaporator. The lipid film was further dried under a stream of argon, followed by hydration with 1 ml of HEPES buffered saline (HBS) (20 mM HEPES, 150 mM NaCl, pH 7.5) containing 200 mM calcein (Sigma). The liposome/calcein dispersion was sonicated in a water-bath sonicator for 10 min, followed by a dozen freeze–thaw cycles. Subsequently, the liposomes were diluted with 3 ml of HBS, passed through syringe filters with pore sizes varying from 0.45 to 0.22 mm, and stored at 41C in the dark under argon. For the liposome disruption assay, a 100 ml aliquot of calcein-loaded liposomes was diluted 1:5 with HBS and dialyzed extensively against HBS in 25,000 MWCO dialysis tubing to remove non-encapsulated calcein. The assay was performed in triplicate, whereby 1 mM (final concentration) protein and 10 ml of purified liposomes were added to 200 ml of pH buffer (20 mM citrate buffer (from 0.1 M citrate buffer: 0.018 M citric acid, 0.082 M sodium citrate), 20 mM MES, 20 mM HEPES, 150 mM NaCl, pH adjusted to desired point). Control samples were set up in parallel where the 10 ml protein was substituted for either water (no protein control) or Triton X-100 (100% liposomal disruption). After 30-min incubation in the dark at 281C, 800 ml of 50 mM HEPES, 150 mM NaCl; pH 7.5 was added for adjustment of the pH to the fluorescence maximum of calcein. Fluorescence was immediately measured in a CARY Eclipse fluorescence spectrophotometer (Varian) in plastic 1 ml cuvettes with excitation at 488 nm and emission at 520 nm. Triplicate values were averaged, water controls subtracted, and the result expressed as a % of the Triton X-100 control (% total liposome disruption). Histone mediated transduction for gene delivery. HMT was performed

as per the protein transduction experiments (above) except that 10 mg DNA (pGGDsRed2-H2A) was added to the engineered histone mix 15 min before incubation with the cells. After the incubation period, cells were not washed; rather, 1 ml fresh DMEM was added, and cells were incubated at 371C for 48 h to express the DsRed2 reporter protein. Cells were imaged live by CLSM, as for mammalian cell transfection, and scored for expression of the reporter gene. Cell viability was determined by manual cell count in the presence of trypan blue as an indicator of deceased cells. Treatment with endocytotic inhibitors. MCF-7 cells were treated with

either 50 mM Chloroquine (Sigma), 10 mM Brefeldin A (Sigma), 2 mM Wortmannin (Sigma), 0.5 M sucrose, or exposed to 41C for 30 min before and during either HMT or transfection utilizing LipofectAMINE 2000. At 5 h after treatment, the cells were washed, the media replaced with fresh RPMI, and the cells incubated at 371C. Cells were imaged live by CLSM, as for HMT, and scored for expression of the reporter gene. FITC-poly-L-lysine-Transferrin (Sigma) was used as a positive control, whereby following 30 min incubation with the various treatments, 1 mg FITC-poly-L-lysine-Transferrin was added to cells. Following a 5-h incubation, cells were imaged live by CLSM.

ACKNOWLEDGMENTS We acknowledge the support of the National Health and Medical Research Council (fellowship no. 334013/no. 384109 and project

www.moleculartherapy.org vol. 15 no. 4, april 2007

Gene Delivery by Histone Transduction

& The American Society of Gene Therapy

grant no. 143710/no. 284205). We also thank Jun Fan for purifying the unlabelled H2A used throughout this study and Julia Young for maintenance and seeding of the ES cells.

REFERENCES 1. 2. 3. 4.

5. 6. 7.

8.

9.

10. 11. 12. 13. 14.

15.

16. 17.

18.

19.

20.

21.

22.

23.

24.

Glover, DJ, Lipps, HJ and Jans, DA (2005). Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet 6: 299–310. Johnson-Saliba, M and Jans, DA (2001). Gene therapy: optimising DNA delivery to the nucleus. Curr Drug Targets 2: 371–399. Wagstaff, KM and Jans, DA (2006). Protein transduction: cell penetrating peptides and their therapeutic applications. Curr Med Chem 13: 1371–1387. Gupta, B, Levchenko, TS and Torchilin, VP (2005). Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev 57: 637–651. Morris, MC, Chaloin, L, Heitz, F and Divita, G (2000). Translocating peptides and proteins and their use for gene delivery. Curr Opin Biotechnol 11: 461–466. Frankel, AD and Pabo, CO (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55: 1189–1193. Green, M and Loewenstein, PM (1988). Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55: 1179–1188. Rosenbluh, J, Hariton-Gazal, E, Dagan, A, Rottem, S, Graessmann, A and Loyter, A (2005). Translocation of histone proteins across lipid bilayers and Mycoplasma membranes. J Mol Biol 345: 387–400. Rosenbluh, J, Singh, SK, Gafni, Y, Graessmann, A and Loyter, A (2004). Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells. Biochim Biophys Acta 1664: 230–240. Pemberton, LF and Paschal, BM (2005). Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6: 187–198. Quimby, BB and Corbett, AH (2001). Nuclear transport mechanisms. see comment. Cell Mol Life Sci 58: 1766–1773. Jans, DA, Xiao, CY and Lam, MH (2000). Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays 22: 532–544. Nigg, EA (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386: 779–787. Jakel, S, Albig, W, Kutay, U, Bischoff, FR, Schwamborn, K and Doenecke, D et al. (1999). The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. EMBO J 18: 2411–2423. Johnson-Saliba, M, Siddon, NA, Clarkson, MJ, Tremethick, DJ and Jans, DA (2000). Distinct importin recognition properties of histones and chromatin assembly factors. FEBS Lett 467: 169–174. Baake, M, Doenecke, D and Albig, W (2001). Characterisation of nuclear localisation signals of the four human core histones. J Cell Biochem 81: 333–346. Langer, T (2000). Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin alpha/beta-mediated process. Histochem Cell Biol 113: 455–465. Mosammaparast, N, Ewart, CS and Pemberton, LF (2002). A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B. EMBO J 21: 6527–6538. Mosammaparast, N, Guo, Y, Shabanowitz, J, Hunt, DF and Pemberton, LF (2002). Pathways mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem 277: 862–868. Hubner, S, Xiao, CY and Jans, DA (1997). The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin. J Biol Chem 272: 17191–17195. Xiao, CY, Hubner, S and Jans, DA (1997). SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence. J Biol Chem 272: 22191–22198. Xiao, CY, Jans, P and Jans, DA (1998). Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T-antigen NLS by importin: effect of conformation. FEBS Lett 440: 297–301. Chan, CK and Jans, DA (2001). Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway. Gene Therapy 8: 166–171. Hariton-Gazal, E, Rosenbluh, J, Graessmann, A, Gilon, C and Loyter, A (2003). Direct translocation of histone molecules across cell membranes. J Cell Sci 116 (Part 22): 4577–4586.

Molecular Therapy vol. 15 no. 4, april 2007

25.

26. 27.

28.

29.

30.

31.

32. 33.

34.

35. 36. 37.

38.

39. 40.

41.

42.

43.

44.

45.

46. 47.

48.

Luger, K, Rechsteiner, TJ, Flaus, AJ, Waye, MM and Richmond, TJ (1997). Characterization of nucleosome core particles containing histone proteins made in bacteria. J Mol Biol 272: 301–311. Luger, K and Richmond, TJ (1998). The histone tails of the nucleosome. Curr Opin Genet Dev 8: 140–146. Wagner, E, Cotten, M, Mechtler, K, Kirlappos, H and Birnstiel, ML (1991). DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety. Bioconjugate Chem 2: 226–231. Rosenkranz, AA, Lunin, VG, Gulak, PV, Sergienko, OV, Shumiantseva, MA and Voronina, OL et al. (2003). Recombinant modular transporters for cell-specific nuclear delivery of locally acting drugs enhance photosensitizer activity. FASEB J 17: 1121–1123. Hashida, H, Miyamoto, M, Cho, Y, Hida, Y, Kato, K and Kurokawa, T et al. (2004). Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool. Br J Cancer 90: 1252–1258. Ignatovich, IA, Dizhe, EB, Pavlotskaya, AV, Akificv, BN, Burov, SV and Orlov, SV et al. (2003). Complexes of plasmid DNA with basic domain 47–57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways. J Biol Chem 278: 42625–42636. Demirhan, I, Hasselmayer, O, Chandra, A, Ehemann, M and Chandra, P (1998). Histone-mediated transfer and expression of the HIV-1 tat gene in Jurkat cells. J Hum Virol 1: 430–440. Balicki, D and Beutler, E (1997). Histone H2A significantly enhances in vitro DNA transfection. Mol Med 3: 782–787. Balicki, D, Putnam, CD, Scaria, PV and Beutler, E (2002). Structure and function correlation in histone H2A peptide-mediated gene transfer. Proc Natl Acad Sci USA 99: 7467–7471. Balicki, D, Reisfeld, RA, Pertl, U, Beutler, E and Lode, HN (2000). Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma. Proc Natl Acad Sci USA 97: 11500–11504. Lundberg, P and Langel, U (2003). A brief introduction to cell-penetrating peptides. J Mol Recogn 16: 227–233. Tung, CH, Mueller, S and Weissleder, R (2002). Novel branching membrane translocational peptide as gene delivery vector. Bioorg Med Chem 10: 3609–3614. Dai, FH, Chen, Y, Ren, CC, Li, JJ, Yao, M and Han, JS et al. (2003). Construction of an EGF receptor-mediated histone H1(0)-based gene delivery system. J Cancer Res Clin Oncol 129: 456–462. Deas, O, Angevin, E, Cherbonnier, C, Senik, A, Charpentier, B and Levillain, JP et al. (2002). In vivo-targeted gene delivery using antibody-based nonviral vector. Hum Gene Ther 13: 1101–1114. Fritz, JD, Herweijer, H, Zhang, G and Wolff, JA (1996). Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum Gene Ther 7: 1395–1404. Moreland, RB, Langevin, GL, Singer, RH, Garcea, RL and Hereford, LM (1987). Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol 7: 4048–4057. Baliga, BC, Colussi, PA, Read, SH, Dias, MM, Jans, DA and Kumar, S (2003). Role of prodomain in importin-mediated nuclear localization and activation of caspase-2. J Biol Chem 278: 4899–4905. Wagstaff, KM and Jans, DA (2006). Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method. Anal Biochem 348: 49–56. Hubner, S, Eam, JE, Wagstaff, KM and Jans, DA (2006). Quantitative analysis of localization and nuclear aggregate formation induced by GFP-lamin A mutant proteins in living HeLa cells. J Cell Biochem 98: 810–826. Lixin, R, Efthymiadis, A, Henderson, B and Jans, DA (2001). Novel properties of the nucleolar targeting signal of human angiogenin. Biochem Biophys Res Commun 284: 185–193. Hubner, S, Smith, HM, Hu, W, Chan, CK, Rihs, HP and Paschal, BM (1999). Plant importin alpha binds nuclear localization sequences with high affinity and can mediate nuclear import independent of importin beta. J Biol Chem 274: 22610–22617. Wagstaff, KM, Dias, MM, Alvisi, G and Jans, DA (2005). Quantitative analysis of protein–protein interactions by native page/fluorimaging. J Fluoresc 15: 469–473. Forwood, JK, Harley, V and Jans, DA (2001). The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1. J Biol Chem 276: 46575–46582. Forwood, JK, Lam, MH and Jans, DA (2001). Nuclear import of Creb and AP-1 transcription factors requires importin-beta 1 and Ran but is independent of importin-alpha. Biochemistry 40: 5208–5217.

731