CNS gene transfer mediated by a novel controlled release ... - Nature

Gene Therapy (2004) 11, 109–114 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt

BRIEF COMMUNICATION

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes Y Li1,2, J Wang3,4, CGL Lee5, CY Wang1,2, SJ Gao1,2, GP Tang1,2, YX Ma1,2, H Yu6, H-Q Mao3, KW Leong3,4 and S Wang1,2,7 1 Institute of Bioengineering and Nanotechnology, Singapore; 2Institute of Materials Research & Engineering, Singapore; 3Tissue and Therapeutic Engineering Lab, Johns Hopkins Singapore, Singapore; 4Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 5Department of Biochemistry, Singapore; 6Department of Physiology, Singapore; and 7Department of Biological Sciences, National University of Singapore, Singapore

Nonviral gene delivery systems based upon polycation/ plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression

mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS. Gene Therapy (2004) 11, 109–114. doi:10.1038/sj.gt.3302135

Keywords: polycation; polyphosphoester; gene transfer; degradability; biocompatibility; brain

Gene transfer into the central nervous system (CNS) offers the prospect of manipulating gene expression for studying neuronal function and eventually for treating neurological disorders. Extensive research has been carried out on CNS gene delivery, using either viral or nonviral vectors. Polymer-based gene delivery systems serve as an alternative to viral gene vectors for their advantages in inducing relatively low toxicity and nearly no immune responses. Other potential advantages of polymer gene delivery systems include capability of dealing with large DNA plasmids, simplicity in preparation, flexibility in use, and cell-type specificity after chemical conjugation of a targeting ligand. Among these polymers, polyethylenimine (PEI) has been extensively studied and shown high transfection efficiency both in vitro and in vivo.1–4 In particular, PEI polymers, especially those with a molecular weight of 25 kDa or Correspondence: Dr S Wang, Institute of Bioengineering and Nanotechnology, 3 Research Link, Singapore 117602, Singapore Received 02 April 2003; accepted 23 June 2003

more, may mediate intracellular DNA delivery and transgene expression in terminally differentiated nondividing neurons.1,5–9 After direct brain injection, PEI/ DNA complexes can produce transgene expression levels comparable to those obtained with the lenti- or adenoviral vectors.5 However, the polymer is highly toxic in various types of cells, including neurons and other cells in the nervous system.6,9 Both PEI and its DNA complexes may enter cell nuclei;10 the accumulation of nonbiodegradable, highly positively charged PEI in the cell nucleus is a concern for its long-term effects on cellular gene expression.11 Recently, a new polymeric gene carrier based on a water-soluble and cationic polyphosphoester, poly(2aminoethyl propylene phosphate) (PPE-EA) has been developed.12 The polymer degrades in phosphate-buffered saline (PBS) at 371C through the cleavage of the phosphate bonds in the backbone. The cytotoxicity and soft tissue response in mouse of PPE-EA is significantly better than PEI and poly L-lysine, another commonly used polycationic gene carrier.12 PPE-EA readily forms

PPE-EA-mediated gene delivery in the brain Y Li et al

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complexes with plasmid DNA, and can slowly biodegrade to provide a sustained release of the plasmid DNA. The polymer can increase the DNA bioavailability by protecting the DNA from degradation and reducing the clearance of the DNA from the injection site, thus offering the possibility of enhanced gene transfer efficiency and prolonged gene expression. Although their transfection efficiency greatly depends on cell type,12 PPE-EA/DNA complexes have resulted in enhanced gene expression in the muscle as compared with naked DNA injections.13 Considering that naked plasmid DNA can mediate gene transfer in the brain, we explored in this study the feasibility of using PPE-EA/DNA complexes for gene transfection in the CNS after intracisternal and intrastriatum injection in mice. We showed in this report that PPE-EA/DNA complexes functioned as a controlled release system in the brain, mediating a prolonged gene transfer that was superior to that offered by either naked DNA or PEI. We showed further a lower cytotoxicity and better nervous tissue compatibility of PPE-EA as compared with PEI. We first examined sizes of PPE-EA/DNA particles and their stability in different solutions by light scattering (Table 1). The particles were prepared in 5% glucose, a solution that shows beneficial effects in formulating homogeneous PEI/DNA complexes and facilitating widespread diffusion of the complexes in the brain after injection into the cerebrospinal fluid (CSF).7 The PPEEA/DNA particles were then diluted with either 5% glucose (a low ionic condition) or 150 mM NaCl (a high ionic, more physiological condition) before size measurement. Under the low ionic condition, the size of the PPEEA/DNA particles changed with N/P ratios, increasing from 99 to 200 nm in the main particle populations as the Table 1

N/P ratio changed from 0.5 to 2. However, the particles were not stable in this 5% glucose solution. After 1-h incubation, larger aggregates were formed, with diameters increasing to 1000 and 3000 nm at N/P ratios of 0.5 and 2, respectively. At high ionic strength, the complex size increased again with an increasing N/P ratio. The complexes tend to be larger, more uniform in size distribution, and less prone to aggregation (Table 1). PEI/DNA complexes at N/P ratio of 15/1, the optimized N/P ratio for gene delivery to the brain in our hands, were also included for comparison (Table 1). The complexes obtained were smaller in the 5% glucose solution, around 90 nm as opposed to 120 nm in the 150 mM NaCl solution. Aggregation occurred in both solutions with time, with approximately 50% of the complexes displaying a size around 400 nm after 1-h incubation. The feasibility of using PPE-EA/DNA complexes for CNS gene delivery was then examined in mice after intracisternal injection using luciferase as a reporter gene. PPE-EA/DNA complexes at different N/P ratios from 0 to 10 were tested. Gene transfer efficiency increased with N/P ratio from 0.2 to 2, and then dropped as the N/P ratio reached 10 (Figure 1a). To further evaluate the gene transfer efficiency of PPE-EA/ DNA complexes, a time course study was carried out using PPE-EA/DNA complexes at N/P ratios of 0.5 and 2, and naked DNA and PEI/DNA complexes at an N/P ratio of 15. At 1 day after injection, PEI/DNA provided the highest transgene expression at 2 105 RLU/brain, about four-fold higher than that produced by PPE-EA/ DNA at an ratio of N/P of 2 and two-fold higher than the expression mediated by naked DNA and PPE-EA/ DNA at an N/P ratio of 0.5 (Figure 1b). At 3 days after injection, the luciferase expression level of PPE-EA/

DNA–polymer complex size (nm)a Peak I Mean7s.d.

(a) 5% glucose PPE-EA (N/P¼0.5) 0h 98.6722.8 1h 163.3728.9 PPE-EA (N/P¼2) 0h 200.4745.5 1h 0 PEI (N/P¼15) 0h 86.9731.1 1h 137.7732.0 (b) 150 mm NaCl PPE-EA (N/P¼0.5) 0h 152.3727.9 1h 171.6720.8 PPE-EA (N/P¼2) 0h 557.87164.1 1h 568.17153.9 PEI (N/P¼15) 0h 118.0735.1 1h 121.5714.9 a

Peak II %

Mean7s.d.

Peak III %

Mean7s.d.

%

58.8 60.4

433.67100.4 0

17.8 0

979.87145.9 932.17151.8

16.7 33.6

51.5 0

1004.37131.9 0

19.3 0

30007393.3 30007299.6

29.0 96.0

82.9 63.2

0 488.0758.9

0 26.9

0 0

0 0

78.2 70.0

0 0

0 0

0 0

0 0

100 100

0 0

0 0

0 0

0 0

65.2 54.0

241.9749.6 375.2794.7

34.8 24.6

0 482.17153.0

0 16.1

A measure of 10 mg of pCAG-luc in 50 ml of 5% glucose was complexed with polymers in 50 ml of 5% glucose. The complexes were diluted either in 5% glucose (a) or 150 mm NaCl (b) to 1 ml for the measurement of particle sizes with N4 Plus Submicron Particles Sizer (COULTER, USA) at room temperature. Scattering light was detected at 901, running for 200 s for each sample (n¼8 for each preparation), and analyzed in the Unimodel Analysis mode. Gene Therapy


111 Brain Stem Cerebellum BG/Diencephalon Cerebral Cortex

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Days Figure 1 Luciferase expression in mouse brain after intracisternal injections of PPE-EA/pCAG-Luc complexes: (a) effects of N/P ratios; (b) time course study. The intracisternal injection in adult male Swiss mice (20–25 g) was carried out according to Reijneveld et al.18 A 10 ml Hamilton syringe connected with a 26-gauge needle was used, and a plastic stopper was set on the needle 4 mm from the tip. A volume of 10 ml of 5% glucose solution containing 1 mg of DNA or 1 mg of DNA condensed by appropriate amounts of PPE-EA or PEI was injected into the cerebellomedullary cistern of each mouse. Mice were killed either 3 days after injection (a) or at different time points indicated (b). Brain samples were collected. The supernatants from tissue homogenates were used for luciferase activity assay in a single-well Luminometer (Berthold Lumat LB 9507, Germany) for 10 s. Values are presented as means7s.d. (n¼6).

DNA at an N/P ratio of 2 increased to 1.8 105 RLU/ brain, while the expression levels of the other three groups remained unchanged. After 10 days, the expression level for PPE-EA/DNA at an N/P ratio of 2 was about the same as day 3, while those of other three groups began to drop. Till 28 days, PPE-EA/DNA at the N/P ratio of 2 still provided a level of transgene expression at 8 104 RLU/brain, similar to that observed at previous time points. This level was significantly higher than those offered by PEI/DNA, naked DNA, and PPE-EA/DNA at an N/P ratio of 0.5. To compare the distribution of the transgene expression, the brain samples collected 3 days after injection were dissected into four parts and analyzed for luciferase activity: brain stem, cerebellum, basal ganglia/diencephalons, and cerebral cortex (Figure 2a and b). Almost all the luciferase activities mediated by naked DNA delivery were located in the brain stem. A similar transgene expression pattern was observed with PPE-EA/DNA

PEI/DNA

Naked DNA

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% of total RLU in the brain

RLU per brain

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105 RLU per tissue

RLU per brain

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Figure 2 Distribution of transgene expression mediated by naked DNA, PPE-EA/DNA complexes (N/P¼0.5 and 2), and PEI/DNA (N/P¼15) after intracisternal injection. A measure of 1 mg of DNA in 10 ml 5% glucose was intracisternally injected into each mouse. Brain samples were collected at day 3 and dissected into four parts: cerebral cortex, basal ganglion (BG)/diencephalon, cerebellum, and brain stem. Luciferase activity assay was carried out as stated in Figure 1. Six mice per group were used. Bars represent means7s.d.

complexes at an N/P ratio of 0.5. With PPE-EA/DNA complexes at an N/P ratio of 2, significant levels of luciferase expression were detected both in the brain stem and the basal ganglia/diencephalons, accounting for about 33 and 65% of the total activity, respectively. In contrast, PEI/DNA complexes appeared to have diffused to the cerebral cortex significantly, with 70% of the luciferase activity found there. All four groups showed very low levels of transgene expression in the cerebellum. Immunostaining of the brain sections using an antibody against luciferase supported the distribution profiles of gene expression described above for the PPE-EA/DNA complexes at N/P ratio 2. Strong signals were observed near the injection site, mainly in the meninges (Figure 3a). The meninges of the brain regions away from the injection site were also positively stained (Figure 3b). For the PEI/DNA complexes, the luciferase was clearly stained even in the meninges of the cerebral cortex (Figure 3c). Gene expression in the parenchyma of the brain was also examined 3 days after intrastriatum, instead of intracisternal, injection of PPE-EA/DNA Gene Therapy


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PPE-EA PEI 120

% Cell Viability

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complexes at an N/P ratio of 2. Many cells close to the injection track were luciferase-positive, several of which displayed typical neuronal morphology (Figure 3d). Knowing that some of the cationic polymers, like PEI and poly L-lysine, exhibit cytotoxicity,11,13 we tested the cytotoxicity of PPE-EA in three different neural cell lines. C17.2 is a multipotent cell line generated via retrovirusmediated V-myc transfer into murine cerebellar progenitor cells. PC12 is a cell line separated from rat pheochromocytoma, inducible to neuronal phenotype by NGF. NT2 cell is a human cell line exhibiting characteristics of committed CNS neuronal precursor cells. Among the three cell lines tested, C17.2 was the most robust, maintaining 90% viability at 1000 mM of PPE-EA, while only 50–70% of PC12 and NT2 cells were viable at 500 mM (Figure 4). All cell lines were more vulnerable to the PEI treatment. For example, at 1000 mM, cell viability ranged from 40 to 90% in the PPE-EAtreated groups, while all cells died in the PEI-treated groups. After neuronal differentiation, C17.2 showed even higher susceptibility to PEI, most of which died at a concentration as low as 165 mM, while close to 80% of the C17.2 neurons were still viable at 1000 mM of PPE-EA (Figure 4b). To further investigate the biocompatibility, PPE-EA and PEI polymers were intracisternally injected into mice cisterna magna at the same doses of charged groups (100 nmol of amino group for both PPE-EA and PEI) and the tissues were collected 7 days after for histological analysis. No significant inflammatory reactions were observed by H&E staining in both PPE-EA- (Figure 5a) and PEI- (Figure 5b) treated animals. Effects of the polymer/DNA complexes were also examined. Consistent with what we observed before,9 TUNEL staining revealed positively stained cells in the PEI/DNA particle treated group (Figure 5d). No such cells were

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Figure 3 Immunostaining with an antibody against luciferase 3 days after intracisternal (a–c) or intrastriatum (d) injection of 1 mg of pCAG-Luc complexed with PPE-EA at an N/P ratio of 2 (a, b, d, bar: 80 mm) or with PEI at an N/P ratio of 15 (c, bar: 80 mm). The inset in (c) shows positively stained cells in arachnoid granulations (bar: 40 mm).

80

80

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250 330 500 1000 M of polymer Figure 4 Viability assay in C17.2, PC12, and NT2 cells. C17.2 cells were also differentiated by plating them onto poly L-lysine (50 mg/ml) and laminin (20 mg/ml) coated plates in serum-free DMEM/F12 media with 1% N2 supplement (Invitrogen, Netherlands) for 2 days. The cells were treated with different concentrations of PPE-EA or PEI diluted in water for 24 h. Cell viability was determined in 96-well microtiter plates by an MTT assay. A volume of 20 ml of sterile filtered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) stock solution in PBS (5 mg/ml) was added to each well reaching a final concentration of 0.5 mg MTT/ml. After 4 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in 100 ml/well DMSO (BDH laboratory Supplies, England) and measured spectrophotometrically in an ELISA plate reader (Model 550, Bio-Rad) at a wavelength of 655 nm. The results are expressed as percentage of control, that is, the untreated sister cultures. Each point represents the means7s.d. of five cultures. The data shown here are representative of two to three independent experiments. 165


Figure 5 Tissue response at day 7 after intracisternal injection of PPEEA, PEI, and their DNA complexes. (a, b) H&E-stained brain stem/cerebellum tissue sections from animals intracisternally injected with PPEEA (a) and PEI (b) polymers containing the same number, 100 nmol, of amino groups. No inflammation responses were observed. (c, d) TUNELstained tissue sections from animals intracisternally injected with 1 mg of DNA complexed with of PPE-EA (c) or PEI (d). For TUNEL staining, 50 ml of TUNEL reaction solution from the TUNEL staining kit (Roche) was added to each paraformaldehyde-fixed section. A volume of 50 ml of the label solution without terminal transferase was used as the negative control. After 60 min incubation at 371C and washing, samples were analyzed under a fluorescence microscope. Note TUNEL-positive cells in (d). The original photographs were taken at 40 magnification for (a) and (b) and 200 magnification for (c) and (d).

detectable in the PPE-EA/DNA complex treated animals (Figure 5c). PPE-EA is a recently developed water-soluble and biodegradable polyphosphoester with positively charged side chains.12,13 PPE-EA condenses DNA efficiently and protects it against nuclease and serum degradation.12 A unique feature of the PPE-EA gene delivery system is controlled release of plasmid from the polymer/DNA complexes, achieved as a consequence of polymer degradation. The released DNA retains structural and functional integrity and may subsequently be taken up by cells to mediate gene expression.12,13 The release rate is adjustable by varying the charge ratio of PPE-EA to DNA. At an N/P ratio of 0.5, complete release of DNA is achieved within 3 days in vitro. Higher charge ratios lead to slower release of DNA from the polymer DNA complexes. It may take 3 days for the onset of DNA release from the complexes at an N/P ratio of 2 to occur.12 This explains a slow increase of gene expression levels during the first 3 days after the injection of the complexes reported here. Complete release of DNA from complexes with a higher N/P ratio takes much longer, most likely because of the stronger grip on the DNA with the increased amount of the polymer in the complexes. This may serve as one mechanism underlying the prolonged gene expression observed in this study. The CSF can only accommodate a small injection volume,

such as the one reported here. To provide a high enough DNA dose, a high concentration of DNA complexes has to be used. This would probably lead to an even slower DNA release. Furthermore, the larger aggregates of PPEEA/DNA particles formed in physiological fluids would have difficulty to be transported from the CSF compartment into the superior sagittal sinus via absorption across the arachnoid granulations and villi. The retention of the biodegradable aggregates along the meningeal surface may facilitate sustained release of plasmid DNA in the CSF, prolonging the subependymal expression of the transgene. Large aggregates of PEI DNA complexes display acute toxicity after tail-vein injection and may kill 50% of the animals within 30 min.14,15 After CSF injection, the aggregates are one possible factor causing cell death in the meninges.9 PPE-EA/DNA complexes also form aggregates at an N/P ratio of 2, but display lower toxicity in neural cells and no apoptotic induction effects after injection into the rat CSF. PPE-EA is designed to have nontoxic building blocks and would ultimately be hydrolyzed within weeks into a-propylene glycol, phosphate, and ethanolamine, which have minimal toxicity profiles.12,13 The biocompatibility of the polymer raises the possibility of repeated injection to achieve even longer period of gene expression. Transient expression of plasmid DNA is a disadvantage of nonviral gene delivery systems in treating many of the neurological disorders, because of their slow pathogenesis. In an attempt to achieve prolonged transgene expression through repeated injection of PEI/DNA complexes into the CSF, we observed a 70% attenuation of gene expression following redosing at a 2-week interval.9 Using a PPE-EA instead of PEI in this kind of administration, one would expect a long-term gene expression at a significantly higher level. The intrathecal injection approach adopted in this study bypasses the blood–brain barrier by delivering reagents directly into the CSF. In other studies, direct injection into the CSF via the lateral ventricles of the rat brain, DNA complexed by cationic liposome16 and recombinant adenovirus vectors17 may mediate transgene expression throughout the entire brain. Given the small size of the mouse model, intracisternal injection is more practical than other intrathecal injection methods. Delicate techniques of intracisternal injection were exploited for inducing leptomeningeal metastases or delivering drugs,18 but seldom used for gene delivery. In this study, intracisternal gene delivery allowed widespread gene expression in the mouse brain, although the distribution pattern was somewhat vehicle-dependent. PEI/DNA complexes produced the most widespread dispersion, followed by PPE-EA/DNA complexes. Naked DNA, probably because of its vulnerability to DNAses, may not be able to diffuse intact far from the injection side. The difference in the distribution of gene expression between PPE-EA/DNA complexes at N/P ratios of 2 and 0.5 is probably attributed to the fact that at the low N/P ratio a significant portion of the DNA is dissociated from the complexes after injection. PEI/DNA complexes used in this study, which were prepared at an N/P ratio of 15, provided a high level of gene expression in the cerebral cortex, a region far away from the injection site. These findings suggest that a higher positive surface charge may facilitate widespread

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distribution of polymer/DNA complexes through the CSF. In summary, the present study has demonstrated that PPE-EA may mediate gene transfer in the CNS, with an efficiency superior to naked DNA and, in terms of prolonged gene expression, to PEI. Different from PEI, PPE-EA is biodegradable and displays low toxicity in the nervous system. To our knowledge, this study is the first one using a controlled DNA release system in the CNS. The results establish PPE-EA as a promising gene carrier applicable to the CNS.

Acknowledgements The work was funded by the Agency for Science, Technology and Research (A* STAR), Singapore to Institute of Bioengineering and Nanotechnology, Institute of Materials Research and Engineering, and Johns Hopkins Singapore.

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