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PEO-PPO-PEO carriers for rAAV-mediated transduction of human articular chondrocytes in vitro and in a human osteochondral defect model Ana Rey-Rico, Janina Frisch, Jagadeesh Kumar Venkatesan, Gertrud Schmitt, Isabel Rial-Hermida, Pablo Taboada, Angel Concheiro, Henning Madry, Carmen Alvarez-Lorenzo, and Magali Cucchiarini ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06509 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 15, 2016

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ACS Applied Materials & Interfaces

PEO-PPO-PEO carriers for rAAV-mediated transduction of human articular chondrocytes in vitro and in a human osteochondral defect model

Ana Rey-Rico a, Janina Frisch a, Jagadesh Kumar Venkatesan a, Gertrud Schmitt a, Isabel Rial-Hermida b, Pablo Taboada c, Angel Concheiro b, Henning Madry a, d, Carmen Alvarez-Lorenzo b, Magali Cucchiarini a, *

a

Center of Experimental Orthopaedics, Saarland University Medical Center,

Homburg, Germany b

Departamento de Farmacia y Tecnología Farmacéutica, R+DPharma Group

(GI-1645), Facultad de Farmacia, Universidade de Santiago de Compostela, Santiago de Compostela, Spain c

Departamento de Física de la Materia Condensada, Facultad de Física,

Universidade de Santiago de Compostela, Santiago de Compostela, Spain d

Department of Orthopaedics and Orthopaedic Surgery, Saarland University

Medical Center, Homburg, Germany

Running title: rAAV/PEO-PPO-PEO systems to target cartilage lesions

* Corresponding author: Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421 Homburg, Germany; Phone:

++

49-6841-1624-987;

Fax:

++

49-6841-1624-988;

[email protected]

1

ACS Paragon Plus Environment

E-mail:


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ABSTRACT

Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed to increase both the stability and the bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defect compared with free vector treatment, without detrimental effects on the biological activity of the cells or on their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current genebased treatments for human OA.

2


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Keywords: Human articular cartilage; osteoarthritis; rAAV gene transfer; poloxamers; poloxamines; micelles.

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1. Introduction

Osteoarthritis (OA) is a major, degenerative disease of the entire joint characterized by complex structural and functional tissue and cell alterations including the gradual and irreversible degradation of the articular cartilage (loss of proteoglycans and type-II collagen), the remodelling of the subchondral bone, and the formation of osteophytes

1-2

due to an impaired homeostasis

3-4

. Thus

far, none of the pharmacological treatments and surgical options available to manage OA has allowed to reproduce the original cartilage integrity in patients. The design of new, effective, and durable approaches is therefore of particular interest to prevent further tissue degeneration and stably repair the site of the lesions. Gene therapy is a valuable tool to achieve these goals as it offers the possibility of directly transferring genes coding for therapeutic factors within the repair tissue. Compared with the short-lived application of recombinant factors, gene therapy allows for the prolonged synthesis of a candidate agent

5-6

. While

protection against cartilage breakdown has been achieved upon delivery of sequences coding for agents with preventive and/or inhibitory activities (interleukin-1 receptor antagonist - IL-1Ra, siRNA inhibitors of NF-κB, thrombospondin-1 - TSP-1, kallistatin, pro-opiomelanocortin - POMC, Dickkopf1 - Dkk-1)

7-14

, restoration of extracellular cartilage matrix (ECM) components

and cells to their native levels and functions has not been reported yet, even when applying sequences for anabolic and/or proliferative factors such as the insulin-like growth factor I (IGF-I)

15-18

, transforming growth factor beta (TGF-β)

4


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15, 19-20

, fibroblast growth factor 2 (FGF-2)

(BMPs)

15

, the transcription factor SOX9

21-22

23-25

beta1,3-glucuronosyltransferase-I (GlcAT-I)

, bone morphogenetic proteins

, proteoglycan 4 (Prg4)

27

26

, or the

. Most of the work originally

performed to rejuvenate OA chondrocytes via gene therapy strategies was based on the use of nonviral retroviruses)

15-17, 22-23

17, 26-27

and classical viral vectors (adenoviruses,

. However, application of these vehicles in vivo remains

impaired by their low, short-term efficiencies (nonviral and adenoviral vectors), by a risk for insertional mutagenesis (retroviral vectors), and by their immunogenic potential (adenoviral vectors) 28. Recombinant vectors based on the human adeno-associated virus (AAV) instead have been reported for their high, extended gene transfer efficacy in articular chondrocytes both in vitro and in their dense ECM in situ (up to 80% for at least 150 days)

29

, probably due to their small size (~ 20 nm) and

maintenance under stable episomal forms, making them now preferred gene delivery vectors for applications in cartilage regenerative medicine

18-21, 25, 30-33

.

Still, the clinical administration of such a vector might be impeded by the presence of neutralizing antibodies against the AAV capsid proteins in patients 34

, especially by those present in the synovial fluid of patients with joint diseases

35

. Administration of rAAV vectors encapsulated in biocompatible materials is a

novel, potent approach to overcome such pre-existing barriers

36

. The design of

viral vector/polymer systems has so far relied on the formulation of virus-based material as i) gel depots for localized implantation, using for example in situ gelling dispersions of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers, ii) polyelectrolyte complexes that provide a cationicallycharged surface to favour gene transfer, iii) microcapsules of biodegradable 5



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polymers for the sustained release of the virus, and iv) polymer conjugates that provide stealth, cell-targeted shells to the virus 37-39. In the present study for the first time to our best knowledge, we examined the possibility of modifying human OA chondrocytes in vitro and in experimental cartilage lesions via rAAV delivered in polymeric micelles of linear poloxamer (PF68) or as X-shaped poloxamine (T908) in light of the advantageous features from these materials already used in the clinics

40

and of our previous work

showing the feasibility of targeting human bone marrow-derived mesenchymal stem cells (MSCs) with such systems 41. We first characterized the virus-micelle interactions by means of isothermal titration calorimetry and then provided evidence that application of an rAAV reporter (lacZ) gene vector via PEO-PPOPEO micelles based on PF68 and T908 enhanced the stability and bioactivity of the gene vector, leading to higher levels of safe transgene expression in human OA chondrocytes in vitro and in a human model of osteochondral defect relative to free vector treatment. These copolymers were further capable of protecting rAAV-mediated gene transfer against experimental neutralization using an AAVspecific antibody directed against the viral capsid. Remarkably, no deleterious effects of the micelles on cell viability and phenotype were noted in vitro and in the experimental osteochondral defects. These findings support the concept of combining rAAV gene transfer techniques with tissue engineering approaches for translational cartilage regeneration

36, 42

, showing a strong value for future

safe and effective delivery of therapeutic candidates in sites of tissue damage to remodel OA cartilage.

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2. Materials and methods

2.1. Materials Pluronic® F68 (PF68) and Tetronic® 908 (T908) were kindly provided by BASF (Ludwigshafen, Germany). The AAV-specific A20 antibody was generously provided by Progen (Heidelberg, Germany). The anti-type-II collagen (II-II6B3) antibody was from DSHB (Iowa, IA, USA). Biotinylated secondary antibodies and the ABC reagent were from Vector Laboratories (Alexis

Deutschland

GmbH,

Grünberg,

Germany).

The

AAVanced™

Concentration Reagent was from System Bioscience (Heidelberg, Germany). The Cy3 Ab Labelling Kit was from Amersham/GE Healthcare (Munich, Germany). The AAV Titration ELISA was from Progen, the Beta-Glo® Assay System from Promega (Mannheim, Germany), and the β-gal staining kit and Cell Proliferation Reagent WST-1 from Roche Applied Science (Mannheim, Germany). The Cytotoxicity Detection KitPLUS (LDH) was from Roche Applied Science. All other reagents were from Sigma (Munich, Germany).

2.2. Cells and osteochondral defect cultures

Articular cartilage was obtained from the joints of OA patients (Mankin score 7-9) undergoing total knee arthroplasty (n = 9). The study was approved by the Ethics Committee of the Saarland Physicians Council. All patients provided informed consent before inclusion in the study, and all procedures were in accordance with the Helsinki Declaration. Human OA chondrocytes

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were isolated as previously described

20

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and used not later than passage three.

Cells were maintained in DMEM, 10% FBS, 100 U/ml penicillin G, 100 µl/ml streptomycin (growth medium) for 12 h at 37ºC under 5% CO2 prior to addition of the various rAAV systems. Osteochondral defects created in human OA cartilage biopsies (n = 7) using a 1-mm drill needle in standardized cylindrical (6-mm diameter) as previously described

43

were incubated in growth medium

prior to addition of the various rAAV systems.

2.3. Plasmids and rAAV vectors

The constructs were derived from pSSV9, an AAV-2 genomic clone

44-45

.

rAAV-lacZ carries the lacZ gene encoding E. coli β-galactosidase (β-gal) under the control of the cytomegalovirus immediate-early promoter

20, 41, 46

. The

vectors were packaged as conventional (not self-complementary) vectors using a helper-free, two-plasmid transfection system in 293 cells with the packaging plasmid pXX2 and the Adenovirus helper plasmid pXX6 as previously described 20

. The vector preparations were purified with the AAVanced™ Concentration

Reagent according to the instructions of the manufacturer and titrated by realtime PCR

20, 41, 46

, averaging 1010 transgene copies/ml (~ 1/500 functional

recombinant viral particles).

2.4. Isothermal titration calorimetry (ITC) Virus-micelle binding studies were performed using a VP-ITC titration microcalorimeter from MicroCal Inc. (Northampton, MA) with a cell volume of 8


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1.436 ml at 25ºC. Samples were degassed in a ThermoVac system (MicroCal) prior to use. The sample cell was filled with PF68 or T908 polymeric micellar (10% sucrose medium) solutions and the reference cell with 10% sucrose medium only. The micellar solution (0.85% copolymer, i.e. 0.34 and 1.01 mM for T908 and PF68, respectively) was introduced into the thermostated cell by means of a syringe and stirred at 286 rpm, which ensured rapid mixing but did not cause foaming on solutions. Each titration consisted of an initial 2 µl injection (neglected in the analysis) followed by 55 subsequent 5-µl injections of rAAV-lacZ viral vector (1010 transgene copies/ml) (also dispersed in 10% sucrose medium) programmed to occur at 600 s intervals, sufficient for the heat signal to return to the baseline. The results of the ITC experiments in terms of the heat of injection normalized by the viral vector concentration added per each injection (Q*) were presented as a function of the rAAV-lacZ to polymer molar ratio. The heats of dilution from titrations of rAAV-lacZ dispersions in sucrose solution were subtracted from the heats from titrations of rAAV-lacZ dispersions in the micelle solution to obtain the net binding heats. All experiments were carried out at least in duplicate with a reproducibility of ± 5%. Raw data of interaction were analyzed as described previously

47

on the

basis of a set of two identical binding sites model by using the Affinimiter software. The two identical binding sites model employs the following fitting equation that incorporates Langmuir isotherm binding equilibria for two independent types of association:

Eq. 1

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where Q* is the heat per injection, ni the binding stoichiometry, Θi the fractional sites

of

macromolecule

occupied

by

ligand,

M

the

macromolecule

concentration, ∆Hi the enthalpy of interaction, V the cell volume, and the subindices 1 and 2 stand for the two sets of sites. One can solve for Θ1 and Θ2 using the equilibria equations for binding constants K1 and K2, with X being the total concentration of ligand and [X] the concentration of unbound ligand, as follows:

Eq. 2 Eq. 3

and

with

Eq. 4

To achieve an accurate fit of all floating parameters to these data, multiple assays were performed starting from random initial parameters. The same values were reached at the minimum χ2 regardless of the values of initialization. Finally, changes in free energy ∆Gi and entropy of binding ∆Si for each site were calculated from the fitted parameter values according to following standard, thermodynamic relations: Eq. 5

Eq. 6 where Rg is the universal gas constant.

10


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2.5. Cy3 labelling

rAAV vectors were labelled using the Cy3 Ab Labelling Kit according to the manufacturer’s recommendations and as previously described

48

. Briefly,

rAAV (1 ml) was mixed with sodium carbonate/sodium bicarbonate buffer (pH 9.3) for 30 min at room temperature, labelled with Cy3 and purified by extensive dialysis against 20 mM HEPES (pH 7.5)/150 ml NaCl

48

. Effective labelling was

monitored in the samples by live fluorescent microscopy with rhodamine filter set (Olympus CKX41, Hamburg, Germany).

2.6. Preparation of copolymer solutions with rAAV vectors

The appropriate amounts of each type of copolymer (PF68 or T908) were added to a given volume of 10% sucrose at 4ºC. The obtained poloxamer and poloxamine solutions were then mixed with rAAV (or Cy3-labelled rAAV; 1010 transgene copies/ml) in equal volumes, incubated on ice for 30 min, and used immediately

41

. The final copolymer concentration into the culture medium was

always 2% (w/v). Transmission electron microscopy (TEM) micrographs were recorded in a PHILIPS CM-12 apparatus (PHILIPS, The Netherlands) after uranyl acetate staining. Dynamic light scattering (DLS) were recorded in a Zetasizer® 3000HS (Malvern Instruments, UK).

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2.7. Delivery of rAAV vectors via polymeric micelles and evaluations

Solutions of copolymers (PF68, T908) containing the vectors (50 µl, containing polymer concentration 16% w/v and 0.25 x 109 transgene copies) were added to the upper compartment of permeable supports (Transwell Permeable Supports; Life Technologies, Darmstadt, Germany pore size: 0.4 µm) in 24-well plates containing DMEM (600 µl in the lower compartment). DMEM (350 µl) was then added to the upper compartment (final polymer concentration 2% w/v and 0.25 x 109 transgene copies). Plates were kept at 37ºC under oscillating agitation (50 osc/min) for 10 days. Aliquots of conditioned culture medium were collected and immediately frozen at -20ºC at preestablished time points in the lower compartment to monitor the diffusion of rAAV delivered via polymeric micelles. Diffusion of free rAAV vectors in absence of polymer was tested under similar conditions. The concentrations of viral particles present in the medium of culture were measured using the AAV Titration ELISA 49.

Bioactivity of the vectors (rAAV-lacZ) delivered via polymeric micelles or in its free form was assessed at the denoted time points by placing aliquots of the conditioned medium from the lower compartment (100 µl) in contact with human OA chondrocytes in monolayer culture (7,500 cells/well in 96-well plates) for 48 h at 37ºC to estimate β-gal activity (lacZ) using the Beta-Glo® Assay System with values expressed as Relative Luminiscence Units (RLU)

41

.

In both types of experiments, control conditions including cells cultured in the presence or absence of copolymers but without vector treatment were used as blanks and subtracted for each condition and time point evaluated. Quantitative 12


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measurements were performed on a GENios spectrophotometer/fluorometer (Tecan, Crailsheim, Germany).

2.8. Gene transfer efficacy using the rAAV/copolymer micelles

Monolayer cultures of human OA chondrocytes (3,000 cells/well in 96well plates) and osteochondral defect cultures were directly incubated with the rAAV/PF68 or rAAV/T908 micelles (20 or 40 µl for monolayer cultures; 100 µl for osteochondral defect cultures; final copolymer concentration 2%) and maintained for up to 10 days at 37ºC with three weekly medium changes

41

.

Control conditions included application of 10% sucrose (control "-"), similar amounts of free rAAV (10 or 20 µl for monolayer cultures; 40 µl for osteochondral defect cultures; 1010 transgene copies/ml) based on doses previously tested

20, 25

in 10% sucrose (v/v; control "+"), and copolymer with

10% sucrose (w/v; copolymer control).

For the neutralization assays, rAAV/PF68 and rAAV/T908 micelles were prepared as described above and subsequently incubated with the A20 antibody (final dilutions of 1:10 and 1:6 based on preliminary experiments) for 2 h at 4ºC

41

. The A20-treated rAAV/copolymer micelles were then added to

monolayer cultures of human OA chondrocytes and osteochondral defect cultures, which were incubated for 24 h as previously described analyses.

13


41

for further


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2.9. Detection of transgene expression

Expression of the transgene (lacZ) was determined by X-Gal staining using a β-gal staining kit and the Beta-Glo® Assay System (lacZ) 41. Quantitative measurements were performed on a GENios spectrophotometer/fluorometer (Tecan). Values from cells cultured in absence of vector with and without the copolymers were used as blanks and subtracted for each condition and time point evaluated.

2.10. Assessment of cell viability

Viability in monolayer cultures was measured using the Cell Proliferation Reagent WST-1, with absorbance values proportional to the cell numbers

41, 50

.

Controls included cells maintained in the presence or absence of a same dose of free vector and a condition with copolymer but without rAAV. Cell survival percentage was calculated as:

cell viability = [sample absorbance/absorbance of the negative control] x 100

Viability in osteochondral defect cultures was determined with the Cytotoxity Detection KitPLUS (LDH) by measuring the release of lactate dehydrogenase (LDH) activity in the supernatants of culture

20, 51

. Absorbance

at 450 nm was measured using a GENios spectrophotometer/fluorometer (Tecan) with the % of cytotoxicity calculated as:

cytotoxicity = [(experimental value - low control)/(high control - low control)] x 100

14


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where low control corresponds to samples without assay treatment and high control to samples placed in the lysis buffer provided by the kit. All measurements were performed on a GENios spectrophotometer/fluorometer (Tecan).

2.11. Histological and immunohistochemical analyses

Cells in monolayer cultures and osteochondral defect cultures were harvested at the denoted time points and fixed in 4% formalin. Fixed cells were stained with alcian blue and excess stain was washed off with double distilled water. The stain was quantified by overnight solubilization in 6 M guanidine hydrochloride to monitor absorbance at 595 nm

52-53

using a GENios

spectrophotometer/fluorometer (Tecan).

Fixed osteochondral defect cultures were further dehydrated in graded alcohols, embedded in paraffin, and sectioned (10 µm). Sections were stained with eosin and safranin O (matrix proteoglycans) as previously described 46

20, 41,

. Expression of type-II collagen was detected using a specific primary

antibody, a biotinylated secondary antibody, and the ABC method with DAB as the chromogen

20, 41, 46

. To control for secondary immunoglobulins, sections

were processed with omission of the primary antibody.

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2.12. Histomorphometry

The intensities of X-Gal staining, safranin O staining and type-II collagen immunostaining were measured at three random sites standardized for their surface or using ten serial histological and immunohistochemical sections for each parameter, test, and replicate condition to allow for calculation of standard deviations (SD). Values were expressed in pixels and as percents of pixels. Analysis programs included SIS AnalySIS (Olympus) and Adobe Photoshop Adobe Systems, Unterschleissheim, Germany) 20, 41, 46.

2.13. Statistical analysis

Each condition was performed in duplicate in three independent experiments. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 23 with p ≤ 0.05 considered statistically significant. Statistical analysis for monolayer cultures was performed by pair-wise comparison of experimental categories using two-tailed t-test. For studies in osteochondral defect cultures, one-way ANOVA analysis with Tukey’s LSD or Games-Howell post-hoc tests was applied to evaluate differences between groups.

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3. Results

3.1. Evaluation of the interactions rAAV/polymer micelles Although Pluronics have been tested as components of gel depots for localized release of viruses

38, 54-55

, there is still a paucity of information about

the interactions that virus can establish with the PEO-PPO-PEO block copolymers. In the case of the X-shaped counterparts with a central ethylenediamine

group

and

four

branches

of

PEO-PPO

(Tetronics),

fragmentation of the PPO component into four short arms may notably affect the performance of the copolymer. The molecule of poloxamine can be envisioned as two PEO-PPO-PEO triblocks covalently linked by the central ethylenediamine group. Therefore, for simplicity purposes T908 is also referred as a PEO-PPO-PEO along the manuscript. Thus, the first step of the present study was to elucidate whether rAAVlacZ could interact with the linear PF68 and the X-shape counterpart T908 when the copolymers were already self-assembled in the form of polymeric micelles. The nature and strength of interactions established between rAAV viral capsids and PF68 and T908 polymeric micelles in solution was investigated using ITC, a quite sensitive technique still barely applied to characterize virus interactions with nanocarriers and physiological membranes 56. The association of rAAV and polymeric micelles was analyzed by titrating an rAAV-lacZ dispersion onto PF68 and T908 dispersions at concentrations above the critical micellar concentration

57

. The net enthalpy of injection of

rAAV-lacZ into the PF68 and T908 micellar dispersions (after subtraction of the

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heat evolved after titration of the viruses into 10% sucrose solution) as a function of rAAV/copolymer molar ratio is shown in Figure S1 (Supporting Information). Solid lines represent the fitting to experimental data. The heat of interaction (Q*) of rAAV with polymeric micelles was initially exothermic, being more negative for T908 than for PF68, and the complexation process then became progressively less negative as the rAAV/block copolymer molar ratio increased, giving rise to an endothermic maximum at [rAAV]/[PF68] of ~ 4 x 1010

and at [rAAV]/[T908] of ~ 2 x 10-9. A slight increase in rAAV concentrations

led to an exothermic decrease which levelled off at larger rAAV/block copolymer molar ratios. The plateau region which corresponds to simple virus dilution effects was observed at molar ratios of ca. 2.5 x 10-9 and 5 x 10-9 for PF68 and T908, respectively. Regarding the thermodynamic characterization, Table 1 summarizes the enthalpy, free energy, entropy, binding constant, and stoichiometry of rAAV/block copolymer interactions derived on the basis of a two-binding site model. Interactions of T908 with rAAV involved binding constants more than one order of magnitude larger than those observed for PF68. Such differences indicate relatively stronger hydrogen bonding and attractive electrostatic interactions of T908 with the virus capsids compared with PF68. The stoichiometry of binding is fractional, corresponding to the interaction of multivalent substrate/ligands, such as rAAV, for which 1/ni would provide the functional valence of the multivalent virus to the copolymers. Regarding the enthalpy of interaction, the large exothermic values of ∆H1 agree with the potential predominance of hydrogen bonding, small dispersion forces of attraction, and electrostatic interactions between T908 and viral capsids being

18


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an average of all different processes occurring during the first injections of rAAV onto the copolymer solution. Conversely, ∆H2 was largely endothermic confirming the important role of desolvation forces at larger rAAV/copolymer molar ratios, which points to the disruption of polymeric micelles and subsequent expulsion of copolymer chains to the solution for the subsequent molecular rearrangements of these polymeric chains upon interaction with the viral capsids. ∆Si values corroborated this picture. ∆S1 values were large and negative confirming the predominant role of hydrogen bonding and electrostatic interactions between polymeric micelles and rAAV capsids at the first stages of the binding process, followed by a distinct entropically-driven mechanism as observed from the relatively large and positive entropy changes. The latter indicates strong changes in solvation, in particular release of water molecules to the solvent, probably corresponding to a polymeric micellar destabilization and subsequent rearrangement of copolymer chains to allow their adsorption onto the capsids or even leading to the formation of rAAV/polymer micelles.

Table 1. Evaluation of the interactions rAAV/polymer micelles. ∆Hi (enthalpy),

∆Gi (free energy), ∆Si (entropy), Ki (binding constant), and ni (stoichiometry) of the interactions between rAAV-lacZ and the PF68 and T908 polymeric micelles at 25°C (mean ± SD, n = 2). Values on first rows of each copolymer correspond to the first class of binding sites (I = 1) and values on the second ones correspond to the second class of binding sites (I = 2).

19



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10-6∆Hi

10-6∆Gi

10-6∆Si

Page 20 of 56

10-13 Ki 109ni

Copolymers (kcal/mol)

(kcal/mol)

(kcal/mol)

(M-1)

-676 ± 15

-40.8 ± 4.1

-2.4 ± 0.9

1.42 ± 0.08

0.21 ± 0.04

244 ± 7

-34.0 ± 3.7

0.7 ± 0.4

0.09 ± 0.01

0.04 ± 0.01

-1212 ± 70

-48.7 ± 3.8

-3.9 ± 0.7

34.60 ± 5.80

0.34 ± 0.02

313 ± 5

-40.2 ± 2.6

1.2 ± 0.4

1.13 ± 0.04

3.40 ± 0.05

PF68

T908

TEM micrographs (Figure S2a, Supporting Information) of rAAV-lacZ in sucrose solution showed 20-30 nm particles as expected from the size values previously reported for rAAV

48

. Much larger objects were recorded for rAAV-

lacZ/PF68 and rAAV-lacZ/T908 micelle dispersions, in good agreement with the formation of rAAV/polymer micelles. Aggregation of the individualized rAAV/polymer micelles may have occurred during processing of the sample for TEM visualization. DLS measurements gave rise to mean values of 195 nm and 225 nm for rAAV-lacZ/PF68 and rAAV-lacZ/T908 systems, respectively.

3.2. Efficacy of diffusion of active rAAV via micelles We then evaluated the ability of the micelles (2% final copolymer concentration [37]) to deliver rAAV vectors through a permeable support system to target human OA chondrocytes in a monolayer culture over time. A good maintenance of the vector concentrations was observed when rAAV was delivered in the micelle formulations compared with diffusion of the vector in its 20


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free form based on doses previously tested

20

(Fig. 1A). By day 2, delivery of

rAAV via the micelles led to 3- and 6-fold increases in vector concentrations using PF68 and T908, respectively, compared with free vector treatment, although statistical significance was not reached (p ≥ 0.130). An analysis of the cumulative percents of viral capsids diffused to the lower compartment medium of the permeable supports revealed higher amounts of rAAV particles delivered via the micelles over time compared with diffusion of free rAAV (Fig. 1B). While delivery of rAAV via PF68 led to the detection of 100% of the total amount loaded in the inserts by day 5, diffusion of rAAV via T908 was faster, with 100% rAAV diffusion by day 2 (p = 0.040 compared with free vector administration at similar time points). In contrast, diffusion of free vector led only to the detection of ~ 75% of the total amount of rAAV loaded after 10 days (p = 0.090 compared with T908 at a similar time point). These data suggest that the micelles increased the vector stability and may also favour vector diffusion, leading to the detection of higher amounts of rAAV capsids over time. We next tested whether the vectors delivered via polymeric micelles were functional and capable of transducing human OA chondrocytes in monolayer culture over time. During the first hours, the rAAV (lacZ) vector formulated in the micelles was particularly active as noted by much higher transgene (lacZ) expression in human OA chondrocytes compared with free vector treatment (45- and 78-fold difference for PF68 and T908, respectively; p ≤ 0.040) (Fig. 1C). After one day, both PF68 and T908 micelles allowed for the enhanced genetic modification of human OA chondrocytes compared with free vector treatment (~ 5-fold difference; p ≤ 0.010), with similar observations over the whole period of evaluation (10 days) although beyond this time point, significant 21



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Page 22 of 56

differences were only reached with T908 (p ≤ 0.010 relative to free vector treatment).

Fig.

1.

Diffusion

of

rAAV

encapsulated

in

PEO-PPO-PEO

micelles

([rAAV]/[PF68]: 4.36 x 10-16 molar ratio; [rAAV]/[T908]: 1.30 x 10-15 molar ratio). The different rAAV/micelles were prepared using rAAV-lacZ and processed to determine (A) the time course concentrations and (B) the accumulated amounts of rAAV-lacZ viral particles (VP) delivered with the micelles over time using the

22


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AAV Titration ELISA, and (C) the bioactivity of rAAV-lacZ released from the micelles with the Beta-Glo® Assay System upon contact with human OA chondrocytes in monolayer culture as described in the Materials and Methods. Free rAAV vector (rAAV-lacZ) was used as a control condition. *Statistically significant compared with free rAAV.

3.3. Efficacy of rAAV-mediated gene transfer and expression in human OA chondrocytes by direct application of rAAV/PEO-PPO-PEO micelles The next step was to examine the ability of the PEO-PPO-PEO micelles to promote rAAV-mediated gene transfer and expression in human OA chondrocytes over time by direct contact with cells in monolayer cultures. Successful rAAV-lacZ gene transfer was achieved in human OA chondrocytes when delivering the vector in the micelles (Fig. 2A). An estimation of the X-Gal staining intensities between days 3 and 10 revealed that delivery of rAAV via the micelles significantly enhanced gene transfer compared with free vector treatment, especially at the highest vector dose employed (up to 2.3-fold difference; p ≤ 0.001) (Fig. 2B). This finding was confirmed by estimating the βgal activities in the cells (up to 1.8-fold increase versus free vector treatment; p ≤ 0.050) (Fig. 2C). While delivery of rAAV via PF68 led to increased levels of transgene expression over time at both vector doses, T908 showed an effect only at the highest vector dose applied (up to 1.5-fold increase between days 3 and 10; p ≥ 0.010) (Fig. 2C). A vector-dose dependent effect was also noted with the micelles, especially after 10 days of treatment (up to 1.2-fold difference between 10 and 20 µl; p ≥ 0.160) (Fig. 2C).

23



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Fig. 2. Transgene expression in rAAV-transduced human OA chondrocytes in the presence of PEO-PPO-PEO micelles. Cells in monolayer culture were prepared and incubated with the rAAV/micelles using rAAV-lacZ as described in the Materials and Methods ([rAAV]/[PF68]: 5.80 x 10-10 molar ratio; [rAAV]/[T908]: 1.73 x 10-9 molar ratio). Cultures were processed at the denoted time points (A) for X-Gal staining (magnification x4, all representative data) with (B) corresponding histomorphometric analyses and (C) using the Beta-Glo® Assay System as described in the Materials and Methods. Control conditions 24


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included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+"). Cell treated with the copolymers in the absence of vector were used as blanks and subtracted for each condition and time point evaluated.*Statistically significant compared with control "+" at similar vector doses and time points.

3.4. Restoration of rAAV uptake in human OA chondrocytes by delivery via PEO-PPO-PEO micelles in conditions of gene transfer inhibition We further evaluated whether delivery of rAAV via PEO-PPO-PEO micelles was capable of overcoming potential neutralization by an AAV-specific (A20) antibody in monolayer cultures of human OA chondrocytes to mimic the natural host immune responses against AAV 34. While the presence of A20 significantly impaired gene transfer via rAAV in human OA chondrocytes relative to transduction conditions where the antibody was omitted (1.4- and 2-fold using 10 or 20 µl vector, respectively; p ≤ 0.050) (Fig. 3A-3C), delivery of rAAV via PF68 or T908 micelles in conditions of neutralization restored gene transfer to levels similar to those achieved upon free vector treatment in absence of A20 (up to 1.9- and 2.3-fold with 10 and 20 µl of vector, respectively; p ≤ 0.040) (Fig. 3A-3C). Interestingly, only T908 showed a dose-dependent effect in restoring rAAV gene transfer in the presence of A20 (p ≤ 0.020) (Fig. 3B and C).

25



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Fig. 3. Restoration of transgene expression in rAAV-transduced human OA chondrocytes using PEO-PPO-PEO micelles in the presence of an AAV-specific neutralizing antibody ([rAAV]/[PF68]: 5.80 x 10-10 molar ratio; [rAAV]/[T908]: 1.73 x 10-9 molar ratio). Cells in monolayer culture were prepared and incubated with A20-treated rAAV/micelles using rAAV-lacZ as described in the Materials 26


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and Methods (A20 dilution 1:10). Cultures were processed after one day (A) for X-Gal staining (magnification x4, all representative data) with (B) corresponding histomorphometric analyses and (C) using the Beta-Glo® Assay System as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+"). Cell treated with the copolymers in the absence of vector were used as blanks and subtracted for each condition and time point evaluated. *Statistically significant compared with free rAAV in the presence of A20.

3.5. Effects of rAAV-mediated gene transfer and expression on the biological activities of human OA chondrocytes upon delivery via micelles Possible deleterious effects of the PEO-PPO-PEO micelles on the viability and activities of human OA chondrocytes were evaluated in monolayer culture over time. No detrimental effect on cell viability was observed when delivering rAAV via PF68 or T908 micelles to the cells at any time point of the analysis as noted when using free vector application (p = 1.000) or in absence of vector treatment (p = 1.000) (Fig. 4A). Also important, modification of human OA chondrocytes via rAAV using the micelles did not alter the cell phenotype at any time point of the evaluation as assessed by an estimation of the levels of alcian blue staining relative to free vector treatment (p ≥ 0.050). Interestingly, a stronger dot-pattern of staining was seen early on, especially when the vector was provided via the polymeric micelles ( p ≤ 0.020) (Fig. 4B and 4C).

27



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Fig. 4. Biological activities in rAAV-transduced human OA chondrocytes in the presence of PEO-PPO-PEO micelles ([rAAV]/[PF68]: 5.80 x 10-10 molar ratio; [rAAV]/[T908]: 1.73 x 10-9 molar ratio). Cells in monolayer culture were prepared and incubated with the rAAV/micelles using rAAV-lacZ as described in the Materials and Methods. Cultures were processed at the denoted time points (A) to monitor cell viability using the WST-1 assay and (B) for alcian blue staining (magnification x10, all representative data) with (C) spectrophotometric

28


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analyses after solubilization as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+"). *Statistically significant compared with control "-".

3.6. Efficacy of rAAV-mediated gene transfer and expression in human osteochondral defect cultures via rAAV/PEO-PPO-PEO micelles In order to translate the approach using a more natural environment, the suitability of the micelles to deliver rAAV to human osteochondral defect cultures was investigated taking into account free vector doses previously tested

25

(Fig. S3A, Supporting Information). Application of Cy3-labelled

rAAV-lacZ first revealed a higher diffusion of the vectors when encapsulated in PF68 and T908 micelles both in the top surface from the region adjacent to the defects and in-depth within the defects compared with free vector treatment (Fig. S3B, Supporting Information). The Cy3 fluorescent signal was well maintained over time when rAAV was delivered via polymeric micelles compared with free vector application showing a quenched fluorescence intensity (Fig. S3B, Supporting Information). No signal was evidenced in the presence of copolymers without Cy3-labeled rAAV at any time point of the analysis (not shown). Successful rAAV-mediated gene transfer was achieved in human osteochondral defect cultures when rAAV-lacZ was administered either in a free form or using the both types of micelles for at least 10 days, the longest time point evaluated (p ≤ 0.020). A histomorphometrical analysis of the percents of

29



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X-Gal staining of the top surface of the samples revealed 1.1-fold increases when rAAV-lacZ was provided via PF68 or T908 micelles relative to free vector treatment although without reaching statistical significance (p ≥ 0.875) (Fig. 5A and 5B). Interestingly, an analysis of the mean intensities of X-Gal staining of the top surface of the samples revealed the highest staining intensity when rAAV-lacZ was supplied via T908 micelles (p ≤ 0.030 compared with other conditions) (Fig. 5C). A similar trend was noted when measuring the mean intensities of X-Gal staining in cross-sectional sections from the osteochondral defect cultures, showing a 1.1-fold increase in staining intensity when rAAVlacZ was delivered in T908 micelles compared with free vector application (p = 0.030) (Fig. 5C).

30


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Fig. 5. Transgene expression in rAAV-transduced human osteochondral defect cultures in the presence of PEO-PPO-PEO micelles ([rAAV]/[PF68]: 2.82 x 10-9 molar ratio; [rAAV]/[T908]: 0.84 x 10-8 molar ratio). Defects were prepared and incubated with the rAAV/micelles using rAAV-lacZ as described in the Materials and Methods. Cultures were processed after 10 days (A) for X-Gal staining

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(macroscopic views of the top surface of the samples; eosin-stained histological cross-sections: magnification x2) (all representative data) with corresponding histomorphometric analyses of (B) the percents of X-Gal staining of the top surface and of (C) the mean intensities of X-Gal staining of the top and eosinstained histological cross-sections as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+"). Statistically significant compared with *control "-" , #control "+", and §rAAVlacZ/PF68.

3.7. Effects of rAAV-mediated gene transfer and expression on the biological activities in human osteochondral defect cultures upon delivery via micelles A next step was to examine whether delivery of rAAV via PEO-PPO-PEO micelles impaired the biological activities in the human osteochondral defect cultures. No deleterious effect on cell viability was reported when rAAV was delivered for 10 days to the osteochondral defect cultures via PF68 or T908 micelles as noted when using the vector in its free form or in absence of vector application (p ≥ 0.250) (Fig. 6A). Remarkably, administration of rAAV via the micelles did not modify the phenotype in the osteochondral defect cultures as monitored by histological and histomorphometric analyses of safranin O staining (Fig. 6B) and of type-II collagen deposition (Fig. 6C) compared with free vector treatment and to a lack of vector delivery (p ≥ 0.210).

32


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Fig. 6. Biological activities in rAAV-transduced human osteochondral defect cultures in the presence of PEO-PPO-PEO micelles ([rAAV]/[PF68]: 2.82 x 10-9 molar ratio; [rAAV]/[T908]: 0.84 x 10-8 molar ratio). Defects were prepared and incubated with the rAAV/micelles using rAAV-lacZ as described in the Materials 33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and Methods. Cultures were processed after 10 days (A) to monitor cell viability using the LDH assay, (B) for safranin O staining with corresponding histomorphometric analyses, and (C) for type-II collagen immunostaining with corresponding histomorphometric analyses as described in the Materials and Methods (magnification x2, all representative data). Control conditions included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+").

3.8. Restoration of rAAV uptake in human osteochondral defect cultures by delivery via micelles in conditions of gene transfer inhibition We finally evaluated the protective effects of the PEO-PPO-PEO micelles against AAV-specific A20-induced neutralization of gene transfer in human osteochondral defect cultures to mimic the natural exposition of the vectors to neutralizing antibodies in patients (Fig. 7). A histomorphometric analysis of the top surface of the osteochondral defect cultures revealed a significant reduction of the percents of rAAVmediated transduction in the presence of A20 (p = 0.040 compared with samples treated with free rAAV in similar conditions without A20) (Fig. 7A and 7B). Delivery of rAAV-lacZ via PF68 and T908 micelles restored the percentages of transgene expression to 4.2- and 5.3-fold (p ≤ 0.010 compared with free rAAV-lacZ treatment in the presence of A20) reaching levels similar to those achieved with free vector in absence of A20 (p ≥ 0.100). No signal was evidenced without vector addition (p ≤ 0.020) (Fig. 7B). An analysis of the intensities of X-Gal staining of the top surface of the osteochondral defect cultures showed that both PF68 and T908 micelles promoted higher intensities 34


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than those noted in samples treated with free rAAV-lacZ in the presence of A20, although only application of T908 allowed to reach statistical significance (p = 0.001) (Fig. 7C). A similar trend was noted when evaluating the cross-sectional intensities of X-Gal staining, with higher values in osteochondral defects treated with the vector delivered in PF68 or T908 micelles compared with free rAAVlacZ in the presence of A20 (p ≤ 0.010) and the highest ones using T908 although statistical significance was not reached compared with free vector treatment in absence of A20 (p = 0.240).

35



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Fig. 7. Restoration of transgene expression in rAAV-transduced human osteochondral defect cultures using PEO-PPO-PEO micelles in the presence of an AAV-specific neutralizing antibody ([rAAV]/[PF68]: 2.82 x 10-9 molar ratio; [rAAV]/[T908]: 0.84 x 10-8 molar ratio). Defects were prepared and incubated with A20-treatedrAAV/micelles using rAAV-lacZ as described in the Materials and Methods. Cultures were processed after one day (A) for X-Gal staining 36


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(macroscopic views of the top surface of the samples; eosin-stained histological cross-sections:

magnification

x2)

with

corresponding

histomorphometric

analyses of the percents of X-Gal staining of the top surface (B) and of the mean intensities of X-Gal staining of the top and eosin-stained histological cross-sections (C) as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (no vector; control "-") and application of free rAAV vector (rAAV-lacZ; control "+"). Statistically significant compared with *control "-" and presence of A20.

37


#

free rAAV in the


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Page 38 of 56

4. Discussion

The use of rAAV gene transfer vectors is a promising strategy to treat a gradual disease like OA as these constructs have been shown to competently and durably modify damaged articular cartilage in situ

18, 20, 29

compared with

other less effective gene delivery methods (nonviral, adenoviral, retroviral vector)

5-6

. Yet, application of rAAV in the clinical situation might be precluded

by the presence of neutralizing antibodies in the patients, especially by those against the viral capsid proteins

34

. An attractive approach to circumvent such

hurdles is to administer rAAV via biocompatible controlled delivery systems 42

. Here, based on our previous evaluations in hMSCs

41

36,

and for the first time to

the best of our knowledge, we tested the benefits of providing rAAV vectors to human OA chondrocytes in vitro and in experimentally created osteochondral defects using polymeric (PEO-PPO-PEO) micelles based on poloxamer PF68 or poloxamine T908. Since the nature and strength of the interactions of rAAV vectors and polymeric micelles have not been previously investigated, ITC analysis was carried out to gain an insight into interactions involved during the formation process of the rAAV/polymer systems we developed. The heat release profiles (Figure S1) ressembled those already observed, for example, when studying protein-drug and polymer-surfactant systems

58

. The initial negative ∆Hi values

may originate from hydrogen bonding formation between rAAV capsids and polymeric micelles. T908 micelles might also electrostatically interact with the negatively

charged

viral

capsid

thanks

to

the

partially

protonated

ethylenediamine core of this copolymer (absent in the lineal PF68) which

38


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explains the initial, more negative Q* values. Strong electrostatic interactions of cationic compounds with negatively-charged AAV particles have been previously reported

59-60

. The steep rise in the endothermic profile is likely

dominated by the dehydration of block copolymer micelles and subsequent solvent reorganization upon their interaction with rAAV capsids, probably creating an adsorbed polymeric layer surrounding them. Also, additional contributions may arise from charge shielding effects of viral capsids upon interaction with micelles. While it is not possible to have a whole picture to depict whether polymeric micelles are directly adsorbed onto the virus capsids or reorganize themselves by releasing copolymer chains which subsequently interacts with the capsids forming some sorts of rAAV/polymer micelles, the presence of only one endothermic maximum followed by a progressive, smooth exothermic heat decrease as the rAAV/copolymer ratio increases suggests that a certain amount of copolymer monomers is expelled to solution as more capsids are present, hence pointing to the need of, at least, a partial disruption of

bare

copolymer-only

micelles,

and

subsequent

polymeric

adsorption/formation of rAAV/polymer micelles. The formation of rAAV/polymer micelles was also supported for the increase in size of the aggregates recorded using TEM and DLS (~ 200 nm), compared with the 20 nm size of single rAAV and PF68 and T908 micelles. The relatively large size of copolymer micelles may be due to the ability of sucrose (10%) to decrease the lower critical solubility temperature of PEO-PPO-PEO which in turn favours self-assembly as reported for other saccharides

61

. It is interesting to note that large aggregates

of retroviruses after polyelectrolyte complexation (e.g. with polylysine) have been reported to be advantageous in terms of diffusion towards the cell surface

39



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Page 40 of 56

and of internalization inside the cells compared with viruses in their free form

37

.

In our case, the formation of rAAV/polymer micelles may provide a chargeshielding effect due to the coating of the negatively charged surface of rAAV with protruding neutral PEO chains which may favour the interaction with the negatively charged membrane of the cells. Subsequent experiments were carried out using rAAV/copolymer molar ratios close to the endothermic maximum of the ITC plot in order to ensure saturation of capsids with copolymer molecules. Our data further indicate that encapsulation of a reporter (lacZ) rAAV in PF68 and T908 micelles enhanced the concentration, stability, and bioactivity of the vectors, resulting in higher levels of lacZ expression in human OA chondrocytes in conditions of vector diffusion compared with free vector treatment. This is in good agreement with earlier observations using these polymeric micelles as nanocarriers of recombinant factors and drugs

57

or when

delivering rAAV via other types of polymers and formulations as blends of sucrose/mannitol protamine and surfactant

62

or cationic polymers

63

. Our

results further demonstrate that administration of rAAV via PF68 or T908 micelles also allowed to successfully modify human OA chondrocytes upon direct contact with the micelles in vitro, without altering the viability of the cells nor their phenotype while achieving again higher levels of transgene expression versus free vector treatment, all concordant with our previous findings using similar systems to target hMSCs

41

. Most importantly, both PF68 and T908

micelles were capable of preventing neutralization of rAAV gene transfer when applying a viral capsid-specific antibody while restoring transgene expression to levels similar to those achieved upon free vector treatment in absence of

40


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neutralization, also in agreement with our previous findings in hMSCs

41

.

Interestingly, T908 showed a dose-dependent, more potent protective effect against neutralization, possibly due to a higher number of shelter PEO units in the PEO shell 41 relative to PF68. Most notably, we provide evidence of the suitability of the current approach to modify cells in a more natural, translational environment by providing rAAV via PEO-PPO-PEO micelles to a human osteochondral defect model. Administration of Cy3-labelled vectors in this system first revealed a stronger signal when using PF68 and T908 compared with free vector treatment, again possibly due to the increased stability of rAAV when delivered via such compounds as already noted in vitro. Successful gene transfer was achieved in the defects upon application of the rAAV/micelles relative to free vector treatment, especially when using T908. Of further note, transfer of rAAV via PF68 or T908 did not impair the biological activities and cell phenotype in the defects, concordant with the findings in vitro and in good agreement with both the work validating the value of the osteochondral defect model

43

and with

our own observations when directly providing rAAV to human OA cartilage samples in situ

20

. Remarkably, formulation in PF68 and T908 micelles also

allowed to both overcome antibody-specific neutralization of rAAV gene transfer and restore transgene expression in the defects to levels similar to those noted upon free vector treatment in absence of neutralization, consistent with the findings in human OA chondrocytes in vitro. Again, T908 was more potent to protect rAAV from neutralization as observed in vitro. Overall, and for the first time to the best of our knowledge, the present work demonstrate the benefits of using PF68- and T908-based micelles as

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effective and stable rAAV delivery systems to promote the genetic modification of human OA chondrocytes in vitro and of experimental human osteochondral defects while affording protection against AAV-specific neutralization. Such a direct approach may avoid the more complex administration of genetically modified cells like performed by Iwai et al. 64 who implanted TGF-β3-transfected cells (chondrocytes, hMSCs) within such defects or by de Vries-van Melle et al. 65

who provided unmodified hMSCs via different types of hydrogels (alginate,

alginate/hyaluronic acid - HA, fibrin, thermoresponsive HA grafted with poly(Nisopropyl acrylamide) side-chains - HA-pNIPAM) in similar models. PEO-PPOPEO micelles may provide powerful, future tools for the delivery of therapeutic rAAV vectors carrying candidate genes for the remodelling and repair of damaged cartilage (IL-1Ra, siRNAs, TSP-1, kallistatin, POMC, Dkk-1, IGF-I, TGF-β, FGF-2, BMPs, SOX9, Prg4, GlcAT-I)

7-27

. Work is ongoing to evaluate

the feasibility of translating the current approach in experimental models of clinically-relevant cartilage injury in vivo as a means to corroborate the findings in vitro and in situ

7-14, 22, 26, 30-33

. Internalization of rAAV/polymeric micelles into

the cells may occur presumably in a independent way of binding to the rAAV receptor

41, 66

, but the exact mechanism is also under active investigation. The

use of such micelles as rAAV carriers opens new avenues of research to develop effective and appropriate treatments for human OA and even for focal cartilage lesions by controlling the duration and localization of the therapeutic gene counteracting the slow and irreversible progression of this disease.

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5. Conclusions

The present study reveals the potential of using controlled delivery systems for rAAV gene transfer vectors to improve the current gene-based treatments for human OA by increasing the temporal and spatial presentation of therapeutic agents into the targets while overcoming the physiological barriers. We showed that encapsulation of rAAV in systems based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as linear poloxamers (PF68) or X-shaped poloxamines (T908) allowed to increase both the stability and bioactivity of the vectors, promoting higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with free vector treatment, without detrimental effects on the biological activities of the cells or on their phenotype. Protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPOPEO micelles, especially when using T908. PEO-PPO-PEO micelles may provide powerful, future tools for the delivery of therapeutic rAAV vectors for the remodelling and repair of damaged cartilage.

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Supporting Information

Heats of interaction of rAAV with PF68 and T908 polymeric micelles; TEM micrographs of rAAV-lacZ, rAAV-lacZ/PF68 micelle dispersion, and rAAVlacZ/T908 micelle dispersion after uranyl acetate staining; design and characterization of rAAV-mediated gene transfer via PEO-PPO-PEO micelles in the human osteochondral defect cultures.

Conflict of Interest

The authors have no conflicts of interest to declare.

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Acknowledgments

This

work

was

supported

by

a

grant

from

Deutsche

Forschungsgemeinschaft (DFG RE 328/2-1 to ARR, HM, MC). The support of MINECO (SAF2014-52632-R and MAT2013-40971-R) Spain and FEDER is also acknowledged. The authors thank R.J. Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC), X. Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA), and E.F. Terwilliger (Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA) for providing the genomic AAV2 plasmid clones, the pXX2 and pXX6 plasmids, and the 293 cell line.

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Graphical abstract 90x60mm (300 x 300 DPI)


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PEO-PPO-PEO Carriers for rAAV-Mediated ... - ACS Publications

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