Determination of Effective Raav-Mediated Gene Transfer Conditions to Support Chondrogenic Differentiation Processes in Human Primary Bone Marrow Aspirates

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ORIGINAL ARTICLE Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates

A Rey-Rico1, J Frisch1, JK Venkatesan1, G Schmitt1, H Madry1,2 and M Cucchiarini1

The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90–94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml − 1 hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.

Gene Therapy (2015) 22, 50–57; doi:10.1038/gt.2014.90; published online 23 October 2014

INTRODUCTION advantage to enhance the cellularity like in the case of large Damage to the articular cartilage is a prevalent, major unsolved cartilage lesions. Nevertheless, ex vivo gene transfer methods problem in a variety of joint disorders,1 including joint trauma2 include steps of cell harvesting and expansion, making them and osteoarthritis.3 Since cartilage is an avascular tissue, it does more complex and laborious. The identification of alternative, not have plain access to chondroprogenitor cells that can support convenient gene delivery procedures is thus under active and participate in regenerative processes.4–7 Therapeutic inter- investigation such as options based on the supply of whole bone 15 ventions such as marrow stimulation techniques by perforation of marrow aspirates instead of isolated progenitor cells. In this the subchondral bone (microfracture, Pridie drilling and abrasion regard, different groups evaluated the feasibility of modifying arthroplasty) have been developed to address this issue, allowing rabbit and sheep bone marrow aspirates via adenoviral gene for the invasion of cells from the bone marrow to repopulate a transfer, reporting marker gene expression in vitro (up to defect and contribute to its repair.7–9 Yet, such options do not 21 days)16,17 and in cartilage defects in vivo (up to 7 days).17 predictably restore an original cartilage structure and function in Although such vectors generally allow only for short-term the lesion as the newly formed tissue is mainly of fibrocartilagi- transgene expression levels and induce undesirable immune – nous nature.7,8,10,11 Although this type of cartilage is clinically responses in the host,4 6,14 those based on the adeno-associated useful, allowing for a reduction of pain and an improved joint virus (AAV) appear more advantageous as recombinant AAV function, it is poorly organized, of lesser mechanical quality and (rAAV) vectors can efficiently, safely and durably transduce usually degrades over time and under loading.12 most cells of the musculoskeletal system among which bone Administration of agents capable of modulating chondrogenic marrow-derived mesenchymal stem cells (MSCs) both in vitro – differentiation pathways in progenitor cells is an attractive and in cartilage defects in vivo.18 22 Furthermore, AAV is a approach to improve the quality of the healing response of nonpathogenic, replication-defective human parvovirus,23 which – cartilage to injury. Gene therapy is a powerful tool to promote the is much less immunogenic than adenoviruses.4 6,14 expression of therapeutic agents in their targets over sustained, As a matter of fact, there is no evidence in the literature relevant periods of time either via direct gene vector application showing that rAAV vectors are capable of genetically modifying or by indirect procedures using genetically modified cells.4–6,13,14 primary human bone marrow aspirates. In the present study, we Although direct approaches are relatively simple to execute, those therefore examined the efficacy and duration of transgene based on indirect procedures are considered safer as no free expression mediated by rAAV-2 vectors in such aspirates and vector particles are introduced in the recipient, having the further tested whether gene transfer with this class of vector modulates

1Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany and 2Department of Orthopaedic Surgery, Saarland University Medical Center, Homburg/Saar, Germany. Correspondence: Professor M Cucchiarini, Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, Homburg/Saar D-66421, Germany. E-mail: [email protected] Received 15 April 2014; revised 23 July 2014; accepted 26 August 2014; published online 23 October 2014 rAAV gene transfer in human bone marrow aspirates A Rey-Rico et al 51 the chondrogenic differentiation potential in transduced samples. samples were collected in the presence of hirudin instead of We performed our evaluations in the absence of heparin, an heparin27 using concentrations previously tested in human cell anticoagulant present at the time of collection that is known to lines24,25, revealing no difference in the aspect of the samples inhibit the attachment of rAAV-2 particles at the surface of human (Figure 2a). cell lines by binding to the cell membrane receptor of the virus We first examined whether hirudin impaired the early adsorp- (a heparin sulfate proteoglycan), consequently impairing their tion of rAAV on primary human bone marrow aspirates. An – functionality.24 26 Instead, we replaced heparin with hirudin (an analysis by X-gal staining of aspirates 2 days following application alternative anticoagulant used in patients with heparin-induced of rAAV-lacZ at a high vector dose (40 μl)18,28 revealed that the thrombocytopenia)27 that does not display such inhibitory presence of this alternative anticoagulant did not prevent rAAV- effects.24,25 Our data show that rAAV are particularly well suited mediated gene transfer and expression compared with control to persistently modify primary human bone marrow aspirates samples, regardless of the concentration of hirudin provided treated with hirudin in contrast with heparin, without impeding (Figure 2b). The transduction efficiencies reached up to 90–97% the chondrogenic differentiation processes. Such processes could compared with control samples (always ⩽ 4%) as noted by be even activated when applying the chondrogenic transcription morphometric analyses performed on macroscopic views and factor SOX9 that was further capable of reducing undesirable histological sections from aspirates (Figures 2b–d). hypertrophic maturation. The use of bone marrow aspirates Evaluation of the efficacy of transgene expression in hirudin- modified by rAAV thus has strong value for therapeutic gene- treated aspirates transduced with rAAV-lacZ over a longer period based approaches to treat articular cartilage defects. of time (21 days) further revealed that sustained, elevated levels of β-gal activities were achieved in extended lacZ-transduced cultures compared with control samples especially when applying RESULTS the anticoagulant at a concentration of 0.5 mg ml − 1 (up to 7.5-fold Inhibitory effects of heparin on rAAV-mediated gene transfer in difference relative to the untransduced aspirates; P ⩽ 0.030; primary human bone marrow aspirates Figure 3: top panels). Interestingly, a plateau of expression was As heparin was reported to strongly interfere with rAAV-2- rapidly reached using the lowest dose applied and well mediated gene transfer in human cell lines,24–26 our goal was to maintained at this concentration of hirudin (P ⩾ 0.329). At lower − first examine whether this anticoagulant displayed similar effects or higher concentrations of anticoagulant (0.2 or 5 mg ml 1), the − in primary human bone marrow aspirates. levels of expression were inferior to those achieved at 0.5 mg ml 1 To test this hypothesis, we evaluated the efficacy of rAAV-lacZ- or the fold increases to the untransduced controls were lower mediated gene transfer in samples treated or not with heparin than in such a condition (1.5-fold using 40 μl rAAV versus no − 1 using increasing doses of vector and compared with untrans- vector treatment at 5 mg ml hirudin), although statistical duced (control) samples. A quantitative analysis of lacZ expression significance was not reached (P ⩾ 0.193). When the samples were by Beta-Glo assay (Promega, Mannheim, Germany) revealed that processed to evaluate possible undesirable effects of the different rAAV transduction was inhibited in heparin-treated samples treatments upon the proliferative activities in the aspirates by at any vector dose applied whereas effective, dose-dependent WST-1 assay (Figure 3: lower panels), neither deleterious effects of β-galactosidase (β-gal) activities were detectable in non- hirudin nor of the vector were seen at any concentration of ⩾ anticoagulated aspirates following administration of rAAV-lacZ anticoagulant or of vector applied (P 0.102), revealing instead (always P ⩽ 0.05; Figure 1). No signal was evidenced in the control higher rates of proliferation in the presence of rAAV (up to 3.6-fold difference, P ⩽ 0.035) in good agreement with previous findings in samples, independently in the presence of heparin (data not isolated human cells transduced with equivalent (low) amounts shown). of vector.29 These results were corroborated by an analysis of cytotoxic events in the samples, showing no deleterious effects of Use of hirudin as an alternative anticoagulant that allows for the the anticoagulant or of the vector treatment at any concentration effective modification of primary human bone marrow aspirates or dose applied compared with conditions without hirudin and/or via rAAV gene delivery vector (always ⩽ 1% after 2 or 21 days, P ⩾ 0.089). Evaluation of To circumvent the inhibitory effects of heparin on the transduc- transgene expression over extended periods of time in hirudin- tion of human bone marrow aspirates with rAAV-2 vectors, treated aspirates transduced with a high dose of rAAV-RFP (rAAV- red fluorescent protein) vector (40 μl) finally demonstrated that a strong red fluorescent signal was still evidenced after 125 days 100,000 (the longest time point evaluated) compared with control no anticoagulant treatments (Figure 4). Applying hirudin at a concentration of − 1 heparin (5,000 I.U./ml) 0.5 mg ml allowed to generate a compact, intense RFP signal in 75,000 the samples, stronger than in the aspirates treated at lower (0.2 mg ml − 1) or higher (5 mg ml − 1) concentrations of anticoagulant fl 50,000 where the levels of uorescence were also more sparse (0.2 mg ml − 1) or more rapidly decreased over time (5 mg ml − 1). RLU/aspirate 25,000 Primary human bone marrow aspirates treated with hirudin maintain their potential for chondrogenic differentiation − 1 0 In light of these findings, we selected hirudin at 0.5 mg ml as 0102040the best suited concentration for effective, safe and prolonged rAAV-lacZ (µl) rAAV-mediated gene transfer and expression in primary human bone marrow aspirates. Application of rAAV-lacZ (40 μl) was Figure 1. Inhibition of rAAV-mediated gene transfer in heparin- treated human bone marrow aspirates. Samples were collected in performed compared with control transduction to test whether heparin or let untreated for transduction with increasing doses of the presence of hirudin and of the vector itself affected the rAAV-lacZ (10, 20 or 40 μl). Transduced samples were processed chondrogenic differentiation processes in aspirates maintained in 2 days after vector administration by Beta-Glo assay as described in defined medium. Successful chondrogenesis was seen in all the Materials and Methods. aspirates as evidenced by intense toluidine blue staining and

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 50 – 57 rAAV gene transfer in human bone marrow aspirates A Rey-Rico et al 52 type-II collagen deposition on histological sections of aspirates 76 ± 3% in untransduced aspirates collected without hirudin or − (Figure 5a). Neither the presence of hirudin nor that of rAAV treated with hirudin 0.5 mg ml 1 and in lacZ-transduced hirudin- altered the potency of the samples for chondrogenesis compared treated aspirates, respectively, P ⩾ 0.277) and of immunostaining with control conditions (absence of hirudin and of vector for type-II collagen (80 ± 2%, 83 ± 2% and 79 ± 1%, respectively, treatment), as confirmed by an histomorphometric analysis of P ⩾ 0.187; Figure 5b). Further analysis of potentially undesirable the intensities of toluidine blue staining (76 ± 2%, 79 ± 2% and effects of the treatments revealed that neither the presence of hirudin nor of the rAAV vectors in the aspirates induced premature hypertrophy and terminal differentiation as observed by moderate immunostaining for type-X collagen on histological sections of a heparin hirudin aspirates (Figure 5a), a result also confirmed by histomorpho- (5,000 I.U./ml) (0.2 mg/ml) (0.5 mg/ml) (5 mg/ml) metric analysis (14 ± 3%, 11 ± 3% and 9 ± 2%, respectively, P ⩾ 0.110; Figure 5b). When a candidate rAAV vector carrying the potent chondrogenic SOX9 transcription factor was provided instead of lacZ at a similar concentration of hirudin, increased effects upon the chondrogenic features in the aspirates were noted by more intense toluidine blue staining and type-II collagen deposition compared with all other conditions (Figure 5a), a finding b hirudin confirmed by histomorphometry (95 ± 2% and 98 ± 1%, respec- ⩽ (0.2 mg/ml) (0.5 mg/ml) (5 mg/ml) tively, P 0.001; Figure 5b), revealing less voids and a more compact structure of the aspirates. Also notably, application of rAAV-FLAG-hsox9 further decreased the expression of type-X collagen relative to all other conditions (4 ± 1%, P ⩽ 0.001; Figures no vector 5a and b). These effects of the SOX9 treatment were associated with enhanced expression levels of the transgene compared with the controls (85 ± 2% versus 10 ± 2%, 11 ± 3% and 10 ± 1%, P ⩽ 0.001; Figure 5a).

rAAV-lacZ DISCUSSION (40 µl) The articular cartilage is an avascular, aneural tissue without lymphatic drainage that has a reduced capacity for self repair. Surgical interventions that make cells from the subchondral bone marrow accessible to a cartilage lesion do not allow for the c no vector rAAV-lacZ (40 µl) production of an original, hyaline cartilage in the repair tissue.7,8,10,11 Active clinical and experimental research is ongoing to improve the course of the healing in such defects, often emphasizing on the implantation of isolated cells known to contribute to the repair process (chondrocytes and MSCs).30–33 Despite critical advances, such transplantation procedures require complex steps of cell extraction and expansion in culture prior to re-administration in the lesions and like for marrow stimulation techniques, they do not restore a native cartilage. In this regard, the use of whole bone marrow aspirates rather than the use of isolated cells, combined with a simple, effective step of gene transfer to activate the reparative processes in the defects is an d no vector attractive strategy for improved, clinically applicable treatments µ rAAV-lacZ (40 l) for cartilage lesions. Interestingly, genetic modiﬁcation of bone (*) (*) (*) (*) marrow aspirates has been attempted in samples from rabbits and 100 sheep by application of immunogenic adenoviral vectors that allowed for relatively short-term transgene expression levels (21 days in vitro and 7 days in vivo).16,17 Instead, vectors derived 75 from the nonpathogenic, low immunogenic AAV human parvovirus are long known for their safe and persistent ability to transduce cells of the musculoskeletal system like chondrocytes

50 Figure 2. Detection of early lacZ expression in hirudin-treated human bone marrow aspirates transduced with rAAV. (a) Macro- scopic views of aspirates collected in hirudin (0.2, 0.5 or 5 mg ml − 1) 25 compared with heparin as described in the Materials and Methods. (b) X-Gal staining of hirudin-treated aspirates at various concentra-

Transduction efficiencies (%) Transduction tions of anticoagulant following application of rAAV-lacZ (40 μl; day 2 post-transduction). (c) Histological evaluation of sections from − 1 0 hirudin-treated aspirates (representative analysis at 0.5 mg ml ) hirudin transduced by rAAV-lacZ (40 μl; day 2 post-transduction; magnifica- (0.2 mg/ml) (0.5 mg/ml) (0.5 mg/ml) (5 mg/ml) tion x20). (d) Transduction efficiencies obtained from b and c, (macroscopy) (macroscopy)(histology) (macroscopy) assessed as described in the Materials and Methods. *Statistically significant compared with absence of vector treatment.

5,000 5,000 5,000 RLU/aspirate RLU/aspirate RLU/aspirate 0 0 0 0102040 0102040 0102040 rAAV-lacZ (µl) rAAV-lacZ (µl) rAAV-lacZ (µl)

hirudin (0.2 mg/ml) hirudin (0.5 mg/ml) hirudin (5 mg/ml)

1 1 1 /aspirate /aspirate 0.5 0.5 /aspirate 0.5 450 nm 450 nm 450 nm OD OD OD 0 0 0 0102040 0102040 0102040 rAAV-lacZ (µl) rAAV-lacZ (µl) rAAV-lacZ (µl) Figure 3. Detection of prolonged lacZ expression in hirudin-treated human bone marrow aspirates transduced with rAAV and effects upon the proliferative activities. Samples were collected in hirudin (0.2, 0.5 or 5 mg ml − 1) and mixed with increasing doses of rAAV-lacZ (10, 20 or 40 μl) or let untransduced as described in the Materials and Methods. The samples were processed to detect the β-gal activities by Beta-Glo assay (top panels) and the cell proliferation indices by WST-1 assay (lower panels) after 21 days. Data are given as relative luminescence units (Beta- Glo assay) or OD450 nm (WST-1 assay) per aspirate as described in the Materials and Methods. *Statistically signiﬁcant compared with absence of vector treatment.

hirudin

(0.2 mg/ml) (0.5 mg/ml) (5 mg/ml)

day 2

day 21

day 125

Figure 4. Detection of RFP expression in hirudin-treated human bone marrow aspirates transduced with rAAV over extended periods of time. The samples were prepared and transduced with rAAV-RFP (40 μl) as described in Figure 3 or let untransduced to detect live fluorescence after 2, 21 and 125 days. Insets show untransduced aspirates (magnification x2, including original magnifications in the insets). and MSCs.18–22,28 Yet, little is known on the potential of rAAV-2 to collected in the presence of heparin, corroborating previous deliver and overexpress gene sequences in bone marrow findings showing that this anticoagulant prevents the adsorption – aspirates. In the present study, we thus evaluated the ability of of rAAV-2 particles at the surface of human cell lines.24 26 Yet, such this class of vector to promote marker gene expression in primary interference could be avoided when heparin was replaced with human bone marrow aspirates over time and tested whether such hirudin,27 as application of this alternative compound allowed for modification interfered with the chondrogenic differentiation early (2 days) and late (up to 21 days) transgene (lacZ) expression processes in transduced samples. in modified aspirates compared with control samples, revealing Our data first demonstrate that a strong inhibition of the very high transduction efficiencies (up to 90–94%), again transduction via rAAV-2 was noted when the aspirates were consistent with the properties of this reagent.24,25 The results of

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 50 – 57 rAAV gene transfer in human bone marrow aspirates A Rey-Rico et al 54 the analyses revealed that applying hirudin at 0.5 mg ml − 1 addition of rAAV upon the proliferative activities in the aspirates allowed for optimal rAAV gene transfer in the aspirates. Of equal were observed. In good agreement, there was no detrimental, importance, neither deleterious effects of hirudin nor of the cytotoxic effect of the treatments or conditions tested over the

no vector, no hirudin no vector, hirudin (0.5 mg/ml) rAAV-lacZ, hirudin (0.5 mg/ml) ,+,# ,+,# (* )(*) rAAV-FLAG-hsox9, hirudin (0.5 mg/ml) 100 (*,+,#)

50 Staining intensities (%) 25

(*,+,#)

0 toluidine blue type-II collagen type-X collagen SOX9 Figure 5. Histological, immunohistochemical and morphometric analyses in chondrogenically induced, hirudin-treated human bone marrow aspirates transduced with rAAV. The samples were prepared and transduced with rAAV-lacZ or rAAV-FLAG-hsox9 (40 μl each vector) as described in Figure 3 or let untransduced for further induction toward chondrogenic differentiation for 21 days as described in the Materials and Methods. (a) The samples were histologically processed for toluidine blue staining and for anti-type-II/-type-X collagen and SOX9 immunostaining (all at magniﬁcation x20). Aspirates collected without hirudin were used as control condition. (b) Morphometric analyses obtained from a, assessed as described in the Materials and Methods. Statistically signiﬁcant compared with conditions *no vector/no hirudin, +no vector/hirudin (0.5 mg ml − 1) and #rAAV-lacZ/hirudin (0.5 mg ml − 1).

Gene Therapy (2015) 50 – 57 © 2015 Macmillan Publishers Limited rAAV gene transfer in human bone marrow aspirates A Rey-Rico et al 55 whole period of evaluation. Most remarkably, a strong, persistent Culture of bone marrow aspirates signal of transgene (RFP) expression was evidenced in the samples The study was approved by the Ethics Committee of the Saarland over extended periods of time (up to 125 days, the longest time Physicians Council. All patients provided informed consent before point examined) at the optimal concentration of hirudin. Finally, inclusion in the study. All procedures were in accordance with the neither administration of rAAV nor of hirudin altered the potential Declaration of Helsinki. Bone marrow aspirates (~15 ml) were obtained in the absence of anticoagulant from the distal femur of patients (n = 12) for chondrogenic differentiation in the aspirates and application of μ a potent candidate gene, the SOX9 transcription factor, was undergoing total knee arthroplasty. Aliquots (150 l) were immediately and rapidly placed in 96-well plates to test the following conditions: capable of enhancing the chondrogenic differentiation processes absence of anticoagulant, treatment with sodium heparin (5000 IU ml − 1,as in the samples while reducing undesirable hypertrophy, in good in use for patients) or treatment with hirudin (0.2, 0.5 or 5 mg ml − 1).24,25 agreement with our previous findings when applying rAAV-FLAG- Aspirates were immediately mixed with the vectors or let untransduced hsox9 to isolated MSCs.22 Taken together, our data show that rAAV and further maintained in DMEM, 100 U ml − 1 penicillin and 100 μlml− 1 vectors are particularly well adapted to deliver gene sequences in streptomycin, 10% fetal bovine serum at 37 °C in a humidified atmosphere primary human bone marrow aspirates in a safe manner over with 5% CO2 for the different experiments at the denoted time points, with persistent periods of time and without impeding the ability to medium changes every 2 to 3 days. commit toward the chondrocyte phenotype. Research is ongoing To evaluate the ability of bone marrow aspirates to undergo chondrogenic differentiation, experiments were performed using opti- to characterize the population of transduced cells that undergo mized treatment and gene transfer conditions in defined medium. such chondrogenic processes in the aspirates. It remains to be Aspirates treated with hirudin (0.5 mg ml − 1) were transduced with rAAV- seen whether too long levels of transgene expression will not be lacZ (40 μl) or let untransduced, and maintained for 21 days in − 1 detrimental to the samples. In such a case, the use of regulatable chondrogenic medium (DMEM, 0.1 μM dexamethasone, 50 μgml ascor- (tetracycline-sensitive) or tissue-specific promoters (SOX9, type-II bic acid, 40 μgml− 1 proline, 110 μgml− 1 pyruvate, 6.25 μgml− 1 insulin, − − − collagen or cartilage matrix oligomeric matrix protein)34–37 rather 6.25 μgml 1 transferrin, 6.25 μgml 1 selenious acid, 1.25 mg ml 1 bovine − 1 − 1 19,22 than the cytomegalovirus immediate-early element may have serum albumin, 5.55 μgml linoleic acid and 10 ng ml TGF-β3) for value to modulate the production of the transgenes. In addition, subsequent analyses. Aspirates without hirudin treatment were used as while AAV-2 has been mostly employed to target MSCs so far, it control conditions. would be also interesting to examine the benefits of applying other serotypes of the virus in the current system. Plasmids and rAAV vectors The next step will be to translate the current strategy in The constructs were derived from pSSV9, an AAV-2 genomic clone.52,53 aspirates from animal species to test the ability of the constructs rAAV-lacZ carries the lacZ gene for Escherichia coli β-gal, rAAV-RFP a 776-bp to efficiently and safely promote transgene expression in cartilage Discosoma sp. RFP cDNA fragment and rAAV-FLAG-hsox9 a 1.7-kb FLAG- tagged human SOX9 (hsox9) cDNA, all under the control of the defects in vivo and to allow for durably, enhanced cartilage repair 18,19,22,28,51,54 fi cytomegalovirus immediate-early promoter. The vectors were upon transplantation of samples modi ed with our SOX9 rAAV packaged as conventional (not self-complementary) vectors using a vector in such clinically relevant experimental models. Encoura- helper-free, two-plasmid transfection system in the 293 cell line (an ging results in vivo have been already reported when directly adenovirus-transformed human embryonic kidney cell line) with the applying the current candidate vector to osteochondral defects in packaging plasmid pXX2 and the adenovirus helper plasmid pXX6 as the knee joints of rabbits, showing the ability of this construct to previously described.54 The vector preparations were purified by dialysis significantly enhance the articular chondrogenic processes in and titered by real-time PCR,18,19,22,28,54 averaging 1010 transgene copies/ bone marrow and the healing of the lesions, with delayed terminal ml (ratio virus particles to functional vectors = 500/1). differentiation and hypertrophy in the newly formed cartilage by opposing effects of the sox9 treatment on RUNX2 and β-catenin rAAV gene transfer expression and with subchondral bone reconstitution by The vectors were directly mixed to the bone marrow aspirates using increased RUNX2 expression in this area.28 Other agents including concentrations previously employed for isolated human MSCs (10, 20 or different transcription factors,22,28,38,39 TGF-β,20,40 bone morpho- 40 μl, that is 2, 4 or 8 × 105 functional recombinant viral particles)19,22 or let genetic proteins,41–45 insulin-like growth factor I,46–48 cartilage- untreated for up to 125 days. derived morphogenetic protein 1 (ref. 49) or zinc-finger protein 145 (ref. 50) might be also good candidates to further enhance Transgene expression chondrogenesis and tissue healing in cartilage lesions. Combined Expression of the lacZ transgene was analyzed by X-gal staining application of various candidates may be even necessary and and visualization under light microscopy (Olympus BX45; Hamburg, again, rAAV will be a powerful system to achieve this goal as Germany)18,19,28 and using the Beta-Glo assay according to the manufacturer’s separate vectors can be simultaneously delivered, allowing recommendations, with normalization of the relative luminescence units for effective co-overexpression of the transgenes applied.51 per aspirate. RFP was detected by live fluorescence using a fluorescent fi 18,19,22,28,54 The current findings provide strong motivation to further microscopy with a 568-nm lter (Olympus CKX41). elaborate gene-based protocols for the modification of bone marrow aspirates as a safe, convenient and clinically relevant Cell proliferation and cytotoxicity analyses option to treat articular cartilage defects. Cell proliferation was assessed using the Cell Proliferation Reagent WST-1 (Roche Applied Science) with OD proportional to the cell numbers, as previously described.19,22 Cytotoxic events were evaluated using the MATERIALS AND METHODS Cytotoxicity Detection Kit (LDH; Roche Applied Science) by measuring the Reagents release of lactate dehydrogenase activity in the supernatants of cultures in accordance with the manufacturer’s instructions.55 Data were generated All reagents were from Sigma-Aldrich (Munich, Germany) except otherwise fl − 1 with a GENios spectrophotometer/ uorometer (Tecan, Crailsheim, Germany) indicated. Sodium heparin (5000 IU ml ) was purchased from B. Braun and calculated as the percentage of cytotoxicity using the formula: fl (Melsungen AG, Germany). Hirudin (Lepirudin, Re udan) was purchased (experimental value − low control/high control − low control) × 100. Where from Celgene (Munich, Germany). The anti-type-II collagen (II-II6B3) low control corresponds to cells without assay treatment and high control antibody was from DSHB (Iowa, IA, USA) and the anti-type-X collagen to cells placed in the lysis buffer provided in the kit. (COL-10) antibody from Sigma-Aldrich. The anti-SOX9 (C-20) antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). The Beta-Glo Assay System was from Promega. The Cell Proliferation Reagent WST-1 and the Histological and immunohistochemical analyses Cytotoxicity Detection Kit (LDH) were from Roche Applied Science Aspirates were harvested and fixed in 4% buffered formalin. Fixed (Mannheim, Germany). aspirates were dehydrated in graded alcohols, embedded in paraffin

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 50 – 57 rAAV gene transfer in human bone marrow aspirates A Rey-Rico et al 56 and sectioned (3 μm). Sections were stained with toluidine blue (matrix 14 Madry H, Cucchiarini M. Advances and challenges in gene-based approaches for proteoglycans) according to routine protocols.18,19,22,28,54 Expression of osteoarthritis. J Gene Med 2013; 15: 343–355. type-II/type-X collagen and SOX9 was detected using specific primary 15 Kim JD, Lee GW, Jung GH, Kim CK, Kim T, Park JH et al. Clinical outcome of antibodies and biotinylated secondary antibodies (Vector Laboratories, autologous bone marrow aspirates concentrate (BMAC) injection in degenerative Alexis Deutschland GmbH, Grünberg, Germany) with the ABC method arthritis of the knee. Eur J Orthop Surg Traumatol 2014; e-pub ahead of print 8 January (Vector Laboratories) and diaminobenzidine as the chromogen.18,19,22,28,54 2014; doi: 10.1007/s00590-013-1393-9. 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Mol Ther 2005; 12: 229–238. were measured at three random standardized sites for each test and 19 Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H. Metabolic activities and replicate condition by using SIS analySIS program (Olympus), Adobe chondrogenic differentiation of human mesenchymal stem cells following Photoshop (Adobe Systems, Unterschleissheim, Germany) and Scion Image 19,54 recombinant adeno-associated virus-mediated gene transfer and overexpression (Scion Corporation, Frederick, MD, USA). The intensities for toluidine of fibroblast growth factor 2. Tissue Eng Part A 2011; 17: 1921–1933. blue staining and for type-II/type-X collagen and SOX9 immunostaining 19,54 20 Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X, Chu CR. Adeno-associated were in pixels per standardized area. viral gene transfer of transforming growth factor-beta1 to human mesenchymal stem cells improves cartilage repair. Gene Therapy 2007; 14:804–813. Statistical analysis 21 Stender S, Murphy M, O'Brien T, Stengaard C, Ulrich-Vinther M, Soballe K et al. Each condition was performed in duplicate in three independent Adeno-associated viral vector transduction of human mesenchymal stem cells. – experiments. Values are expressed as mean ± s.d. The t-test and Mann– Eur Cell Mater 2007; 13:93 99. Whitney rank sum test were employed where appropriate. P values o0.05 22 Venkatesan JK, Ekici M, Madry H, Schmitt G, Kohn D, Cucchiarini M. SOX9 gene were considered statistically significant. transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells. Stem Cell Res Ther 2012; 3:22–36. CONFLICT OF INTEREST 23 Berns KI, Linden RM. The cryptic life style of adeno-associated virus. Bioessays 1995; 17: 237–245. 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