Abstract
Articular cartilage injuries have an inadequate aptitude to reproduce the original structure and functions of this highly specialized tissue. As most of the currently available options also do not lead to the restoration of the original hyaline cartilage, novel treatments are critically needed to address this global problems in the clinics. Gene therapy combined with tissue engineering approaches offers effective tools capable of enhancing cartilage repair experimentally, especially those based on the controlled delivery of the highly effective, clinically adapted recombinant adeno-associated viral (rAAV) vectors. This work presents an overview of the most recent evidence showing the benefits of using rAAV vectors and biocompatible materials for the elaboration of adapted treatments against cartilage injuries.
Introduction
The adult articular cartilage, the tissue that allows for load transmission and joint mobility, has a limited capacity for self-healing due to an absence of blood and chondroregenerative cells supply [1]. While a number of clinical options are available to treat cartilage injuries resulting from trauma or during osteoarthritis (OA) (marrow-stimulating techniques like microfrature; cell or tissue transplantation by autologous chondrocyte implantation or by administration of mesenchymal stem cells – MSCs; replacement surgery) [2,3], none definitely allow for an effective, consistent full regeneration of the native hyaline cartilage structure (type-II collagen, proteoglycans) with full biomechanical integrity [4–9], urging for the development of novel therapeutic regimens. Gene therapy is an attractive strategy that allows for the durable production of therapeutic factors in sites of cartilage lesions [8,10–13]. In this regard, the use of human recombinant adeno-associated viral (rAAV) gene vectors may be best suited for clinical translation in the patient as these safe vehicles have the ability to directly modify most of the cells involved in the cartilage repair processes (differentiated articular chondrocytes, progenitor cells) at very high efficiencies and over extended periods of time compared with other less adapted, less efficient systems (nonviral vectors, adenoviral or retro-/lentiviral vehicles) [4–8,10–13], making them preferred systems to treat human disorders [10,12,13].
rAAV-mediated gene delivery for cartilage repair: Current strategies
Biology of rAAV vectors
AAV is a non-pathogenic, replication-defective human single-strand DNA (ssDNA) parvovirus [14] that can be modified to generate recombinant constructs upon removal of all viral coding sequences to replace them by a therapeutic (or gene marker) cassette [15]. As a result, rAAV vectors are much less immunogenic and toxic than adenoviral and herpes simplex vectors that maintain original sequences even in new vector generations [16] (Fig. 1(A)). While most work has been performed with the serotype 2 of the virus (AAV-2), up to 12 total serotypes have been cloned thus far to expand cell targeting [8,10]. rAAV vectors are small particles (∼20 nm) that can target dividing and quiescent cells at very high efficacy (up to 100%) [17,18] so direct gene delivery strategies are made possible in vivo despite the presence of the dense extracellular cartilage matrix [18]. Self-complementary AAV (scAAV) that bypass the need for DNA synthesis may provide interesting alternatives to ssDNA vectors for cartilage repair [19].
rAAV-mediated gene transfer for cartilage repair. (A) Organization of rAAV vectors. These gene vehicles carry two inverted terminal repeats (ITRs) surrounding a transgene cassette composed of a heterelogous promoter, the gene of interest, and an intron/polyA signal. (B) Direct versus indirect delivery of rAAV vectors in cartilage lesions. Abbreviations: rAAV, recombinant adeno-associated viral vectors; OA, osteoarthritis.
Gene transfer of rAAV can be performed either directly in the recipient (in vivo setting) or indirectly by implantation of genetically modified cells (ex vivo approach) in sites of cartilage lesions (Fig. 1(B)).
In vivo rAAV delivery is convenient and cost-effective but associated with various hurdles:
a large number of cells in damaged cartilage is necessary for effective targeting and transgene expression at therapeutic levels;
vector dissemination to non-target tissues may occur early on, potentially followed by vector DNA clearance or to a contralateral effect of the gene treatment in nonmodified joints (trafficking of the vectors or of vector-modified cells) [20,21].
While ex vivo application of rAAV may address such issues, it represents a costly procedure that includes complex, arduous steps of cell collection and expansion that may be replaced by the manipulation of tissue biopsies (marrow and peripheral blood aspirates, fat, muscle) [11,22–27].
The most critical issue that still needs to be addressed to allow for an optimal use of rAAV vectors in the clinics is the existence of humoral and cellular responses in patients (neutralizing antibodies against the AAV capsid proteins, activation of the Toll-like Receptor (TLR) 9/MyD88, interferon-1 cascade in plasmacytoid dendritic cells) [28–30] that may impede effective rAAV-mediated therapeutic gene delivery and overexpression.
rAAV- and tissue engineering-based approaches for cartilage repair. rAAV vectors may be delivered in cartilage lesions by controlled delivery fromsolid scaffolds or hydrogels to prevent neutralization by pre-existing humoral or cellular immune responses in the recipient. Abbreviations: rAAV, recombinant adeno-associated viral vectors; OA, osteoarthritis.
Controlled rAAV vector delivery systems using biocompatible materials (solid scaffolds, hydrogels) may provide efficient, novel tools to overcome such humoral and cellular immune responses in vivo [10,12,13] (Fig. 2). Thus far, focus was given on hydrogels for cartilage research due to the adapted release pattern of these systems via diffusion processes. Compounds like fibrin glue (FG) [31], self-assembling peptide hydrogels [32], alginate [33], poloxamers and poloxamines alone [34,35] or combined with alginate [33] have been tested to achieve a controlled release profile of rAAV in cells relevant of cartilage repair mechanisms (human MSCs – hMSCs) (Table 1).
Combined rAAV and tissue engineering systems for cartilage repair
Abbreviations: FG, fibrin glue; RAD16-I, (RADA)4 peptide; HA, hyaluronic acid; PF68, poloxamer F68; T908, poloxamine 908; TGF-β, transforming growth factor beta; lacZ, E. coli β-galactosidase; RFP, red fluorescent protein; sox9, sex determining region Y-box 9; hMSCs, human mesenchymal stem cells; OCD, osteochondral defect.
Specifically, FG has been initially employed as a means to release an rAAV encoding the transforming growth factor beta (TGF-β) construct to enhance the cartilage-specific gene expression in hMSCs [31]. Hydrogels formed using self-assembling peptides like those based on RAD16-I in a pure form or combined by hyaluronic acid (HA) were also produced to effectively deliver marker rAAV vectors as novel tools to genetically modify hMSCs [32]. Alginate structures were also prepared for a similar purpose either alone or mixed with poloxamers or poloxamines [33]. Such copolymers composed of “smart” poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) as tri-blocks are particularly promising compounds capable of forming polymeric micelles and of undergoing sol-to-gel transition upon heating. In addition, recent work demonstrated the benefits of encapsulating rAAV in linear poloxamer PF68 and X-shaped poloxamine T908 micelles as effective and safe carrier systems to durably transduce hMSCs to levels similar to or even higher than those noted upon direct vector application (up to 95% of gene transfer efficiency) [34]. Such copolymers were also capable of restoring the transduction of hMSCs with rAAV in conditions of gene transfer inhibition like in the presence of heparin or of a specific antibody directed against the AAV capsid proteins, enabling effective therapeutic delivery of the chondrogenic sex determining region Y-box 9 (sox9) sequence leading to an enhanced chondrocyte differentiation of the cells. Furthermore, such micelles were also capable of promoting the effective and stable genetic modification of human OA chondrocytes in vitro and in an experimental model of human osteochondral defect ex vivo without detrimental effects on the biological activity of the cells or their phenotype while affording protection against neutralization by AAV-specific antibodies [35], showing overall the strong potential of these systems for the development of new therapeutic regimens against OA or to treat focal cartilage defects.
Combining tissue engineering and rAAV-based gene transfer procedures may provide novel, powerful tools to address the remaining limitations and obstacles of the current therapeutic options to an effective clinical treatment of cartilage lesions in the patient. Different systems have been tested to release rAAV vectors in cells relevant of the chondroreparative processes. Yet, little is known thus far on their potentiality in adapted animal models in vivo even though most may effectively act as supportive matrices with a proper mechanical strength required for the functionality of the tissue. Active work is ongoing to address this issue in order to generate workable, off-the-shelf systems for enhanced cartilage repair.
Footnotes
Acknowledgement
This work was supported by a grant from Deutsche Forschungsgemeinschaft (DFG RE 328/2-1).
Conflict of interest
The author has no conflict of interest to report.