Advanced Gene Therapy Strategies for the Repair of ACL Injuries

The anterior cruciate ligament (ACL), the principal ligament for stabilization of the knee, is highly predisposed to injury in the human population. As a result of its poor intrinsic healing capacities, surgical intervention is generally necessary to repair ACL lesions, yet the outcomes are never fully satisfactory in terms of long-lasting, complete, and safe repair. Gene therapy, based on the transfer of therapeutic genetic sequences via a gene vector, is a potent tool to durably and adeptly enhance the processes of ACL repair and has been reported for its workability in various experimental models relevant to ACL injuries in vitro, in situ, and in vivo. As critical hurdles to the effective and safe translation of gene therapy for clinical applications still remain, including physiological barriers and host immune responses, biomaterial-guided gene therapy inspired by drug delivery systems has been further developed to protect and improve the classical procedures of gene transfer in the future treatment of ACL injuries in patients, as critically presented here.


Introduction
Injuries in the anterior cruciate ligament (ACL) of the knee are common, representing an important socioeconomical burden as they may occur both in young individuals following sport activities and in the aging population by chronic degeneration and result in diminished musculoskeletal functions and potentially leading to osteoarthritis [1][2][3].
To address these issues, novel therapeutic strategies were established to improve the mechanisms of ACL repair based on the further use of tissue engineering tools including adapted scaffolds (structural templates), cell-based material (reparative elements), bioreactors (environmental and loading control systems), and biological stimuli (regulatory factors and cues) [2,8,37,, yet application of these systems in experimental settings was met with only limited success and did not allow for full and durable ACL repair.
In this regard, gene therapy may provide adapted tools for an improved, prolonged healing of ACL lesions by transfer of candidate genetic sequences with a gene carrier (vector) to extend the therapeutic activities of the gene products (growth and transcription factors, signaling molecules, therapeutic ribonucleic acids-RNAs) in sites of ACL In this regard, gene therapy may provide adapted tools for an improved, prolonged healing of ACL lesions by transfer of candidate genetic sequences with a gene carrier (vector) to extend the therapeutic activities of the gene products (growth and transcription factors, signaling molecules, therapeutic ribonucleic acids-RNAs) in sites of ACL injury . However, clinical gene therapy is still hindered by different physiological barriers in the recipient, including physical obstacles (body fluids, dense joint extracellular matrices) and biological inhibitors (pH/enzymatic environment of the joint, neutralizing host immune responses, rate-limiting intracellular steps, gene dissemination to non-target locations) [83,[94][95][96][97][98]. A very innovative approach to tackle these problems is to provide therapeutic gene vectors via biomaterial-guided delivery in sites of ACL injury as an offthe-shelf system, allowing for a safe, stabilized, and protected controlled release of the gene vehicles using biocompatible scaffolds (cargos) [82,83,87,90,[99][100][101][102][103][104]. The goal of this work is, therefore, to provide an overview of the most advanced gene therapy procedures that aim at enhancing the repair of ACL injuries.

ACL Function and Structure
The knee joint contains four ligaments that play key roles in kinematics and maintaining knee stability, connecting bones to each other, including two lateral ligaments that provide stability in the frontal plane and the anterior and posterior cruciate ligaments that support stability in the sagittal plane. Located in the center of the knee, the ACL forms part of the "central pivot" with the posterior cruciate ligament. The ACL is fundamental to connect the anterior part of the tibia to the posterior part of the femur, opposing forward displacement and excessive internal rotation of the tibia relative to the femur and stabilizing the knee during rotational movements [17,[105][106][107] (Figure 1). The ACL is a dense, cable-like tissue (27-32 mm in length, 10 mm in breadth, 4-10 mm in width, and 44.4-57.5 mm 2 cross-sectional area) [46, 105,108] that is highly organized, with an abundant extracellular matrix (ECM) mainly composed of collagens in fiber bundles (predominantly type-I collagen and types-III, -IV, -V, and -VI collagens; 70-80% dry weight) with elastin, fibronectin, thrombospondin, and proteoglycans (organization and lubrication of collagen fibril bundles) also surrounding ECM-producing cells (fibroblasts) in a hypocellular structure containing water (55-70%). The ACL has a hierarchical, sequentially assembled organization with increasing diameter and mechanical strength that includes collagen molecules (triple-helix polypeptide chains, <2 nm in diameter) that crosslink to form microfibrils (3.5 nm in diameter) that arrange themselves into subfibrils (10-20 nm in diameter) and then in fibrils (50-500 nm in diameter) that form fibers in fascicles (50-300 μm, with a crimp pattern repeated every 45-60 μm) that mutually cross- The ACL is a dense, cable-like tissue (27-32 mm in length, 10 mm in breadth, 4-10 mm in width, and 44.4-57.5 mm 2 cross-sectional area) [46, 105,108] that is highly organized, with an abundant extracellular matrix (ECM) mainly composed of collagens in fiber bundles (predominantly type-I collagen and types-III, -IV, -V, and -VI collagens; 70-80% dry weight) with elastin, fibronectin, thrombospondin, and proteoglycans (organization and lubrication of collagen fibril bundles) also surrounding ECM-producing cells (fibroblasts) in a hypocellular structure containing water (55-70%). The ACL has a hierarchical, sequentially assembled organization with increasing diameter and mechanical strength that includes collagen molecules (triple-helix polypeptide chains, <2 nm in diameter) that crosslink to form microfibrils (3.5 nm in diameter) that arrange themselves into subfibrils (10-20 nm in diameter) and then in fibrils (50-500 nm in diameter) that form fibers in fascicles (50-300 µm, with a crimp pattern repeated every 45-60 µm) that mutually crosslink to make a subfascicular unit running parallel to the long axis of the tissue, also containing proteoglycans and elastin and surrounded by a vascularized epiligament sheath to form the ligament [7,45,46,49,105,[109][110][111][112] (Figure 2). link to make a subfascicular unit running parallel to the long axis of the tissue, also containing proteoglycans and elastin and surrounded by a vascularized epiligament sheath to form the ligament [7,45,46,49,105,[109][110][111][112] (Figure 2). The cells in the ACL are interspersed between the collagen fibril bundles along the long axis, with a spindle-shaped form at an immature stage and a more elongated shape when they age. The ACL originates from the lateral plate mesoderm and its development is regulated by signaling from various growth factors (transforming growth factor beta-TGF-β-via TGFBR-receptor signaling and small mothers against decapentaplegic proteins-Smads; fibroblast growth factor-FGF-via FGFR-receptor signaling and extracellular signal-regulated kinase-ERK/mitogen-activated protein kinase-MAPK) and transcription factors (scleraxis-SCX-a basic helix-loop-helix factor; Mohawk-MKX-encoded by an atypical, three-amino-acid loop homeobox gene; early growth response factor 1-Egr1-a zinc finger transcription factor) that promote the differentiation of mesenchymal progenitor cells into ligament cells [11,105,[113][114][115][116][117][118][119][120] (Figure 3).  The cells in the ACL are interspersed between the collagen fibril bundles along the long axis, with a spindle-shaped form at an immature stage and a more elongated shape when they age. The ACL originates from the lateral plate mesoderm and its development is regulated by signaling from various growth factors (transforming growth factor beta-TGF-β-via TGFBR-receptor signaling and small mothers against decapentaplegic proteins-Smads; fibroblast growth factor-FGF-via FGFR-receptor signaling and extracellular signal-regulated kinase-ERK/mitogen-activated protein kinase-MAPK) and transcription factors (scleraxis-SCX-a basic helix-loop-helix factor; Mohawk-MKXencoded by an atypical, three-amino-acid loop homeobox gene; early growth response factor 1-Egr1-a zinc finger transcription factor) that promote the differentiation of mesenchymal progenitor cells into ligament cells [11,105,[113][114][115][116][117][118][119][120] (Figure 3). link to make a subfascicular unit running parallel to the long axis of the tissue, also containing proteoglycans and elastin and surrounded by a vascularized epiligament sheath to form the ligament [7,45,46,49,105,[109][110][111][112] (Figure 2). The cells in the ACL are interspersed between the collagen fibril bundles along the long axis, with a spindle-shaped form at an immature stage and a more elongated shape when they age. The ACL originates from the lateral plate mesoderm and its development is regulated by signaling from various growth factors (transforming growth factor beta-TGF-β-via TGFBR-receptor signaling and small mothers against decapentaplegic proteins-Smads; fibroblast growth factor-FGF-via FGFR-receptor signaling and extracellular signal-regulated kinase-ERK/mitogen-activated protein kinase-MAPK) and transcription factors (scleraxis-SCX-a basic helix-loop-helix factor; Mohawk-MKX-encoded by an atypical, three-amino-acid loop homeobox gene; early growth response factor 1-Egr1-a zinc finger transcription factor) that promote the differentiation of mesenchymal progenitor cells into ligament cells [11,105,[113][114][115][116][117][118][119][120] (Figure 3).

Clinical Aspects: Pathology, Natural Healing, and Current Treatments
Ligament injuries are common, especially in the ACL (25-50% of knee ligamentous injuries; incidence of 1 per 3000 inhabitants per year in the United States and in Europe) [1]. ACL injuries are mainly derived from sport activities (65%) in the young population (70% of affected patients between 20-35 years old) and occur via chronic tissue degeneration in the aging population, both resulting in diminished musculoskeletal functions and potentially leading to osteoarthritis [1,35]. In the United States only, approximately 300.000 ACL reconstruction surgeries are performed annually, costing approximately USD 30 billion [2,3,10,60], clearly representing a significant public health problem.
The natural healing of ACL injuries is slow and inefficient, leading to the formation of scar tissue that does not naturally reproduce the original biological and mechanical properties of the tissue due to the limited intrinsic ability of the ACL to fully heal [2,[4][5][6][7][8]10,11,[13][14][15][16]46,54,121]. Three major cascade phases of ligament healing have been defined in response to injury, including (1) an inflammatory stage within the first days and weeks of injury (serous fluid accumulation, fragilization of the area, formation of a fibrin clot, invasion by monocytes/leukocytes/macrophages, debris removal, release of pro-angiogenic and proliferative growth factors and cytokines), (2) a proliferative stage by 8 weeks (blood vessel formation, fibroblast proliferation with collagen matrix production to fill up the injury), and (3) a remodeling phase between 1 and 2 years (decrease in cellularity, matrix realignment for adapted response to mechanical forces) [5,8]. The lack of proper ACL healing is due to the intra-articular conditions (synovial fluid, intra-articular movements) that hinder the stable formation of a fibrin/platelet scaffold (a prerequisite to primary healing), to an insufficient availability of reparative factors (growth factors and cytokines, wound filling compounds such as fibrinogen/fibronectin), to the lack of continuity between collagen fibers between the new and the old matrices leading to reduced tissue mechanical properties, and to the absence of vascularization of this ligament [2,5,8,10].
While the use of biomaterial scaffolds with or without augmentation (cells, biological/mechanical stimuli) proved beneficial at least to a certain point experimentally, these Int. J. Mol. Sci. 2022, 23, 14467 8 of 33 systems have again been unable to support long-lasting ACL repair, showing the critical need for improved treatment strategies to effectively manage this clinical problem. In this regard, gene therapy may provide powerful tools to adeptly heal sites of ACL injury.

Classical Gene Therapy for the Repair of ACL Injuries
Gene therapy is based on the transfer of genetic sequences in target cells, tissues/organs, and live organisms with gene vehicles as a means to prolong the therapeutic effects of one or various gene products relative to the application of recombinant factors with short half-lives 236,237]. Candidate factors for gene therapy may include growth and transcription factors, signaling molecules, as well as therapeutic RNAs that may be either suppressive (i.e., interfering) sequences such as oligodeoxyribonucleotide (ODNs), antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and long non-coding RNAs (lncRNAs) or activating sequences such as messenger RNAs (mRNAs). Gene therapy can be performed either via a direct (in vivo) administration of the gene vector or via an indirect (ex vivo) supply of cells/grafts that are genetically modified in vitro prior to reimplantation in the recipient.

Gene Transfer Vectors
Gene vehicles include both nonviral and viral systems with specific characteristics that make them more adapted for in vivo or for ex vivo therapy ( Table 2). Nonviral vectors are non-replicative, non-immunogenic, safe systems without size limitation, but they exhibit a relatively low gene transfer efficiency for only very short periods of time (<40% for few weeks) since the genes being carried are kept as episomes that necessitate cell division for effective expression, making these vectors more suitable for ex vivo therapy [238,239]. Viral vectors that employ the ability of viruses to penetrate various cell types include adenoviral, herpes simplex viral (HSV), retro-l/lentiviral, and recombinant adenoassociated virus (rAAV) vectors. Adenoviral and HSV vectors are capable of directly modifying dividing and quiescent cells at elevated efficiencies (~100%), making them adapted for in vivo therapy, but they support only short-term (episomal) transgene expression (between some days to 1-2 weeks) while activating host immune responses [98,[240][241][242]. Retroviral vectors have the ability to integrate in cellular genomes, allowing for long-term transgene expression, but they have low efficiencies (<20%), making them more suited for ex vivo therapy, and can only modify dividing cells, with possible insertional mutagenesis [243], while being also potentially immunogenic [98]. As an alternative, lentiviral vectors can also target quiescent cells, but they derive from the pathogenic human immunodeficiency virus (HIV) and may also lead to insertional mutagenesis [244]. rAAVs are small (~20 nm), safe vectors devoid of viral sequences and that are capable of modifying both dividing and quiescent cells at elevated efficiencies over extended periods of time (~100% for months to years) due their maintenance as stably expressed episomes [245,246], making them adapted for in vivo therapy, but they may raise immune responses, in particular by pre-existing neutralizing antibodies directed against the viral capsid proteins [98,[247][248][249].

Candidate Therapeutic Factors
Therapeutic factors amenable to gene transfer to repair ACL injuries include growth factors, transcription factors, anti-inflammatory agents, matrix components, and signaling molecules.

Applications of Classical Gene Therapy for ACL Repair
Therapeutic gene therapy in the goal of ACL repair has been performed in relevant experimental models in cell culture in vitro, in tissue (explant) culture in situ, and in relevant animal models using both direct (in vivo) and indirect (ex vivo) gene transfer approaches.

Limitations of Classical Gene Therapy for ACL Repair
While experimental work showed the potential benefits of classical gene therapy for ACL repair, a number of critical limitations need to be carefully addressed before initiating relevant approaches for translational regenerative therapy in the field of clinically adapted repair in patients.
ACL gene therapy may be first hindered by pre-existing physical obstacles and biological barriers to effective and safe therapeutic gene transfer, including the presence of inhibitory factors in the joint (body fluids, i.e., synovial fluid; clinical compounds, i.e., heparin), the local pH and/or enzymatic environment, and the dense extracellular matrix of the tissue itself that may impair the penetration of the vectors before reaching the target cells [82,83,85,87,90,349]. Another impairment is associated with the existence of innate and/or adaptive responses from the immune system of the recipient (antibodies, cellular helper, and cytotoxic T cells) that may be directed against the viral particles and/or the transgene sequence (neutralization processes) [98,247,[350][351][352][353]. Various cellassociated steps may also limit the rate and occurrence of therapeutic gene expression such as the effective uptake of the gene vector (presence/amount of the specific cell membrane receptor/co-receptor to a particular vector type), its successful internalization and transport in the cell (endosomal escape and nuclear entry), as well as its active processing (levels of adapted cell activity, transgenic genome conversion) [94][95][96]241,[354][355][356]. Other challenges to address also involve inherent features of the vectors that may potentially affect the efficacy of the therapeutic treatment (vector dissemination to non-target sites in the joint, toxicity of viral vector proteins, genotoxicity upon a possible transgene integration in the host genome, transformation risk) [97].
A variety of strategies have been developed to tackle such issues, including (1) the use of alternative routes of vector injection, vector doses, clinical components (passive hirudin versus inhibitory heparin), immunosuppressive agents, and/or alternative (viral) vector serotypes, (2) the modification/masking of viral particles to evade host immune responses (chemical conjugation with polyethylene glycol-PEG, tropism/viral capsid tailoring by inclusion of substitute peptide sequences/epitopes, viral capsid engineering using chimeric/hybrid/mosaic/shuffled/variant/mutant/decoy vectors or viruslike particles-VLPs, vector-like microvesicles, vexosomes), and (3) the modification of the vector genome to circumvent the rate-limiting steps of its intracellular processing (tissue-specific/activatable/disease-responsive promoters, hybrid/self-complementary vectors, optimized coding/noncoding sequences in the transgene cassettes, artificial chromosomes) [82,83,85,87,90,96,245,[357][358][359][360][361]. Still, even though such approaches led to some improvements in the efficacy of gene transfer, they remain complex (capsid/vector genome modification and engineering) or do not allow for sufficient and adapted therapeutic effects and outcomes, especially in vivo, while the variability of the host immune responses between patients has not been carefully taken into account with any of these techniques. Overall, these observations show the critical need to develop novel, suitable tools to address such challenges for clinical gene therapy, in particular for human ACL repair.

Biomaterial-Guided Gene Therapy for the Repair of ACL Injuries
The administration of therapeutic gene vectors using biomaterials already employed in tissue engineering research is an attractive approach to tackle the current obstacles of clinical human gene therapy and regenerative medicine [82,83,[85][86][87]90,93,[102][103][104]362,363] and might be applied in the goal of human ACL repair [90,91].

Principles
Biomaterial-guided gene therapy is a groundbreaking, convenient therapeutic concept based on the controlled delivery of gene transfer vectors from biocompatible scaffolds (hydrogels, solid scaffolds) originating from approaches developed to apply drugs and recombinant agents with such systems in human medicine [139,[364][365][366][367][368][369]. Biomaterial-guided gene therapy instead combines the application of gene vehicles using materials (cargos) that may mimic the properties of the ACL tissue while strengthening it [99][100][101][370][371][372][373][374][375][376][377][378][379][380][381][382] in order to allow for a spatiotemporal, safe expression of therapeutic genetic sequences in the recipient via an off-the-shelf, cell-free (patient-independent) compound. Biomaterials may be derived from natural (biocompatible, biodegradable) or synthetic (reproducible) components, or both, to either encapsulate gene vectors to achieve a polymeric vector release (gradient release by degradation of the polymer loaded with the vectors during scaffold formation for hydrogels) or to immobilize the gene vectors to achieve a substrate-mediated release (release of the vectors incorporated at the surface of preformed scaffolds for solid scaffolds) [383]. Gene therapy guided via controlled release from biomaterials may allow for the stabilization of the gene vectors against degradation, to enhance the residence time of the therapeutic genes being carried and the effects of their products, to minimize vector dissemination to nontarget sites and the vector doses needed to be applied in patients, and to mask potentially immunogenic viral particle epitopes and protect the vectors from host immune responses [82,83,85,87,90,102].

Conclusions
To address the unsolved problem of achieving long-lasting, safe, and mechanically competent ACL repair in patients, as none of the currently available clinical options (ACL reconstruction, engineering, augmentation) can competently afford it thus far, advanced strategies were developed to improve the intrinsic mechanisms of tissue repair in prevalent ACL injuries based on gene therapy procedures using durable therapeutic gene transfer (growth/transcription factors, anti-inflammatory agents, matrix components, signaling molecules, therapeutic RNAs) in vectors in experimental systems in vitro and in situ and relevant animal models in vivo via classical gene transfer methods (direct gene vector administration, indirect implantation of genetically modified cells and tissues). While such experimental approaches met undeniable success, they may be limited by the existence of physical and biological barriers in patients such as the joint environment (inhibitory soluble factors, dense extracellular matrix, immune host responses) and rate-limiting steps to effective therapeutic gene expression (ACL cell-associated rate-limiting steps, vector dissemination and toxicity). Besides active work using complex vector engineering techniques, a more convenient strategy, namely biomaterial-guided gene therapy, has emerged to tackle these issues via the application of biocompatible materials as cargos for therapeutic gene vectors, allowing for their spatiotemporal, safe, and prolonged controlled release and expression in the recipient while mimicking the properties of the ACL tissue and strengthening it. With promising results reported in experimental systems in vitro and in situ and in relevant animal models in vivo using this highly innovative procedure, work is now needed to confirm its workability in large preclinical animal models before envisaging a possible translation in individuals that first requires approval by regulatory organizations [83,[392][393][394]. With this in mind, it still remains to be determined which biomaterial (type, production method possibly including three-dimensional-3D-bioprinting to mimic the structural features of the ACL) [395][396][397][398][399], vector (class, dose), and gene (single sequence or combination of genes, potential use of direct genome editing such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system) [400][401][402][403][404][405][406][407][408][409] will be the most appropriate to effectively and permanently heal ACL lesions in order to be accessible to the patients in a near future.