Serotype-specific transduction of canine joint tissue explants and cultured monolayers by self-complementary adeno-associated viral vectors.

A formal screening of self-complementary adeno-associated virus (scAAV) vector serotypes in canine joint tissues has not been performed to date. Selecting appropriate serotypes is crucial for successful treatment due to their varying levels of tissue tropism. The objective of this study is to identify the most optimal scAAV vector serotype that maximizes transduction efficiencies in canine cell monolayer cultures (chondrocytes, synoviocytes, and mesenchymal stem cells) and tissue explant cultures (cartilage and synovium). Transduction efficiencies of scAAV serotypes 1, 2, 2.5, 3, 4, 5, 6, 8, and 9 were evaluated in each culture type in three different vector concentrations by encoding a green fluorescent protein. It was found that scAAV2 and 2.5 showed the overall highest transduction efficiency among serotypes with dose-response. Since possible immune response against conventional AAV2 was previously reported in dogs, the chimeric scAAV2.5 may be more suitable to use. Evaluation of the safety and efficacy of the scAAV2.5 vector with an appropriate therapeutic gene in vivo is indicated.

Gene therapy approaches for equine osteoarthritis.

With an intrinsically low ability for self-repair, articular cartilage injuries often progress to cartilage loss and joint degeneration resulting in osteoarthritis (OA). Osteoarthritis and the associated articular cartilage changes can be debilitating, resulting in lameness and functional disability both in human and equine patients. While articular cartilage damage plays a central role in the pathogenesis of OA, the contribution of other joint tissues to the pathogenesis of OA has increasingly been recognized thus prompting a whole organ approach for therapeutic strategies. Gene therapy methods have generated significant interest in OA therapy in recent years. These utilize viral or non-viral vectors to deliver therapeutic molecules directly into the joint space with the goal of reprogramming the cells' machinery to secrete high levels of the target protein at the site of injection. Several viral vector-based approaches have demonstrated successful gene transfer with persistent therapeutic levels of transgene expression in the equine joint. As an experimental model, horses represent the pathology of human OA more accurately compared to other animal models. The anatomical and biomechanical similarities between equine and human joints also allow for the use of similar imaging and diagnostic methods as used in humans. In addition, horses experience naturally occurring OA and undergo similar therapies as human patients and, therefore, are a clinically relevant patient population. Thus, further studies utilizing this equine model would not only help advance the field of human OA therapy but also benefit the clinical equine patients with naturally occurring joint disease. In this review, we discuss the advancements in gene therapeutic approaches for the treatment of OA with the horse as a relevant patient population as well as an effective and commonly utilized species as a translational model.

Corrigendum: Gene therapy approaches for equine osteoarthritis.

[This corrects the article DOI: 10.3389/fvets.2022.962898.].

Single and repeated intra-articular injections in the tarsocrural joint with allogeneic and autologous equine bone marrow-derived mesenchymal stem cells are safe, but did not reduce acute inflammation in an experimental interleukin-1ß model of synovitis.

BACKGROUND: Allogeneic and autologous bone marrow-derived mesenchymal stem cells (BMDMSCs) have been administered in equine joints for their anti-inflammatory effects. However, allogeneic BMDMSC offer multiple clinical and practical advantages. Therefore, it is important to determine the relative effectiveness of allogeneic vs autologous BMDMSCs. OBJECTIVES: The objective of the study was to compare the inflamed joint response to autologous vs allogeneic BMDMSCs injections, and to determine if either treatment generated an anti-inflammatory effect. STUDY DESIGN: Randomised controlled study. METHOD: Bone marrow was harvested from eight horses. Autologous BMDMSCs and pooled allogeneic BMDMSCs were culture expanded, cryopreserved and thawed immediately prior to administration. Ten million autologous BMDMSCs were administered with 75 ng rIL-1ß into one tarsocrural joint and the contralateral tarsocrural joint received allogeneic BMDMSC plus 75 ng rIL-1ß. Repeat injections were performed with the same treatment administered into the same joint. Four additional horses received 75 ng rIL-1ß alone in a single tarsocrural joint. Clinical parameters (lameness, joint circumference and joint effusion) and synovial fluid parameters, including nucleated cell count (NCC), differential cell count, total protein (TP), prostaglandin E2 (PGE2 ) and C-reactive protein (CRP), were measured at baseline, 6, 12, 24, 72, 168 and 336 hours post-injection. RESULTS: No difference was detected between autologous and allogeneic treatment groups with respect to subjective lameness, joint effusion, joint circumference, NCC, TP, differential cell count, CRP or PGE2 . Neither autologous nor allogeneic treatments resulted in an improvement in clinical or cytological parameters over that elicited by rIL-1ß alone. MAIN LIMITATIONS: A single dose of rIL-1ß was evaluated and resulted in a severe synovitis which may have been too severe to observe a BMDMSC-mediated effect. CONCLUSIONS: This study revealed that allogeneic and autologous BMDMSCs resulted in an equivalent clinical and cytological response. Allogeneic and autologous BMDMSCs were equally ineffective in reducing the inflammatory response from acute rIL-1ß-induced joint inflammation in horses.

Can Extracorporeal Shockwave Promote Osteogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells In Vitro?

Both bone marrow-derived mesenchymal stem cells (BMDMSCs) and extracorporeal shockwave (ESW) have shown promise for enhancing fracture repair. If exposure of BMDMSCs to ESW enhances osteogenic differentiation, these therapies may be combined in vivo or used as a method for preconditioning BMDMSCs. The objective of this study was to determine the effect of ESW on the osteogenic ability of equine BMDMSCs. We hypothesized that ESW would promote osteogenesis evidenced by increased gene expression, alkaline phosphatase (ALPL) expression, slide morphologic score, and protein expression. BMDMSCs were evaluated from six horses. BMDMSCs were culture expanded to passage 3, dissociated, then placed in conical tubes. Treatment cells ("shocked") were exposed to 500 pulses at 0.16 mJ/mm2 energy. Cells were then reseeded and grown in either growth medium or osteogenic medium. Cellular proliferation and trilineage potential were determined. Cellular morphology was scored and cells were harvested at 1, 3, 7, 14, and 21 days for rtPCR gene expression of osteogenic markers [osteonectin (ONT), osteocalcin (OCN), ALPL, collagen type 3 (COL3), and runt-related transcription factor 2 (RUNX2)]. Media supernatants were evaluated for secretion of BMP-2, VEGF, TGFß, and PGE2 and cellular lysates were evaluated for ALPL production. There was no difference between the proliferative ability of shocked cells versus unshocked cells in either growth medium or osteogenic medium. ALPL production was greater in shocked cells maintained in osteogenic medium versus unshocked cells in osteogenic medium at day 3 (P < 0.005). Independent of media type, ESW caused a decrease in VEGF and TGFß production at day 3. No significant increases in gene expression were identified by rtPCR. Exposure of BMDMSCs to ESW does not result in negative effects. An initial significant increase in ALPL was detected but no persistent osteogenic effect was observed with cell expansion.

Genetic modification of scAAV-equine-BMP-2 transduced bone-marrow-derived mesenchymal stem cells before and after cryopreservation: An "off-the-shelf" option for fracture repair.

Optimizing the environment of complex bone healing and improving treatment of catastrophic bone fractures and segmental bone defects remains an unmet clinical need both human and equine veterinary medical orthopaedics. The objective of this study was to determine whether scAAV-equine-BMP-2 transduced cells would induce osteogenesis in equine bone marrow derived mesenchymal stem cells (BMDMSCs) in vitro, and if these cells could be cryopreserved in an effort to osteogenically prime them as an "off-the-shelf" gene therapeutic approach for fracture repair. Our study found that transgene expression is altered by cell expansion, as would be expected by a transduction resulting in episomal transgene expression, and that osteoinductive levels could still be achieved 5 days after recovery, and protein expression would continue up to 14 days after transduction. This is the first evidence that cryopreservation of genetically modified BMDMSCs would not alter the osteoinductive potential or clinical use of allogeneic donor cells in cases of equine fracture repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1310-1317, 2019.

Autologous and Allogeneic Equine Mesenchymal Stem Cells Exhibit Equivalent Immunomodulatory Properties In Vitro.

The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared with those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. The immunosuppressive activity of the two cell types was evaluated by adding autologous or allogeneic BMDMSCs to activated lymphocytes and assessing suppression of lymphocyte proliferation and IFNγ production. Fifty-six allogeneic and 12 autologous combinations were evaluated. Studies were also done to elucidate mechanisms by which equine mesenchymal stem cells (MSCs) suppress lymphocyte function. Potential mechanisms evaluated included production of prostaglandin E2 (PGE2), nitric oxide, transforming growth factor-beta, and indoleamine 2,3-dioxygenase. We found that autologous and allogeneic BMDMSCs both induced mild but equivalent levels of spontaneous lymphocyte activation in vitro. In in vitro assays assessing the ability of BMDMSCs to suppress activated lymphocytes, both allogeneic and autologous BMDMSCs suppressed T cell proliferation and IFNγ production to an equal degree. The primary mechanism of equine BMDMSC suppression of T cells was mediated by PGE2. We concluded that allogeneic and autologous BMDMSCs are equivalent in terms of their immunomodulatory properties, and stimulated peripheral blood mononuclear cells appear to trigger the immunosuppressive properties of MSCs. Therefore, both cell types appear to have equal potency in modulating inflammatory processes related to acute or chronic musculoskeletal injuries in the horse.

Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis.

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken ß-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200-800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.Molecular Therapy - Nucleic Acids (2013) 2, e70; doi:10.1038/mtna.2012.61; published online 5 February 2013.

Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells.

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).