Low back pain affects 80% of the population with half of cases attributed to intervertebral disc (IVD) degeneration. However, the majority of treatments focus on pain management, with none targeting the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of the pro-inflammatory cytokine IL-1, which orchestrates the catabolic degeneration of the IVD. Our objective here is to determine the ability of PCRX-201 to infect human nucleus pulposus (NP) cells and tissue to increase the production of IL-1Ra and assess downstream effects on catabolic protein production. Degenerate human NP cells and tissue explants were infected with PCRX-201 at 0 or 3000 multiplicities of infection (MOI) and subsequently cultured for 5 days in monolayer (n=7), 21 days in alginate beads (n=6) and 14 days in tissue explants (n=5). Cell culture supernatant was collected throughout culture duration and downstream targets associated with pain and degeneration were assessed using ELISA. IL-1Ra production was increased in NP cells and tissue infected with PCRX-201. The production of downstream catabolic proteins such as IL-1β, IL-6, MMP3, ADAMTS4 and VEGF was decreased in both 3D-cultured NP cells and tissue explants. Here, we have demonstrated that a novel gene therapy, PCRX-201, is able to infect and increase the production of IL-1Ra in degenerate NP cells and tissue in vitro. The increase of IL-1Ra also resulted in a decrease in the production of a number of pro-inflammatory and catabolic proteins, suggesting PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. At present, no treatments for IVD degeneration target the underlying pathology. The ability of FX201 to elicit anti-catabolic responses is promising and warrants further investigation in vitro and in vivo, to determine the efficacy of this exciting, novel gene therapy

Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (. ClinicalTrials.gov. Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (. ClinicalTrials.gov. Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress. Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540

The purpose of this study was to develop a cell-based VEGF gene therapy in order to accelerate fracture healing and investigate the effect of VEGF on bone repair in vivo. Twenty-one rabbits were studied. A ten millimeter segmental bone defect was created after twelve millimeter periosteal excision in the middle one third of each tibia and each tibia was plated. Primary cultured rabbit fibroblasts were transfected by use of SuperFect (Qiagen Inc) with pcDNA-VEGF. 5.0 X 106 cells in 1ml PBS were delivered via impregnated gelfoam into the fracture site. Experimental groups were:. Transfected fibroblasts with VEGF (n=7),. Fibroblasts alone (n=7), and. PBS only (n=7). The animals were sacrificed and fracture healing specimens collected at ten weeks post surgery. Radiology: Fracture healing was defined as those with bone bridging of the fracture defect. After ten weeks, fourteen tibial fractures were healed in total including six in group one, four in group two and four in group three. The VEGF group had an earlier initial sufficient volume of bridging new bone formation. Histological evaluation demonstrated ossification across the entire defect in response to the VEGF gene therapy, whereas the defects were predominantly fibrotic and sparsely ossified in groups two and three. Numerous positively stained (CD31) vessels were shown in the VEGF group. MicroCT evaluation showed complete bridging for the VEGF group, but incomplete healing for groups two and three. Micro-CT evaluation of the new bone structural parameters showed that the amount of new bone (volume of bone (VolB) x bone mineral density (BMD)), bone volume fractions (BVF), bone volume/tissues (BV/TV), trabecular thickness (Tb.Th), number (Tb.N) and connectivity density (Euler number) were higher; while structure model index (SMI), bone surface/bone volume (BS/BV), and trabecular separations (Tb.Sp) were lower in the VEGF group than the other groups. P-Values < 0.05 indicated statistical significance (ANOVA, SPSS) in all parameters except for SMI (0.089) and VolBx-BMD (0.197). These results indicate that cell-based VEGF gene delivery has significant osteogenic and angiogenic effects and demonstrates the ability of cell based VEGF gene therapy to enhance healing of a critical sized defect in a long bone in rabbits

Introduction: New biotechnologies create opportunities for gene therapy to promote rotator cuff healing. We have previously demonstrated that genetically engineered mesenchymal stem cells (MSCs) over expressing BMP-2 and SMAD8 signaling molecule differentiate to tenocytes in vitro and in vivo. Therefore, we hypothesized that rotator cuff defect could be regenerated using genetically engineered MSCs. Method: Nonviral methods were utilized to establish genetically engineered MSCs that co-express BMP-2 and the Smad8 signaling molecule. A previously validated animal model was utilized to examine rotator cuff healing. A 2mm x2mm full thickness defect was created in the infraspinatus tendon of 8 nude rats. A collagen-I biomembrane (TissueMend) containing 3 x 10. 6. engineered cells was sewn into the defect. An identical control procedure was repeated on the contralateral side with biomembrane containing non-engineered MSCs. Results: 4 weeks post implantation the area of implantation was isolated and analyzed by light microscopy and histochemical staining. Analysis of the engineered implants revealed the formation of dense connective tissue with parallel-organized fibers and spindle shaped cells, unlike the control samples. Proton Double Quantum Filtered Magnetic Resonance Imaging technique of the rotator cuff tendons demonstrated an increased presence of organized collagen fibers within the engineered rotator cuff tissue when compared with either native rotator cuff or those treated with non-engineered MSCs. Conclusion: This is the first report showing rotator cuff tendon repair using genetically engineered MSCs. Moreover these findings may have considerable importance for tendon healing and may indicate a clinical gene therapy platform to augment surgical repair

Mesenchymal Stem Cells (MSCs) are key regulators in senile osteoporosis and in bone formation and regeneration. MSCs are therefore suitable candidates for stem cells mediated gene therapy of bone. Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is a highly osteoinductive cytokine, promoting osteogenic differentiation of MSCs. We hypothesized that genetically engineered MSCs, expressing rhBMP2, can be utilized for targeted cell mediated gene therapy for local and systemic bone disorders and for bone/cartilage tissue engineering. Engineered MSCs expressing rhBMP-2 have both autocrine and paracrine effects enabling the engineered cells to actively participate in bone formation. We conditionally expressed rhBMP2 (tet-controlled gene expression, tet-off system) in mouse and human mesenchymal stem cells. RhBMP2 expressing clones (tet-off and adeno-BMP2 infected MSCs), spontaneously differentiated into osteogenic cells in vitro and in vivo. Engineered MSCs were transplanted locally and tracked in vivo in radial segmental defects (regenerating site) and in ectopic muscular and subcutaneous sites (non-regenerating sites). In vitro and in vivo analysis revealed rhBMP2 expression and function, confirmed by RT-PCR, ELISA, western blot, immunohistochemistry and bioassays. Secretion of rhBMP2 in vitro was controlled by tetracycline and resulted in secretion of 1231 ng/24 hours/106 cells. Quantitative Micro-CT 3-Dimentional reconstruction revealed complete bone regeneration regulated by tetracycline in vivo, indicating the potential of this platform for bone and cartilage tissue engineering. Angiogenesis, a crucial element in tissue engineering, was increased by 10-folds in transplants of rhBMP2 expressing MSCs (tet-off), shown by histomorphometry and MRI analysis (p< 0.05). In order to establish a gene therapy platform for systemic bone disorders, MSCs with tet-controlled rhBMP-2 expression, were injected systemically (iv). These engineered MSCs were genetically modified in order to achieve homing to the bone marrow. Systemic non invasive tracking of engineered MSCs was achieved by recording topographical bioluminescence derived from luciferase expression detected by a coupled charged CCD imaging camera. For clinical situations that require immuno-isolation of transplanted cells, we developed an additional platform utilizing cell encapsulation technique. Immuno-isolated engineered MSCs, with tet-controlled rhBMP-2 expression, encapsulated with sodium alginate induced bone formation by paracrine effect of secreted rhBMP-2. Finally, we have characterized a novel tissue-engineering platform composed of engineered MSCs and biodegradable polymeric scaffolds, creating a 3D bone tissue in rotating Bioreactors. Our results indicate that engineered MSCs and polymeric scaffolds can be utilized for ex vivo bone tissue engineering. We therefore conclude that genetically engineered MSCs expressing rhBMP-2 under tetracycline control are applicable for: a) local and systemic gene therapy to bone, and b) bone tissue engineering. Our studies should lead to the creation of gene therapy platforms for systemic and local bone diseases in humans and bone/cartilage tissue engineering

Background; We investigated, as a neoadjuvant to surgical therapy, the effect of a gene therapy of the primary tumour on the progression of minimal residual disease to overt liver metastases. The gene construct coding for the immunostimulatory molecules GM-CSF and B7-1 was delivered to the growing tumour by electroporation in Balb/C mice. Methods; JBS fibrosarcomas were induced subcutaneously and were randomised at 80mm3 to control and treatment groups. One day prior to treatment, the portal circulation was seeded with tumour cells. Gene delivery was assessed by in vivo imaging, cytokine measurement and anti–tumour cytotoxicity (in vitro and in vivo). Responses were determined by liver examination. Results; Gene expression and cytokine production was evident in treated tumours. Development of liver metastases was inhibited by neoadjuvant therapy in all 8 animals, in comparison to none of the control animals (n = 6) (average liver weight=0.99 g vs. 1.748 g. p< 0.03.) Metastases were confirmed histologically. Cytotoxicity studies and rechallenge confirmed development of specific T cell antitumour responses after gene therapy. Conclusions; Immunogene therapy of the primary tumour induces effective anti–tumoural responses and inhibits the development of liver metastases. This strategy could be developed for neoadjuvant therapy of some human cancers

Elderly patients with a high mortality risk for revision surgery are severely handicapped by a loosened hip prosthesis. Loosening is mainly caused by particle-induced osteolysis leading to the formation of a synovium-like interface tissue. As an alternative to revision surgery we have investigated the possibility of removing the tissue using a gene therapy approach and thereafter stabilizing the prosthesis with percutaneous cement injection. First we demonstrated that transduction of interface cells with a gene coding for E.coli nitroreductase (NTR) resulted in a 60-fold increase in sensitivity to the prodrug CB1954 that is converted to a toxic metabolite by NTR. Given these in-vitro data, we explored if intra-articular administration of this adenoviral vector encoding NTR followed by the prodrug was able to kill sufficient tissue in-vivo to allow refixation of the prosthesis by cement. We report the first three patients from a phase 1 study of 12 patients with a loosened hip who are experiencing debilitating pain and have significant comorbidity. On day 1 the vector is injected into the hip joint and on day 3 the prodrug is injected. On day 10 three holes are drilled in the femur and one in the acetabulum. Biopsies are taken from the periprosthetic space and low viscosity cement (Osteopal, Biomet Merck, Sjöbo, Sweden) is injected under fluoroscopic guidance. The first three patients have been included in the study and five more are planned for treatment before June 2005. The patients are females of 86, 72, and 79 years old. There were no adverse effects from vector injection (3x10 exp 9 particles). Six hours after prodrug injection the patients experienced nausea, (WHO grade 1) a commonly reported reaction to this prodrug. There was vomiting in two patients. Hip pain increased, but this was anticipated as this therapy will increase prosthesis loosening. 16 ml of cement was subsequently injected into the periprosthetic space in the first patient and 18 ml in the second. The patients were ambulated the day after surgery. The first two patients have a follow-up of twelve and six weeks. There was no pain in the hip. The maximum walking distance had increased from 5 to 30 meters in the first patient. The current study is the first to use in vivo intra-articular adenoviral mediated gene transfer in a clinical setting. Our preliminary results suggest that gene therapy and cement injection for hip prosthesis refixation is clinically feasible

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised β-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised β-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches

Aims: Recent advances in our understanding of intervertebral disc biology have led to develop novel treatments for intervertebral disc degeneration (IDD). With the ability to provide sustained delivery of a potentially therapeutic agent, gene therapy has shown much promise in regard to the treatment of IDD. The aims of this study are (part 1) to test efficacy in delaying course of IDD by intrediscal injection of adenoviral vectors carrying human BMP-2 and (part 2) to describe the application of an inducible system in order to modulate transgene expression. Methods: (Part 1) IDD was induced in 13 NZW rabbits by anterolateral stab. Three weeks post-stab, saline with or without virus was injected directly into stabbed lumbar discs. Group 1 (n=8) received Ad/hBMP-2 while group 2 (n=5) received saline only. Rabbits were followed longitudinally with MRIs and X-rays preoperatively for up to 12 weeks post-stab. ELISAs were done to confirm BMP-2 production. (Part 2) Human nucleus pulposus cells (NPC) were transduced with an adenoviral vector that expresses GFP under the control of a tetracycline (Ad/GFP. tet. ). Cells were cultutred with and without tetracycline. Transgene expression was assessed by detecting GFP signal with both the FACS and the fluorescent microscope. Results: (Part 1) By 12 weeks, the saline-injected discs had lost 49% of their MRI Index, in contrast to only a 25% decrease for the Ad/hBMP-2 treated discs. X-rays demonstrated no obvious bony intervertebral fusion in either group. ELISAs confirmed vigorous hBMP-2 production 3 weeks after therapeutic gene transfer. (Part 2) NPC expressed GFP after transduction. GFP positivity was not observed two days after administration of tetracycline. The cells expressed GFP again three days after removal of tetracycline. Discussion: The results of this study demonstrate the efficacy of vector-mediated BMP-2 gene transfer to alter the course of IDD in a reproducible animal model, as well as the potential to control transgene expression, improving safety

Purpose: To determine if an adenovirus vector expressing BMP-7 can alter the progression of post-traumatic osteoarthritis. Method: Preliminary dose-response studies were done in ovine metacarpal-phalangeal joints using 10^9, 10^10, and 10^11 virus particles (VP). In-vitro transfection efficiency studies were done using ovine synovial cells, chondrocytes and HEK293 cells. In-vivo studies were conducted in 16 sheep that underwent surgery to create bilateral contusive impact injuries to the medial femoral condyle. One week later 10^9 VP were injected into one joint of each sheep, while four sheep remained untreated bilateral controls. Three months later the sheep were sacrificed for assessments including histological scoring, cartilage glycosaminoglycan assays, and immunostaining for Col2 3/4 short collagen fragments that are generated by metalloproteinases during OA progression. Results: Transfection with 10^9 VP produced slightly longer expression than higher concentrations of VP. HEK293 cells expressed BMP-7 quickly but synoviocytes and chondrocytes expressed this protein at 48 and 96 hours. Knee joints that received Ad5-BMP-7 produced up to 2.5 ng of BMP-7 between day seven and 21. These joints had reduced cartilage degneration at the injury sites and less centrifugal progression of OA across the femoral condyle. Histological scores were reduced as was Col2 C3/4 short immunostaining. Conclusion: BMP-7 has a homeostatic role in cartilage and can be used therapeutically. 1. Ad5-BMP-7 transfection of synovial tissue produced sufficient BMP-7 to stop the progression of degenerative changes after trauma that would usually lead to OA. Adenoviral vectors can create inflammation and neutralizing antibodies but these complications were minimized by using a low (10^9) dose. Human trials using similar vectors are ongoing and the outcome of these will determine whether gene therapy will become a useful tool when patients are at risk of post-traumatic OA

Aims: The present study examined the effect of ade-novirus-mediated recombinant human BMP-2 (RAd-BMP-2) gene therapy combined with bioactive glass (BG) microspheres in promotion of new bone formation. Methods: Harlan Dawley female rats (n=72) underwent unilateral surgery of right or left tibia in a random order. A round cortical window (. −. 2.8 mm) was drilled into the anteromedial cortex of the proximal tibia. A smaller unicortical hole (. −. 1.0 mm) was drilled 5 mm distally. Bone marrow was removed and the medullary space between the cortical holes was þlled with BG microspheres. Adenoviral vectors RAdBMP-2 carrying the BMP-2 gene or RAdLacZ harbouring the E. coli LacZ reporter gene were injected locally into the medullary spaces. The control defects were þlled with BG microspheres only. Empty control defects were left to heal without any þlling. The rats were killed 4 days, 2 and 8 weeks after surgery and the tibias were harvested for analyses. At each time point, six animals were used for pQCT, radiography, BEI-SEM and histomorphometric analyses. Results: All BG-þlled defects showed a time-related increase of intramedullary new bone. At 8 weeks, there was signiþcantly more new bone in defects treated with BG and RAdBMP-2 gene than in defects left to heal without þlling (p=0.003) (BG + RAdBMP-2: 25.0 ± 6.0% and empty control defects: 12.3 ± 3.8%). Also defects þlled with BG only showed higher new bone formation than empty control defects, but this was not statistically signiþcant (p=0.10) (BG: 19.9 ± 7.3%). Conclusions: The current study showed that local BMP-2 gene therapy enhances new bone formation on bioactive glass microspheres

Aims. Gene therapy research in the field of orthopaedics and traumatology have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and cartilage defects. In particular, several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, using in vitro and ex vivo genetically modified cells to be implanted in the animal, produced promising results as they enable the production of physiologic doses of an osteoinductive gene product into selected anatomical sites. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the new osteo-genic factor Lim Mineralization Factor-3 (Ad-LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Methods. Primary dermal fibroblasts cultures were established using a 1cm. 2. biopsy of shaved skin obtained from the abdomen of each rat after anesthesia. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3 or both, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP at the same MOI were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, resuspended in sterile PBS, let adsorbed on a Hydroxyapatite/Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: 9 rats were treated with cells infected with AdLMP-3, 9 rats with cells infected with AdBMP-2 (positive controls), 9 rats with cells transduced with Ad-eGFP and 9 rats with untreated cells (controls). 3 Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology (Von kossa and Alizarin staining). Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed his-tologically and radiographically. On the contrary none of the controls showed bone formation at latest time point. Conclusions. Recently, Lim Mineralization Proteins (LMP), coded by three different splice variants (LMP-1, LMP-2, LMP-3) of the same gene, have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 (hLMP-3) contributes actively to bone formation, acting at least in part, through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mes-enchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have analyzed the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Engineered cells produced bone healing when implanted by the use of a scaffold in a rodent model, validating the in vivo osteoinductive properties of hLMP-3

Objectives. Previously, we reported the improved transfection efficiency of a plasmid DNA-chitosan (pDNA-CS) complex using a phosphorylatable nuclear localization signal-linked nucleic kinase substrate short peptide (pNNS) conjugated to chitosan (pNNS-CS). This study investigated the effects of pNNS-CS-mediated miR-140 and interleukin-1 receptor antagonist protein (IL-1Ra) gene transfection both in rabbit chondrocytes and a cartilage defect model. Methods. The pBudCE4.1-miR-140, pBudCE4.1-IL-1Ra, and negative control pBudCE4.1 plasmids were constructed and combined with pNNS-CS to form pDNA/pNNS-CS complexes. These complexes were transfected into chondrocytes or injected into the knee joint cavity. Results. High IL-1Ra and miR-140 expression levels were detected both in vitro and in vivo. In vitro, compared with the pBudCE4.1 group, the transgenic group presented with significantly increased chondrocyte proliferation and glycosaminoglycan (GAG) synthesis, as well as increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and TIMP metallopeptidase inhibitor 1 (TIMP-1) levels. Nitric oxide (NO) synthesis was reduced, as were a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 (ADAMTS-5) and matrix metalloproteinase (MMP)-13 levels. In vivo, the exogenous genes reduced the synovial fluid GAG and NO concentrations and the ADAMTS-5 and MMP-13 levels in cartilage. In contrast, COL2A1, ACAN, and TIMP-1 levels were increased, and the cartilage Mankin score was decreased in the transgenic group compared with the pBudCE4.1 group. Double gene combination produced greater efficacies than each single gene, both in vitro and in vivo. Conclusion. This study suggests that pNNS-CS is a good candidate for treating cartilage defects via gene therapy, and that IL-1Ra in combination with miR-140 produces promising biological effects on cartilage defects. Cite this article: R. Zhao, S. Wang, L. Jia, Q. Li, J. Qiao, X. Peng. Interleukin-1 receptor antagonist protein (IL-1Ra) and miR-140 overexpression via pNNS-conjugated chitosan-mediated gene transfer enhances the repair of full-thickness cartilage defects in a rabbit model. Bone Joint Res 2019;8:165–178. DOI: 10.1302/2046-3758.83.BJR-2018-0222.R1

Lim mineralisation protein (LMP) is a new positive regulator of the osteoblast differentiation programme. In humans three “splice variants” of LMP have been identified: LMP-1, LMP-2 and LMP-3. Recent studies demostrated that LMP-1 gene acts as a transcriptional factor inside the cells and is able to induce expression of specific bone genes, like the bone morphogenetic proteins (BMPs), to improve bone formation in vitro and ectopic bone formation in vivo and seems to induce spinal fusion in several animal models.

In order to evaluate the osteoinductive properties of the shorter variant of LMP, plasmidic and adeno-viral vectors expressing the optimised sequence of human LMP-3 have been generated. The osteogenic activity of LMP-3 was evaluated in vitro with experiments of transfection and infection of mesenchymal stem cells, fibroblasts and pre-osteoblasts; in vivo we investigated whether direct gene transfer of LMP-3 in the triceps muscles of immunocompetent mice was able to induce ectopic bone formation. All the animals were studied by histology and X-rays at different time points. In all the experiments BMP-2 was used as positive control.

These experiments demonstrated that the “gene transfer” of LMP-3 in fibroblasts and pre-osteoblasts stimulates production of specific bone proteins, such as osteocalcin, osteopontin and bone sialoprotein, and induces bone mineralisation in vitro. It was also demonstrated that LMP-3 is able to induce, in a dose-dependent manner, bone mineralisation and expression of specific bone genes (BMP-2, OSX, RunX2, alkaline phosphatase) in mesenchymal stem cells. Finally, the experiments showed that direct gene transfer of LMP-3 in the triceps muscle of mice induces ectopic bone formation in all the animals treated more efficiently than BMP-2.

These data demonstrate that gene transfer of LMP-3 could be used, more efficiently than BMP-2, in inducing bone formation in several cell lines and in vivo, establishing the osteoinductive ability of LMP-3. Thus, LMP-3 could represent, in the near future, a therapeutic alternative in several clinical conditions.

Aims: This in vitro study investigates the use of Collagen/PRP Hydrogels as a biological matrix for containing genetically modified human ACL cells, and supporting transgene expression. Methods: Adenoviral vectors encoding marker genes (green fluorescent protein (GFP)) and bioactive) where used to infect cultured human ACL cells?genes (TGF- ex vivo. The cells were seeded in Collagen/PRP Hydrogels and maintained in culture. To expression over time, ELISA was performed at days 4, 8, 15, 23,?measure TGFand 29. GFP positive cells within the gel were viewed by fluorescence microscopy at the same time points. After 29 days, the cultures were fixed, sectioned and various sections were stained with H& E, toluidine blue to detect proteoglycans and by immunhistocemistry for collagen type I and II. Results: Collagen/PRP Hydrogels were transgenes for up to 29 days.?able to support expression of GFP and TGF- expressing gel/cell constructs produced an abundant?Compared to controls, TGF- amount of type I collagen, consistent with the ligament phenotype and appeared more cellular. Little or no proteoglycan staining was observed in either group. Conclusion: These results demonstrate that genetically modified human ACL cells can support persistent transgene expression in vitro, sufficient to stimulate growth of ligamentlike tissue within a Collagen/PRP Hydrogel. The high levels of transgene expression suggest that the Collagen/PRP Hydrogel can function as an effective gene delivery system for tendon repair in vivo.

Objective To decide whether recombined rat transforming growth factor beta-1 gene and insulin-like growth factor-1 gene have positive influences on ACLT-induced osteoarthritis-like changes in NZW rabbit articular cartilage. Methods Twenty-four NZW rabbits, with osteoarthritis caused by anterior cruciate ligament transection£. . ACLT£©, were distributed to 4 groups randomly and another six rabbits were taken as normal control group (group 1). Chondrocytes which had been transfected with TGF-¦Â1 gene, IGF-1 gene (group 3–5) were injected into the knee of these NZW rabbits. Experimental control group (group 2) was only suffered ACLT but nothing injected. After 4, 8 weeks, rabbits were sacrificed and evaluated by morphological grades, histological examination, examination of in situ hybridization, immunohistochemistry, and transmission electron microscopy (TEM). Results The data of morphological grades showed that the normal control showed a significant difference compared with experimental control group (P< 0.01). The groups with injected chondrocytes carring TGF-¦Â1 gene and double genes (group 3,5) had a significant difference compared with experimental control group (P< 0.05). The in situ hybridization and immunohis-tochemistry examination showed the same results as above, and the group carring double genes (group 5) had a significant difference with that single gene (group 3,4) (P< 0.05). After 8 weeks, the examination data showed that all groups lower than the data of 4 weeks except the normal control group and experimental control group (P< 0.05). Ultrastructural examination indicated that the ultrastructure of experimental control group was more turbulent than that of normal control group. The ultra-structure of the gene therapy groups was more normal than that of experimental control group after gene therapy, but it turned to be turbulent again after 8 weeks. Conclusion It is effectual on osteoarthritis to inject chondrocytes carring recombined TGF-¦Â1,IGF-1 genes into NZW rabbits knee joints. It was obvious that the therapy effect of double genes was better than single gene. The fact that gene expression was decreased gradually after 4 weeks makes out that gene therapy is limited by time. These results suggest that therapeutic TGF-¦Â1 and IGF-1 gene transfer may be applicable for the treatment of OA

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production. 1. The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use. 2. Furthermore, BMP-7 was removed from the market. 3. These complications are related to the high doses used (1.5-40 miligrams per surgery). 2. compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range). 4. In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones. 5. Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA. 6. BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration. Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790

Messenger RNA (mRNA) is a new class of drug that can be used to express a therapeutic protein and, in contrast to DNA, is safer and inexpensive. Among its advantages, mRNA will immediately begin to express its encoded protein in the cell cytoplasm. The protein will be expressed for a period of time, after which the mRNA is degraded. There is no risk of genetic damage, one of the concerns with plasmid DNA (pDNA) used in traditional gene therapy approaches. Nevertheless, mRNA application in tissue regeneration and regenerative medicine remains limited. In this case, mRNA must overcome its main hurdles: immunogenicity, lack of stability, and intracellular delivery. Research has been done to overcome these limitations, and the future of mRNA seems promising for tissue repair. 1,2. This keynote talk will address questions including: What are the opportunities for mRNA to improve outcomes in musculoskeletal tissue repair, in particular bone and cartilage? What are the key factors and challenges to expediting this technology to patient treatment (beyond COVID-19 vaccination)?. Acknowledgements: E.R.B thanks the cmRNAbone project funded by the European Union's Horizon 2020 research and innovation program under the grant agreement no. 874790 and the NIH R01 AR074395 from NIAMS for funding her mRNA work