Periodic Reporting for period 1 - CARTHAGO (Cartilaginous tissue regeneration by non-viral gene therapy; taking the hurdles towards efficient delivery)
Période du rapport: 2020-09-01 au 2022-08-31
Diseases of the musculoskeletal system impose a substantial burden on Western societies, which is ever increasing with ageing of the population. Amongst the diseases with most impact are osteoarthritis (OA) and chronic low back pain (CLBP) caused by intervertebral disc (IVD) degeneration, involving the cartilaginous tissues of these organs. OA and CLBP are in fact the no 5 and no 1 causes of loss of disability-adjusted life years. Moreover, absenteeism poses a substantial economical burden on society. Despite the severity of the problems, medical solutions currently are limited and consist mainly of highly invasive surgery that replaces or immobilises the joint. Significant effort worldwide is aimed at helping the joint and IVD heal themselves by regenerative medicine, using stem cells or growth factors to regenerate the tissue, yet very few of these strategies have entered the clinic; Non viral gene therapy has the potential to overcome these hurdles, due to its high specificity and hence safety, longevity of effects, and cost-effectiveness. However, the main challenge for application of gene therapy in this field is that the tight extracellular matrix of cartilaginous tissue does not contain blood vessels for transport and distribution of the therapeutic nucleic acids. This poses a challenge to the delivery of gene activity modifying agents, in addition to prevention of extracellular and intracellular degradation of th nucleic acids. Furthermore, in the public opinion, as with many other innovative medical treatments, gene therapy is conceived as a potentially hazardous strategy, and awareness and adequate information of the public will be fundamental to enhance well-informed adoption. Hence the overall objectives of CARTHAGO were to
1) Develop smart nucleic acid carriers that are biochemically tailored to enhance penetration into the dense tissues of the joint and IVD, and biochemically modified nucleic acids that result in enhanced protein production
2) Implement non-linear ultrasound as novel tool to a) enhance tissue permeation of nucleic acids, either naked or within carrier systems, in cartilage and intervertebral disc tissue b) locally trigger dissociation and release of systemically administered albumin-bound oligonucleotides.
3) Apply systemic administration of gene modifying agents using albumin as an endogenous or preformulated carrier in combination with ultrasound triggered local dissociation, even paving the way for non-invasive self-administered local treatment on demand, even for small joints that cannot be accessed by injection.
4) Implement statistical approaches for efficient nucleic acid-therapeutics design and hence treatment development.
5) Develop live imaging of cartilage and IVD regeneration in vivo, using in vivo click chemistry.
6) Development of a draft for the first educational framework incorporating ethics in biomedical development.
1) Develop smart nucleic acid carriers that are biochemically tailored to enhance penetration into the dense tissues of the joint and IVD, and biochemically modified nucleic acids that result in enhanced protein production
2) Implement non-linear ultrasound as novel tool to a) enhance tissue permeation of nucleic acids, either naked or within carrier systems, in cartilage and intervertebral disc tissue b) locally trigger dissociation and release of systemically administered albumin-bound oligonucleotides.
3) Apply systemic administration of gene modifying agents using albumin as an endogenous or preformulated carrier in combination with ultrasound triggered local dissociation, even paving the way for non-invasive self-administered local treatment on demand, even for small joints that cannot be accessed by injection.
4) Implement statistical approaches for efficient nucleic acid-therapeutics design and hence treatment development.
5) Develop live imaging of cartilage and IVD regeneration in vivo, using in vivo click chemistry.
6) Development of a draft for the first educational framework incorporating ethics in biomedical development.
Extensive progress has been made in the development of the various novel delivery tools. The biocompatibility and high transfection efficiency of the nanogels are promising for intra-articular delivery. Intracellular GSH concentrations should be considered when testing bioreducible PAA NPs in the oxidative stressed, GSH-deficient OA synovial joints. Benzylamine-terminated G3 PEG-GATGE dendritic block copolymers were synthesised in very good yields. These successfully formed dendriplexes with siRNA at NP ratios of 5, 10, 20, 40 and 80, showing excellent complexation efficiencies with this nucleic acid, and appropriate nanosizes, polydispersity indexes and zeta potential for nucleic acid delivery application. In addition, the prepared dendriplexes have shown excellent cell association/internalization results and higher internalization efficacy than the positive control in a chondrocyte cell line (C28/I2). 2’Guanidium modified siRNA was synthesized with both one and two modifications on the overhang of both sense and antisense strands, and retained the original transfection activity of unmodified siRNA. When there is antisense strand modification, we observed a decrease in activity. Also HA-cysteine and HA-CHO derivatives for different hydrogels were developed. We have successfully optimised the ratios and the concentration of the polymeric nanoparticles using QbD approach. For the hydrogels, we have also successfully identified the molecular weights of hyaluronic acid and the molar ratios of the coupling agent to be used to get targeted rheological properties with less degree of modification. For dendriplexes, we have performed the multivariate data analysis to identify how the physicochemical properties like particle size, PDI, zeta potential and complexation capacity influence the cytotoxicity, cell internalisation and the gene silencing. For WP3, targeting ligands have been incorporated in albumin constructs and nanogels and will be tested in relevant in vitro systems. Together with the activities in WP4, US protocols were tested in agarose gels as model for cartilage, showing a moderate enhancement of penetration. For cartilage tissue, a system was set up to subsequently test the obtained US settings in cartilage.
In 2D culture models of joint cells, effective transfection was shown for the nanogels. Also agarose gels containing nucleus pulposus cells as model for the NP showed successful transfection, albeit at a lower rate. Transfection of IVD and human ostearthritic and bovine healthy cartilage tissue has been undertaken, but is being met with some difficulties. Furthermore YAP was shown to be a promising target for siRNA delivery in OA. Ultrasound protocols were optimised for delivery of NA in agarose gels as tissue phantoms and showed clear enhancement of lipofectamin-loaded labelled RNA. Finally proof of principle for proteoglycan-specific incorporation of click-chemistry labelled buidling blocks was provided in cartilage tissue, paving the way towards validation of the novel building blocks and associated click chemistry to be developed in WP5. A systematic review on the ethics of gene therapy has been initiated in WP6. In addition the foundation was layed for an educational framework by estalishing focus group discussions with ESRs, which stimulated ethical reflection on their research.
In 2D culture models of joint cells, effective transfection was shown for the nanogels. Also agarose gels containing nucleus pulposus cells as model for the NP showed successful transfection, albeit at a lower rate. Transfection of IVD and human ostearthritic and bovine healthy cartilage tissue has been undertaken, but is being met with some difficulties. Furthermore YAP was shown to be a promising target for siRNA delivery in OA. Ultrasound protocols were optimised for delivery of NA in agarose gels as tissue phantoms and showed clear enhancement of lipofectamin-loaded labelled RNA. Finally proof of principle for proteoglycan-specific incorporation of click-chemistry labelled buidling blocks was provided in cartilage tissue, paving the way towards validation of the novel building blocks and associated click chemistry to be developed in WP5. A systematic review on the ethics of gene therapy has been initiated in WP6. In addition the foundation was layed for an educational framework by estalishing focus group discussions with ESRs, which stimulated ethical reflection on their research.
At the end of the project, we envisage novel treatments developed for tissue regeneration in OA and CLBP. With the novel treatments, patients will receive efficient long term treatments allowing an active life and improved well-being, health care costs and costs related to work absenteeism will be reduced. Moreover, the newly developed technologies can in addition be applied in other fields of health sciences, including US to enhance particle penetration, click chemistry to visualise tissue production and albumin as endogenous carrier for nucleic acid based drugs. Finally, responsible research and innovation in the EU will be enhanced by a novel framework joining ethics and biotechnology research to be applied in future EU research projects.