Final Report Summary - NINIVE (Non invasive nanotranducer for in vivo gene therapy)
A European research team led by researchers from the medical science laboratory, group of nanotechnology of the Scuola Superiore Sant'Anna in Italy, commenced work in December 2006 on the important Sixth Framework Programme (FP6) project 'NINIVE' (NMP4-CT-2006-033378). The multidisciplinary team involved in the project included scientists from the Center for Drug Delivery Research of the ULSOF (United Kingdom), the MPI-FKF fuer Festkoerperforschung (Germany), Thales research & technology (France), the Institute of Neuroscience of CNR (Italy) and Nanothinx S.A. (Greece). The main objective of the NINIVE project (please see http://www.ninive-project.org/ online) is the development of a non-viral vector for a safe and efficient gene transfection and targeted drug delivery. The system proposed by NINIVE is based on use of Carbon nanotubes (CNTs) which consist exclusively of carbon atoms arranged in a series of condensed benzene rings rolled-up to form single or multi-walled tubular structures. This novel nanomaterial belongs to the family of fullerenes, an allotropic form of carbon. CNTs have nanometric dimensions and unique physicochemical properties which make CNTs unique materials with several potential applications especially in the biomedical field. Specifically, in the NINIVE project CNTs act as transporters of genes in addition to being functionalised with appropriate ligands to enable specific binding to receptors over-expressed by the cells targeted for transfection. A solution containing myriads of these coated and functionalised nano-vectors is administered by local injection in the target tissue. These nano-vectors home on and bind to the intended target cells. In the NINIVE project, two strategies of cell transfection are investigated:
(i) cellular up-take of the gene-carrying nano-vectors by endocytosis; and
(ii) transfer of genes from the nano-vectors to the recipient target cells via electroporation (i.e. permeabilisation of cell membrane by application of short-duration electric pulses). With this mechanism, cell permeabilisation is induced by CNTs on exposure to external electromagnetic fields by exploiting their nanotransducers properties.
Following confirmation of the proof of efficacy by in-vitro studies, the NINIVE system as a platform for targeted gene delivery now requires validation by in vivo pre-clinical studies, followed by phase I clinical trials involving patients with a specific neurological disorder. This ambitious project developed by the multidisciplinary NINIVE team covering a broad range of scientific expertise in the fields of nanobiotechnologies, communication technologies and neuroscience has achieved all the objectives outlined in the proposal including efficacy of the NINIVE vector as transporter of genes and its biocompatibility.
All the milestones planned in the project have been succesfully concluded. The Group of Medical and Ethical Experts (GMEE) was constituted. The experts are Dr Caterina Cinti, Dr David Klatzmann, Dr Luis Mir, Dr Ulrich Lauer, Dr Francois Lachapelle. They were engaged to overview the project and provide advisements. For the expert recruitment, the consortium followed strictly the European Commission guidelines.
The state of the art was deeply analysed by all partners. As results of this deep research, we concluded that nowadays there are thousands of publications in the literature concerning the use of carbon nanotubes in biomedicine, and many of them concern the CNT ability to translocate through plasma membranes, allowing their use for the delivery of therapeutically active molecules. Despite this strong interest, there are very few examples of exploitation of the CNT unique electrical, optical, thermal, and spectroscopic properties in a biological context. We concluded that the real breakdown of NINIVE project over the current research is the discovery, not yet achieved, of a new methodology, which exploits CNT physical properties to perform an enhanced and effective in vivo gene and drug therapy and no competitors have been identified yet. Concerning the biocompatibility studies, the analysis of the existing literature pointed out the need of procedure standardisation for biological testing. Also nanotube characterisation should be accurate, as biocompatibility results are closely dependent from the CNT properties, in particular length.
The medical requirements and specifications of gene therapy via electroporation and technical specifications of NINIVE tool for gene therapy have been identified by the consortium and described in the dedicated deliverable. The results achieved in this task came out from the exchange of know-how among the partners with background in nanotechnology (SSSA, NTX and Thales), nanoscience (CDDR, MPI), and biology (CNR). This work provided the necessary input to propose the design of the NINIVE vector. In parallel, CNR provided the model of brain disease to try an experimental gene therapy in rodents using the NINIVE vectors.
The aspects related to how NINIVE nanotransducers are envisaged to work, such as the diffusion within the target tissue, the mechanisms of targeted cell binding, cellular uptake via endocytosis and via electroporation were investigated by SSSA, in order to identify base models by using both analytical and finite-element modelling. The architecture of the NINIVE vector was proposed. The basic idea is to cover the nanotube surface with charged functional groups (-NH3+) able to bind DNA. These charged groups are provided by a low density (25 kDa) PEI covalently binded to the weakly oxidised nanotube surface (ldPEI-CNT). The other molecules (i.e. fluorescent molecule for nanotube tracking and ligand for cell binding) are attached to the nanotube by covalent binding to the amino groups. The final molecules chosen for this application were a chemokine and alexa fluor. Several recent evidences suggest that chemokines have a neurotransmitter / neuromodulatory role on brain functions similar to several neuropeptides. In the stroke (animal model for the NINIVE study), among the molecules that allow trophic support to the neurons in the penumbra surrounding the infarct area, the chemokine CXCL12 (previously known as SDF-1) is expressed.
This chemokine is quickly internalised by the neurons that express its receptor CXCR4. So, we believe that NINIVE vectors can be functionalised with this small (10 KDa) protein to specifically target the neurons located in the penumbra. Moreover, the internalisation of the CXCL12 receptor, CXCR4, could help the localisation of the nanotubes.
Finally, alexa-ldPEI-CNTs were produced which are a valid tool to follow the pathway and the diffusion of the nanotubes after injection. The localisation of these fluorescent nanotubes was followed in vivo by fluorescent / confocal microscopy.
All the methods, methodologies, and technologies required for the development and the characterisation of the vector were identified and proved to be effective. The main out coming of all this activity was the development of the Standard operating procedures (SOP). These SOPs represent the summary and the conclusion of the first year of experimental activity performed in the project. They establish the material and methods of the project and assure the homogeneity of protocols used by partners in their experimental activity.
(i) cellular up-take of the gene-carrying nano-vectors by endocytosis; and
(ii) transfer of genes from the nano-vectors to the recipient target cells via electroporation (i.e. permeabilisation of cell membrane by application of short-duration electric pulses). With this mechanism, cell permeabilisation is induced by CNTs on exposure to external electromagnetic fields by exploiting their nanotransducers properties.
Following confirmation of the proof of efficacy by in-vitro studies, the NINIVE system as a platform for targeted gene delivery now requires validation by in vivo pre-clinical studies, followed by phase I clinical trials involving patients with a specific neurological disorder. This ambitious project developed by the multidisciplinary NINIVE team covering a broad range of scientific expertise in the fields of nanobiotechnologies, communication technologies and neuroscience has achieved all the objectives outlined in the proposal including efficacy of the NINIVE vector as transporter of genes and its biocompatibility.
All the milestones planned in the project have been succesfully concluded. The Group of Medical and Ethical Experts (GMEE) was constituted. The experts are Dr Caterina Cinti, Dr David Klatzmann, Dr Luis Mir, Dr Ulrich Lauer, Dr Francois Lachapelle. They were engaged to overview the project and provide advisements. For the expert recruitment, the consortium followed strictly the European Commission guidelines.
The state of the art was deeply analysed by all partners. As results of this deep research, we concluded that nowadays there are thousands of publications in the literature concerning the use of carbon nanotubes in biomedicine, and many of them concern the CNT ability to translocate through plasma membranes, allowing their use for the delivery of therapeutically active molecules. Despite this strong interest, there are very few examples of exploitation of the CNT unique electrical, optical, thermal, and spectroscopic properties in a biological context. We concluded that the real breakdown of NINIVE project over the current research is the discovery, not yet achieved, of a new methodology, which exploits CNT physical properties to perform an enhanced and effective in vivo gene and drug therapy and no competitors have been identified yet. Concerning the biocompatibility studies, the analysis of the existing literature pointed out the need of procedure standardisation for biological testing. Also nanotube characterisation should be accurate, as biocompatibility results are closely dependent from the CNT properties, in particular length.
The medical requirements and specifications of gene therapy via electroporation and technical specifications of NINIVE tool for gene therapy have been identified by the consortium and described in the dedicated deliverable. The results achieved in this task came out from the exchange of know-how among the partners with background in nanotechnology (SSSA, NTX and Thales), nanoscience (CDDR, MPI), and biology (CNR). This work provided the necessary input to propose the design of the NINIVE vector. In parallel, CNR provided the model of brain disease to try an experimental gene therapy in rodents using the NINIVE vectors.
The aspects related to how NINIVE nanotransducers are envisaged to work, such as the diffusion within the target tissue, the mechanisms of targeted cell binding, cellular uptake via endocytosis and via electroporation were investigated by SSSA, in order to identify base models by using both analytical and finite-element modelling. The architecture of the NINIVE vector was proposed. The basic idea is to cover the nanotube surface with charged functional groups (-NH3+) able to bind DNA. These charged groups are provided by a low density (25 kDa) PEI covalently binded to the weakly oxidised nanotube surface (ldPEI-CNT). The other molecules (i.e. fluorescent molecule for nanotube tracking and ligand for cell binding) are attached to the nanotube by covalent binding to the amino groups. The final molecules chosen for this application were a chemokine and alexa fluor. Several recent evidences suggest that chemokines have a neurotransmitter / neuromodulatory role on brain functions similar to several neuropeptides. In the stroke (animal model for the NINIVE study), among the molecules that allow trophic support to the neurons in the penumbra surrounding the infarct area, the chemokine CXCL12 (previously known as SDF-1) is expressed.
This chemokine is quickly internalised by the neurons that express its receptor CXCR4. So, we believe that NINIVE vectors can be functionalised with this small (10 KDa) protein to specifically target the neurons located in the penumbra. Moreover, the internalisation of the CXCL12 receptor, CXCR4, could help the localisation of the nanotubes.
Finally, alexa-ldPEI-CNTs were produced which are a valid tool to follow the pathway and the diffusion of the nanotubes after injection. The localisation of these fluorescent nanotubes was followed in vivo by fluorescent / confocal microscopy.
All the methods, methodologies, and technologies required for the development and the characterisation of the vector were identified and proved to be effective. The main out coming of all this activity was the development of the Standard operating procedures (SOP). These SOPs represent the summary and the conclusion of the first year of experimental activity performed in the project. They establish the material and methods of the project and assure the homogeneity of protocols used by partners in their experimental activity.
