Gene therapy for Friedreich’s ataxia based on the modification of viral and non-viral vectors to improve their delivery across the blood brain barrier.
Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Universidad Autónoma de Madrid; CIBER de Enfermedades Raras (CIBERER).
IRB Barcelona; Universitat de Barcelona.
Background and state of the art:
As a monogenic disease, Friedreich´s ataxia (FA) seems amenable to be treated through gene therapy. The feasibility of a gene therapy approach for FA through the introduction of correct copies of the frataxin gene using distinct lentiviral, adenoassociated and herpesviral vectors has been demonstrated in cell models and in an in vivo mouse models. Among viral vectors, herpes simplex type 1 (HSV-1)-derived vectors are unique by their ability to accommodate the whole genomic locus of Fxn. The use of whole genomic loci has important advantages for gene therapy (when compared with the use of cDNAs controlled by exogenous promoters) since genomic loci contain all the endogenous regulatory sequences controlling their expression (also in response to different physiological signals) as well as the signals controlling their alternative splicing and may therefore generate all the different protein isoforms in a physiological manner.
However, the delivery of gene vectors to neurons in all the affected sites of the nervous system constitutes the major challenge for a successful gene therapy approach in the case of FA.
In this context, the development of novel DNA nanocarriers carrying the whole genomic locus of Fxn and equipped with a higher capacity to cross the Blood Brain Barrier (BBB) may constitute an important breakthrough in the development of a gene therapy for FA.
The BBB is a highly restrictive barrier that prevents the passage of the vast majority of compounds from the blood to the nervous system. Interestingly, it has recently been shown that peptides can act as efficient vectors (BBB-shuttles) to carry a range of molecules with therapeutic properties of interest (cargoes) into the nervous system, independently of the physico-chemical properties of the cargo. In this context, Giralt’s group has recently demonstrated the efficiency of peptídic BBB shuttles by showing the delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor.
Accordingly, this project aims to generate new modified viral vectors and viral-free synthetic nanoparticles for highly efficient DNA delivery into the nervous system that may facilitate gene therapy for Friedreich’s ataxia (FA). More specifically, we plan a strategy where a DNA encoding for frataxin will be actively delivered through the blood-brain barrier (BBB) with the help of novel nanocarriers including modified viral vectors and synthetic nanoparticles.
This project aims to generate new molecular tools, including modified viral vectors and viral-free nanoparticles, for highly efficient DNA delivery into the nervous system that may facilitate gene therapy for Friedreich’s ataxia (FA).
The project addresses the design and development of novel gene delivery systems formed by either a modified viral vector or a synthetic nanoparticle (based on PLGA-PEG nanoparticles) which will cross the blood-brain barrier (BBB) thanks to novel peptides able to carry cargoes through the BBB (which are referred to as BBB-shuttles). These peptidic BBB shuttles will decorate the surface of both viral vectors and synthetic nanoparticles. To reach the target cells, the surface of these gene delivery nanocarriers will also be decorated with homing peptides (HPs) that will target the constructs to neurons, and cell-penetrating peptides (CPPs) that will allow the transport across cell membranes.
This project tries to go significantly beyond the state of the art in gene delivery to the nervous system by combining the properties of two well-known DNA nanocarriers (such as viral vectors and PLGA-PEG nanoparticles) with the functional targeting properties of novel peptides that function as BBB-shuttles, HPs and CPPs.
Herpesviral vectors have demonstrated to be efficient gene delivery systems with the ability to accommodate whole genomic loci (which favor physiological gene expression as well as the generation of different protein isoforms arising from alternative splicing), but lack the ability to cross the BBB. Modification of these viral vectors with BBB-shuttles may render them able to be transported through the BBB.
Synthetic PLGA-PEG nanoparticles are highly advantageous as delivery vehicles since they are very simple to prepare and scale-up and have generally less pro-inflammatory effects than viral particles. PLGA-PEG -based nanoparticles are accepted by regulators as a safe material and are being used as drug delivery vehicles. These nanoparticles will be decorated with several peptides acting as BBB-shuttles, HPs and CPPs in order to improve their gene-delivery efficiency to the nervous system.
In this project we will compare the gene transfer efficiency, toxicity and therapeutic activity of the different gene delivery nanocarriers in suitable human cell and mouse models of FA in order to find the most promising candidate for further development.
To reach this goal the project assembles two partners that will contribute complementary knowledge and technology. Diaz-Nido’s group at Centro de Biología Molecular Severo Ochoa has expertise in experimental models and molecular therapy strategies for neurodegenerative diseases with an emphasis on Friedreich’s ataxia. Giralt’s group at IRB Barcelona has a long-term expertise in the field of peptide chemistry and its application to biomedicine and is an international reference in this field. Of major relevance to this project are his recent developments on peptidic BBB shuttles and drug delivery systems. This unique blend of expertise is crucial for the success of this project which goes from the synthesis of novel DNA nanocarriers to their biological evaluation in cell and mouse models of FA.
Specific Aims of the Project
The project is focused on generating novel molecular tools to alleviate the neurodegenerative component of FA by means of therapeutic approaches that take advantage of active targeting to cross the blood-brain barrier (BBB). More specifically, we plan a strategy where a DNA encoding for frataxin will be delivered through the BBB with the help of modified viral vectors and PLGA-PEG nanoparticles. To reach the target cell, the surface of these delivery nanosystems will be decorated with peptides able to transport cargoes though the BBB (which are referred to as BBB-shuttles), homing peptides (HPs) that will target the constructs to neurons, and cell-penetrating peptides (CPPs).
The concrete aims of the present project are as follows:
Aim1.- Development of a gene therapy system for FA based on modified viral vectors.
Aim2.- Development of a viral-free system for FA gene therapy based on PLGA-PEG nanoparticles.
Aim3.- Biological examination and comparison of the efficiency of the different modified vectors in cell and mouse models.