Research / Scientific departments / Neuroscience / Units and Groups / AAV-MPS clinical program

The AAV-MPS clinical program


The objective of the programme is to set up a gene therapy approach for the CNS in a LSD called Sanfilippo syndrome or mucopolysaccharidosis type III (MPSIII), a disease with prominent early onset neurological manifestations. The considered treatments consist in intracerebral stereotactic injections of viral vectors derived from adeno-associated viruses (AAVs), which code for the missing enzyme. There are two major biochemical forms of the disease with identical clinical manifestations: MPSIIIA is a defect of N-sulfoglycosamine sulfohydrolase (SGSH), MPSIIIB a defect of a-N-acetylglucosaminidase (NAGLU).
Our focus on Sanfilippo syndrome was decided according to the following considerations:
- CNS early onset disorders in Sanfilippo syndrome are responsible for cognitive and behavioural manifestations leading to progressive mental retardation and neurodegeneration. In contrast, manifestations outside the CNS are mild. Efficient therapeutic procedures aimed at lessening, slowing or preventing neurological manifestations would therefore presumably improve patient wellbeing and life expectancy, and the quality of life of their close relatives.
- Clinical manifestations of Sanfilippo syndrome have a unique cause, the deficiency of a lysosomal enzyme, which interrupts the degradation of heparan sulfate (HS) and provokes the accumulation of HS oligosaccharides, the supposedly toxic products for the brain. Although largely not understood, the link between molecular and cognitive dysfunctions is well demonstrated, making it possible to decipher the cascade of secondary pathological events leading to progressive loss of brain plasticity.
 - The possibility of suppressing the primary cause of neurodegeneration in a young brain is the best situation to assessing to which extent brain plasticity and efficient brain functions can be restored by treatment.
- With respect to these considerations and to the difficulty of conceiving and realising a clinical trial in Hurler syndrome or mucopolysaccharidosis type I (MPSI), the other lysosomal disease which we initially saw as a potential target for gene therapy, all efforts have been focused on Sanfilippo syndrome, and gene therapy for Hurler syndrome will be considered only after previous demonstration of safety and efficacy in Sanfilippo patients.

Preclinical studies were performed within a consortium, which is animated and supervised by J-M Heard in collaboration with Pr. M. Tardieu, a neuro-paediatrician at Bicêtre hospital, and Dr. M. Eliaszewicz, the Medical Director of the Institut Pasteur. It is supported by the Association Française contre les Myopathies (AFM), the Institut Pasteur and the INSERM. AAV vectors batches were prepared at the vector core of the University of Nantes (P. Moullier) and by the Dutch company Amsterdam Molecular Therapeutics (AMT). MPSI dogs were bred and tissue pathology was examined at the Ecole Nationale Vétérinaire de Nantes (M-A Colle), where surgery was performed by neurosurgeons from the CHU of Nantes (S. Raoul, Y. Lajat). MPSIIIB dogs were bred and treated at Iowa State University (M. Ellinwood, K. Kline). Specific biochemical assays were performed at the Hospices civils de Lyon (I. Maire, R. Froissart and M-T Vanier). Epidemiology studies involved B. Héron, M. Tardieu, the UK MPS society, and groups of paediatricians in France, UK and Greece. Biomarkers studies are performed by the teams of J. Ausseil (Université d’Amiens), R. Daniel (CNRS, Université d’Evry) and I. Blasig (Leibniz Institute, Berlin). The conception of the clinical trial organisation, the preparation of the dossiers for submission to regulatory agencies and the interactions with CROs were performed by the Pole Intégré de Recherche Clinique (PIRC) of the Institut Pasteur (C. Delval and C. Artaud).

Data generated during these preclinical studies provided rationales for two gene therapy trials in MPSIIIA and MPSIIIB, respectively. Pr. M. Tardieu and Pr. M. Zerah, the head of a paediatric neurosurgery unit at Necker hospital, are the principal investigator and the neurosurgeon of these two studies, respectively.  

Overview of recent achievements

- We completed preclinical studies in 35 dogs, demonstrating the safety of the gene therapy procedure and providing evidence for biochemical and histological correction in the brain of two animal models relevant to the human disease, the MPSI and the MPSIIIB dogs (1, 4).

- We performed a retrospective epidemiological studies of children born with MPSIII in France, UK and Greece between 1990 and 2006, providing data about incidence and measurable clinical markers of the major events of the natural history of the disease (7).

- We initiated a study of biological markers to be detected and measured in patient’s cerebro-spinal fluid (CSF). During an ongoing initial stage CSF collected in the dogs that received or did not received gene therapy are analysed. This study will be completed by the investigation of candidate biomarkers in human samples stored in dedicated biobanks. HS oligosaccharides are biomarkers of the disease. They are identified and quantified in the CSF by mass-spectrometry. Quantitative proteomic studies are performed in parallel for the detection of proteins associated with neuroinflammation and/or neurodegeneration in the CSF. 

- We validated a procedure for large-scale manufacturing of the AAV5-hNAGLU vector in insect cells according to GMP requirements. The biological activity of vectors produced according to this method was documented in MPSIIIB mice and MPSIIIB dogs. The clinical vector batch has been manufactured and will be released when certification and stability studies are completed.

- Regulatory toxicology and biodistribution studies confirmed previous observations in affected mice and dogs, showing excellent general tolerance and absence of local inflammatory response at the sites of vector deposit in the brain of healthy rats and healthy dogs.

- The Institut Pasteur decided to be the sponsor of a phase I/II clinical trial in MPSIIIB patients and the Institut Pasteur clinical research office (PIRC) has prepared documents for submission to French regulatory authorities (ANMS). Orphan drug designation was delivered by the EMA.

- The company Lysogene decided to be the sponsor of a phase I/II gene therapy clinical trial in MPSIIIA, using a protocol identical to that designed for MPSIIIIB, and the same clinical investigators (Pr. M. Tardieu and Pr. M. Zerah). However, AAV vectors used in the MPSIIIA trial were different from those prepared for MPSIIIB (AAV11 coding for sulfamidase versus AAV5 coding for NAGLU), and manufactured differently (triple DNA transfection versus baculovirus infection) by different providers (Weill Cornell Medical College versus Unicure). Four patients (age 5-6 years) have been enrolled in the MPSIIIA trial and treated in 2011-2012. The surgical procedure was well tolerated and undesired side effects have not been observed during the current follow-up period of 6 to 13 months.
Gene therapy trial in patients with Sanfilippo syndrome type B

Study objectives are the assessment of a therapeutic approach consisting of intracerebral deposits of AAV vector inducing the expression of missing lysosomal enzyme NAGLU. Primary end-points are the evaluation of clinical, radiological, biological and immunological tolerances. The secondary end-point are the definition of exploratory tests that will become evaluation criteria for efficacy studies and the measurement of biological markers of disease severity in patient cerebro-spinal fluid (CSF).
They study will be a phase I/II, open label, non-randomized single centre investigation of intracerebral administration of adeno-associated viraAAV vectors. Vector suspensions will be simulta deposited in both hemispheres simultaneously at 16 sites through eight burr holes in the white matter adjacent to putamen and in the cerebellum during a single surgical session. Combined immunosuppression is justified by previous studies in children receiving recombinant enzyme therapy and by previous investigations in affected dogs. It will consist of tacrolimus tarted 15 days before surgery and maintained throughout the study, combined with mycophenolate mofetil for the initial 3 months and associated with prednisolone during 10 days after surgery.
The studies will concern four children with mild clinical expression of the disease, older than 18 months and younger than 5 years, with mutation in the NAGLU gene confirmed by molecular analysis, vital parameters in normal range, preserved ability at independent walking, absence of brain atrophy. These children do not have alternative therapeutic option.
Trial oversight committees will be set up to ensure regulatory, ethical, and good running of the study. They will consist of a clinical trial committee chaired by the principal investigator, an Independent Data Safety Monitoring Committee (IDMC), and a trial follow-up board including representatives of the sponsors and of family organizations.
Clinical, radiological and biological data will be recorded at regular intervals over the one-year study period, and over an unlimited period after the study has been completed. Biological samples, including CSF, will be collected at defined time-points according to a defined schedule and stored in a specific biobank. Primary safety end-points will be reached according to clinical parameters, which are expected to remain stable over a 1-year interval, MRI, which is not expected to show inflammation at the injection site, and usual biological parameters, which should remain in the normal range. Immunological tolerance will be defined as the absence of circulating T-cells responding to enzyme-born epitopes. Secondary end-points will be reached according to the detection of the corrective enzyme and the modification of biological markers of disease severity in collected CSF samples.
After a one-year follow-up conclusion about tolerance will be drawn according to primary endpoints, and secondary endpoints will provide preliminary indications about efficacy. Patients will then be enrolled in two-year continuation studies with the objective to conclude about treatment efficacy. Clinical efficacy will be measured according to clinical markers, as they have been defined in the retrospective epidemiological study of the natural history of Sanfilippo syndrome that we previously performed (7). Biochemical efficacy will be measured according to biomarkers present in patient CSF. Disease severity markers are heparan sulfate oligosaccharides and molecules associated to inflammation or extra-cellular matrix damages. They have been defined using samples collected during our previous dog studies and are currently being validated in human CSF.       
These studies will provide unique information on the possibility of restoring brain plasticity in affected children. An ethical challenge with regard to LSDs is diagnosis through systematic screening of newborns in at-risk, or in the general population at preclinical stages. Indication that benefit can be expected from treatment at preclinical stage will have major impact on the current debate on this issue.
Immune reaction is central to human gene therapy. Exploration of immune responses in patients receiving immunosuppressant will specify whether tolerance to specific antigens of the therapeutic enzymes develops with time in these conditions. Results will produce indicators of the risk of potential deleterious immune reaction once immunity is restored. These data will be useful for managing human gene therapy studies in the future.
Rare diseases have the potential to serve as launching platforms for innovative medicine. Demonstration of success in these rare diseases will pave the way for expansion to related disorders. Through collaborations with teams at the University of Cambridge (UK) and at the San Rafaele Institute in Milan (IT), we are associated to the preparation of two other gene therapy trials in LSDs, targeting Tays-Sachs disease and Hurler syndrome, respectively.