Characterizing novel gene therapy vectors to identify a potential promising new treatment for Fragile X Syndrome
Creators: name: Kaminioti-Dumont, Laura Christina
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: Fragile X Syndrome
FMR1
mouse model
gene therapy viral vector
gene therapy

description: INTRODUCTION: Fragile X Syndrome (FXS) is an X-chromosome linked, neurodevelopmental disorder, which is caused by the silencing of the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene that encodes the Fragile X Messenger Ribonucleoprotein (FMRP) protein. FMRP is ubiquitously expressed and is especially abundant in the brain and testes. There are multiple isoforms of FMR1 but the precise function of the protein remains to be fully characterized. FXS is the most common form of inherited intellectual disability and the leading single gene cause of autism, affecting approximately 1 in 4000 males and 1 in 8000 females worldwide. FXS is considered a good candidate for gene therapy as it is a monogenic disorder with a severe manifestation and has no disease modifying treatment options. AIM: The aim of this project was to test novel AAV9/FMR1 gene therapy vectors both in-vitro and in-vivo, to establish efficacy and safety of these vectors and to assess if re-expression of FMRP can prevent the development of FXS-associated phenotypes in a mouse model of FXS. METHODS: Three novel single stranded AAV9/FMR1 (FXS4, FXS5 &amp; FXS6) gene therapy vectors (designed by the Cobb lab) underwent quality control assessments and were evaluated in two in-vitro models, Neuro-2a cells and HEK-293 cells prior to testing in a mouse model of FXS. The most promising vector (FXS6) was identified and used for larger therapeutic prevention trials. This promising novel cassette delivers a dominant human FMR1 isoform, isoform 7, and includes native elements of the human FMR1 promoter and 3’UTR sequence. Neonatal wild-type (WT) and Fmr1-/y mice were dosed with either the FMR1 gene therapy vector or excipient control via intracerebroventricular injections. At two months post injection, mice were evaluated using a battery of behavioural tests. In addition, mice were assessed for seizure threshold and severity using an auditory stimulus protocol. Following the in-life phase, mouse brains were assessed for vector biodistribution, transduction efficiency and vector derived FMRP protein levels. RESULTS: All three vectors passed the quality control tests and proceeded with virus production. In-vitro testing revealed transcript being produced during Neuro-2a cell transfections using all three constructs. However, protein analysis revealed limited vector-derived protein expression and low transduction efficiency with all but one construct, FXS4. Initial pilot in-vivo testing using FXS4 resulted in surprisingly low transduction efficiency. Nevertheless, following further pilot in-vivo testing of all three vectors, only one was identified as promising, FXS6, indicated by high cell transduction levels. The initial assessment trial of the lead ssAAV9/FMR1 vector (FXS6), achieved relatively moderate and variable transduction efficiency ranging between 20-60% of positive nuclei in the cortex, hippocampus, thalamus, and inferior colliculus expressing vector derived FMRP. Fmr1-/y mice showed a profound genotype effect when assessed for audiogenic seizures with high a penetrance of seizures (&gt;80% of subjects) in vehicle treated Fmr1-/y mice relative to WT littermates (10-25%). Cohorts treated with lead ssAAV9/FMR1 vector showed no improvement in seizure incidence or severity. No statistically significant improvement was observed in the marble burying test and open field test. An important caveat of this trial was the mouse model used, which had a mutation in a gene causing retinal degeneration and thus questioning the behavioral data obtained in this study. Additionally, the dose injected (5*1011 vg/mouse) and the resulting transduction efficiency didn’t correlate with the literature, putting into question the quality of the virus. To address these issues, a secondary therapeutic prevention trial was completed, using a sighted Fmr1-/y mouse model and new virus manufactured by a separate vendor. The second therapeutic prevention trial using FXS6, assessed mice dosed at 5 different doses of the vector ranging from 5*109 -3*1011 vg/mouse. The gene therapy vector was well tolerated in wild-type mice and did not produce any overt adverse neurological observations at any of the doses tested. Cohorts treated with the vector showed a strong and dose dependent reduction in the occurrence and severity of induced seizures with the higher doses rescuing the induced seizure burden to WT levels. Behavioral analysis revealed improvements in marble burying for Fmr1-/y mice treated with the two highest doses but no clear treatment effect in the open field or elevated plus maze tests. Ex-vivo analysis of brain samples from treated mice revealed a dose dependent increase in the brain transduction efficiencies (2-20% with the lowest dose and 68-92% with the highest dose, dependent on the brain region). Vector derived FMRP expression, validated by western blot, also varied depending on the brain regions, and was measure at between approximately 5% and 200% of the WT level with the lowest dose and the highest dose respectively. CONCLUSION: The work done throughout this project illuminates the potentials of gene therapy as a promising therapeutic avenue for complex monogenic disorders that have no effective treatment. The main findings of the project demonstrate the effectiveness and safety of an AAV9/FMR1 gene therapy vector utilizing native regulatory elements in ameliorating disease-related phenotypes in mice modeling FXS. Future studies aim to assess neurological and behavioral outcomes in the more robust rat model of the disorder as well as utilize new and emerging technologies in the field of gene therapy.