Assessing the potential for gene therapy in a mouse model of SYNGAP1 haploinsufficiency-associated disorder

Abstract

SYNGAP1 (Synaptic Ras GTPase activating protein 1) haploinsufficiency-associated disorder or intellectual developmental disorder, autosomal dominant 5 (MRD5) is caused by autosomal dominant loss-of-function mutations in the SYNGAP1 gene. De novo SYNGAP1 mutations represent one of the most common causes of non-syndromic intellectual disability (NSID) (OMIM #603384). The most common symptoms include moderate-to-severe intellectual disability (ID), epilepsy and autism spectrum phenotypes.

SYNGAP1 protein is a component of the postsynaptic density complex where it acts as one of the key effectors of N-methyl-D-aspartate (NMDA) receptor downstream pathways. It is involved in regulatory pathways such as the trafficking of the α-amino-3-hydroxy-5-mehyl-4-isoxazolepropionic acid (AMPA) receptors, regulation of AMPA receptor translation and the regulation of several forms of synaptic plasticity. With the aim to study SYNGAP1 protein function and its involvement in the pathophysiology of the disease, several rodent models have been created and extensively characterised. These studies revealed phenotypes which partially resemble the symptomatology observed in humans such as seizure susceptibility, hyperactivity and impulsivity. It has been shown that early restoration of normal SYNGAP1 protein levels prevents the onset of haploinsufficiency-associated phenotypes in the mouse. This, and the monogenic origin of the disorder, makes SYNGAP1 a candidate target for gene replacement therapeutic approaches.

With the aim to study SYNGAP1 protein function and its involvement in the pathophysiology of the disease, several rodent models have been created and extensively characterised. These studies revealed phenotypes which partially resemble the symptomatology observed in humans such as seizure susceptibility, hyperactivity and impulsivity. It has been shown that early restoration of normal SYNGAP1 protein levels prevents the onset of haploinsufficiency-associated phenotypes in the mouse. This, and the monogenic origin of the disorder, makes SYNGAP1 a candidate target for gene replacement therapeutic approaches.

I hypothesised that restoration of SYNGAP1 levels via Adeno Associated Virus (AAV)-mediated gene transfer during early postnatal development, will prevent the appearance or ameliorate the severity of behavioural phenotypes observed in the mouse model of SYNGAP1 haploinsufficiency. Therefore, the aim of this project was to evaluate molecular therapies to test this hypothesis.

Therefore, the aim of this project was to evaluate molecular therapies to test this hypothesis.

To this end, I first defined a robust battery of behavioural tests in order to confirm and identify deficits to use as endpoints to evaluate the efficacy of gene therapy products. Well-established and novel phenotypes were evaluated in wild-type and Syngap1+/- mice at different postnatal developmental time points.

For the first generation of candidate therapeutic constructs two different promoters were used, the minimal murine Mecp2 promoter (MeP) 229 and the synthetic JeT promoter. MeP229 regulated constructs were expressing three different Myc-tagged human SYNGAP1 isoforms, Aα2, Aα1 and Bα2 while the JeT regulated construct the Myc-tagged human SYNGAP1 isoform Aα1. In vitro evaluation of protein expression revealed low levels of plasmid-derived SYNGAP1 produced from the MeP229 regulated constructs while a higher amount of Myc-tagged SYNGAP1 protein was detectable from the JeT regulated cassette via both fluorescence immunocytochemistry and immunoblotting.

Protein expression from the vectorised ssAAV9/JeT-hSYNGAP1_Aα1-Myc was confirmed in the brain in vivo at 5 weeks post intracerebroventricular injection. A therapeutic dose escalation efficacy study assessing ssAAV9/JeT-hSYNGAP1_Aα1-Myc was then conducted in Syngap1⁺/⁻ mice (1E10, 5E10 and 1E11 vg/mouse). Overall, these data revealed no amelioration of hyperactivity or anxiety phenotypes, but a modest trend toward the amelioration of the risk-taking behaviour was observed in a platform departure test. Post-hoc immunoblot analysis confirmed a dose-dependent expression of the vector-derived SYNGAP1. However absolute levels of SYNGAP1 protein measured in different brain areas were below physiological levels of the native protein. Subcellular localisation analysis in whole hippocampal synaptosomal preparation showed that the vector-derived protein was correctly translocated to the synaptic compartment, mimicking endogenous patterns of protein localisation.

With the aim to improve the previous generation construct, a new therapeutic cassette was designed that expressed a codon-optimised FLAG-tagged version of the human SYNGAP1 Aα1 isoform, under the control of the human SYNAPSIN1 (hSYN1) promoter. In vitro expression of the hSYN1-FLAG-hSYNGAP1(opt)_Aα1 cassette was verified by immunostaining of transfected HEK293A cells and in vivo expression of ssAAV9/hSYN1-hSYNGAP1(opt)_Aα1 was verified in the brain at 5 weeks post intracerebroventricular injection. Behavioural analysis, at 7 and 15 weeks of mice treated with 5E10 and 1E11 vg/mouse, delivered via intracerebroventricular injections, recapitulated what was observed with the JeT-hSYNGAP1_Aα1 expressing vector. There was no treatment effect on hyperactivity or the anxiety phenotype, but an improvement in the platform departure test was observed. Moreover, I showed the utility of the Motion Sequence test, a novel machine learning system that allows analysing 3D modular elements of mouse behaviour in an ethologically conserved manner. The test identified a clear genotype effect between wild-type and Syngap1⁺/⁻ vehicle-treated mice however, there was no treatment effect observed in the vector-treated Syngap1⁺/⁻ group. Post-hoc analysis showed expression of the transgene in the hippocampus and cortex, however, vector-derived SYNGAP1 protein accounted only for a small portion of the total SYNGAP1. Whole hippocampus synaptosomal preparation showed correct localisation of the FLAG-tagged protein.

In conclusion, I identified a series of robust disease relevant genotype effects to utilise as outcome measures in preclinical gene therapy trials. I have also shown the many challenges to overcome to develop an effective gene replacement treatment of SYNGAP1 haploinsufficiency-associated disorder. The very limited therapeutic effect observed in these studies could be due to the low expression level of the vector-derived protein, the unequal distribution of AAV9 across the brain and the exclusion of other SYNGAP1 isoforms which could be necessary for a therapeutic effect. The identification of the most important isoform is mandatory for the further development of the next generation vectors.