Gene Therapy in the Nervous System: Failures and Successes.

Genetic disorders, caused by deleterious changes in the DNA sequence away from the normal genomic sequence, affect millions of people worldwide. Gene therapy as a treatment option for patients is an attractive proposition due to its conceptual simplicity. In principle, gene therapy involves correcting the genetic disorder by either restoring a normal functioning copy of a gene or reducing the toxicity arising from a mutated gene. In this way specific genetic function can be restored without altering the expression of other genes and the proteins they encode. The reality however is much more complex, and as a result the vector systems used to deliver gene therapies have by necessity continued to evolve and improve over time with respect to safety profile, efficiency, and long-term expression. In this chapter we examine the current approaches to gene therapy, assess the different gene delivery systems utilized, and highlight the failures and successes of relevant clinical trials. We do not intend for this chapter to be a comprehensive and exhaustive assessment of all clinical trials that have been conducted in the CNS, but instead will focus on specific diseases that have seen successes and failures with different gene therapy vehicles to gauge how preclinical models have informed the design of clinical trials.

Progressive behavioral deficits in DJ-1-deficient mice are associated with normal nigrostriatal function.

Loss-of-function mutations in the DJ-1 gene account for an autosomal recessive form of Parkinson's disease (PD). To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1(-/-)) mice. Younger (<1 year) DJ-1(-/-) mice were hypoactive and had mild gait abnormalities. Older DJ-1(-/-), however, showed decreased body weight and grip strength and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1(-/-) mice, which was consistent with similar results between DJ-1(-/-) and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems.

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome.

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVß6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.

SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits.

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.

Disc1 variation leads to specific alterations in adult neurogenesis.

Disrupted in schizophrenia 1 (DISC1) is a risk factor for a spectrum of neuropsychiatric illnesses including schizophrenia, bipolar disorder, and major depressive disorder. Here we use two missense Disc1 mouse mutants, described previously with distinct behavioural phenotypes, to demonstrate that Disc1 variation exerts differing effects on the formation of newly generated neurons in the adult hippocampus. Disc1 mice carrying a homozygous Q31L mutation, and displaying depressive-like phenotypes, have fewer proliferating cells while Disc1 mice with a homozygous L100P mutation that induces schizophrenia-like phenotypes, show changes in the generation, placement and maturation of newly generated neurons in the hippocampal dentate gyrus. Our results demonstrate Disc1 allele specific effects in the adult hippocampus, and suggest that the divergence in behavioural phenotypes may in part stem from changes in specific cell populations in the brain.

DJ-1 is essential for long-term depression at hippocampal CA1 synapses.

Mutations in DJ-1 cause inherited Parkinson's disease (PD) in several families. The normal function of DJ-1 is unknown, but mice lacking DJ-1 exhibit a deficit in dopaminergic signaling in the striatum. Since the hippocampus contains relatively high levels of DJ-1, and PD patients are often cognitively impaired, we evaluated the effects of DJ-1 deficiency on the plasticity of hippocampal CA1 synapses. LTP was slightly impaired and LTD was abolished in DJ-1-/- mice, whereas DJ-1+/- mice exhibited no alterations in synaptic plasticity. The dopamine receptor D2/3 agonist quinpirole rescued LTD in DJ-1-/- mice, suggesting a role for impaired dopaminergic signaling in the hippocampal LTD deficit.