AAV9-Mediated Expression of SMN Restricted to Neurons Does Not Rescue the Spinal Muscular Atrophy Phenotype in Mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by mutations or deletions in the survival of motor neuron 1 (SMN1) gene and characterized by the degeneration of motor neurons and progressive muscle weakness. A viable therapeutic approach for SMA patients is a gene replacement strategy that restores functional SMN expression using adeno-associated virus serotype 9 (AAV9) vectors. Currently, systemic or intra-cerebrospinal fluid (CSF) delivery of AAV9-SMN is being explored in clinical trials. In this study, we show that the postnatal delivery of an AAV9 that expresses SMN under the control of the neuron-specific promoter synapsin selectively targets neurons without inducing re-expression in the peripheral organs of SMA mice. However, this approach is less efficient in restoring the survival and neuromuscular functions of SMA mice than the systemic or intra-CSF delivery of an AAV9 in which SMN is placed under the control of a ubiquitous promoter. This study suggests that further efforts are needed to understand the extent to which SMN is required in neurons and peripheral organs for a successful therapeutic effect.

Intravenous administration of scAAV9-Hexb normalizes lifespan and prevents pathology in Sandhoff disease mice.

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.

Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase.

Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

A New AAV10-U7-Mediated Gene Therapy Prolongs Survival and Restores Function in an ALS Mouse Model.

One of the most promising therapeutic approaches for familial amyotrophic lateral sclerosis linked to superoxide dismutase 1 (SOD1) is the suppression of toxic mutant SOD1 in the affected tissues. Here, we report an innovative molecular strategy for inducing substantial, widespread, and sustained reduction of mutant human SOD1 (hSOD1) levels throughout the body of SOD1G93A mice, leading to therapeutic effects in animals. Adeno-associated virus serotype rh10 vectors (AAV10) were used to mediate exon skipping of the hSOD1 pre-mRNA by expression of exon-2-targeted antisense sequences embedded in a modified U7 small-nuclear RNA (AAV10-U7-hSOD). Skipping of hSOD1 exon 2 led to the generation of a premature termination codon, inducing production of a deleted transcript that was subsequently degraded by the activation of nonsense-mediated decay. Combined intravenous and intracerebroventricular delivery of AAV10-U7-hSOD increased the survival of SOD1G93A mice injected either at birth or at 50 days of age (by 92% and 58%, respectively) and prevented weight loss and the decline of neuromuscular function. This study reports the effectiveness of an exon-skipping approach in SOD1-ALS mice, supporting the translation of this technology to the treatment of this as yet incurable disease.

Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice.

Systemic delivery of self-complementary (sc) adeno-associated-virus vector of serotype 9 (AAV9) was recently shown to provide robust and widespread gene transfer to the central nervous system (CNS), opening new avenues for practical, and non-invasive gene therapy of neurological diseases. More recently, AAV of serotype rh10 (AAVrh10) was also found highly efficient to mediate CNS transduction after intravenous administration in mice. However, only a few studies compared AAV9 and AAVrh10 efficiencies, particularly in the spinal cord. In this study, we compared the transduction capabilities of AAV9 and AAVrh10 in the brain, the spinal cord, and the peripheral nervous system (PNS) after intravenous delivery in neonatal mice. As reported in previous studies, AAVrh10 achieved either similar or higher transduction than AAV9 in all the examined brain regions. The superiority of AAVrh10 over AAV9 appeared statistically significant only in the medulla and the cerebellum, but a clear trend was also observed in other structures like the hippocampus or the cortex. In contrast to previous studies, we found that AAVrh10 was more efficient than AAV9 for transduction of the dorsal spinal cord and the lower motor neurons (MNs). However, differences between the two serotypes appeared mainly significant at low dose, and surprisingly, increasing the dose did not improve AAVrh10 distribution in the spinal cord, in contrary to AAV9. Similar dose-related differences between transduction efficiency of the two serotypes were also observed in the sciatic nerve. These findings suggest differences in the transduction mechanisms of these two serotypes, which both hold great promise for gene therapy of neurological diseases.

scAAV9 intracisternal delivery results in efficient gene transfer to the central nervous system of a feline model of motor neuron disease.

On the basis of previous studies suggesting that vascular endothelial growth factor (VEGF) could protect motor neurons from degeneration, adeno-associated virus vectors (serotypes 1 and 9) encoding VEGF (AAV.vegf) were administered in a limb-expression 1 (LIX1)-deficient cat-a large animal model of lower motor neuron disease-using three different delivery routes to the central nervous system. AAV.vegf vectors were injected into the motor cortex via intracerebral administration, into the cisterna magna, or intravenously in young adult cats. Intracerebral injections resulted in detectable transgene DNA and transcripts throughout the spinal cord, confirming anterograde transport of AAV via the corticospinal pathway. However, such strategy led to low levels of VEGF expression in the spinal cord. Similar AAV doses injected intravenously resulted also in poor spinal cord transduction. In contrast, intracisternal delivery of AAV exhibited long-term transduction and high levels of VEGF expression in the entire spinal cord, yet with no detectable therapeutic clinical benefit in LIX1-deficient animals. Altogether, we demonstrate (i) that intracisternal delivery is an effective AAV delivery route resulting in high transduction of the entire spinal cord, associated with little to no off-target gene expression, and (ii) that in a LIX1-deficient cat model, however, VEGF expressed at high levels in the spinal cord has no beneficial impact on the disease course.

A single intravenous AAV9 injection mediates bilateral gene transfer to the adult mouse retina.

Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.

Intramuscular scAAV9-SMN injection mediates widespread gene delivery to the spinal cord and decreases disease severity in SMA mice.

We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing "survival of motor neuron" (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.

A rare recessive distal hereditary motor neuropathy with HSJ1 chaperone mutation.

OBJECTIVE: Distal hereditary motor neuropathies (dHMN) form a clinically and genetically heterogeneous group of disorders, characterized by muscle weakness and atrophy predominating at the distal part of the limbs, due to the progressive degeneration of motor neurons in the spinal cord. We report here a novel rare variant of dHMN with autosomal recessive inheritance in a large Jewish family originating from Morocco. The disease is characterized by a predominance of paralysis at the lower limbs and an early adulthood onset. We performed a genetic study in this family to identify and characterized the causing mutation. METHODS: Homozygosity mapping strategy and sequencing of the candidate genes were performed. Expression studies were made on patient fibroblasts. Functional experiments were performed on a cellular model of motor neuron disease. RESULTS: We mapped the disease to the 2q34-q36.1 chromosomal region and identified a homozygous splice mutation in the gene HSJ1 (DNAJB2) decreasing the expression of the 2 main isoforms HSJ1a and HSJ1b. Overexpression of both HSJ1a and HSJ1b reduced inclusion formation induced by the mutated SOD1-A4V in a neuronal cellular model. INTERPRETATION: HSJ1 is a neuronal enriched member of the HSP40/DNAJ co-chaperone family. Previous studies have shown that HSP40 proteins play a crucial role in protein aggregation and neurodegeneration in several neuronal types, in animal models and human diseases. Interestingly, this mutation causing a loss-of-function of HSJ1 is linked to a pure lower motor neuron disease, strongly suggesting that HSJ1 also plays an important and specific role in motor neurons.

Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice.

Spinal muscular atrophy (SMA) is the most common genetic disease leading to infant mortality. This neuromuscular disorder is caused by the loss or mutation of the telomeric copy of the 'survival of motor neuron' (Smn) gene, termed SMN1. Loss of SMN1 leads to reduced SMN protein levels, inducing degeneration of motor neurons (MN) and progressive muscle weakness and atrophy. To date, SMA remains incurable due to the lack of a method to deliver therapeutically active molecules to the spinal cord. Gene therapy, consisting of reintroducing SMN1 in MNs, is an attractive approach for SMA. Here we used postnatal day 1 systemic injection of self-complementary adeno-associated virus (scAAV9) vectors carrying a codon-optimized SMN1 sequence and a chimeric intron placed downstream of the strong phosphoglycerate kinase (PGK) promoter (SMNopti) to overexpress the human SMN protein in a mouse model of severe SMA. Survival analysis showed that this treatment rescued 100% of the mice, increasing life expectancy from 27 to over 340 days (median survival of 199 days) in mice that normally survive about 13 days. The systemic scAAV9 therapy mediated complete correction of motor function, prevented MN death and rescued the weight loss phenotype close to normal. This study reports the most efficient rescue of SMA mice to date after a single intravenous injection of an optimized SMN-encoding scAAV9, highlighting the considerable potential of this method for the treatment of human SMA.