Mehdi Gasmi

Delivery of neurturin by AAV2 (CERE‐120)‐mediated gene transfer provides structural and functional neuroprotection and neurorestoration in MPTP‐treated monkeys

We tested the hypothesis that gene delivery of the trophic factor neurturin could preserve motor function and protect nigrostriatal circuitry in hemiparkinsonian monkeys.

AAV2-mediated delivery of human neurturin to the rat nigrostriatal system: Long-term efficacy and tolerability of CERE-120 for Parkinson's disease

Neurturin (NTN) is a neurotrophic factor with known potential to protect and restore the function of dopaminergic substantia nigra neurons whose degeneration has been most closely linked to the major motor deficits in Parkinsons disease (PD). CERE-120, an adeno-associated virus serotype 2 (AAV2)-based gene delivery vector encoding human NTN, is being developed as a potential therapeutic for PD. In a series of preclinical studies reported herein, CERE-120 delivery to the striatum produced a dose-related neuroprotection of nigrostriatal neurons in the rat 6-hydroxydopamine (6-OHDA) lesion model. Long-lasting efficacy of CERE-120 was evidenced by substantia nigra cell protection, preserved fiber innervation of the striatum, and behavioral recovery for at least 6 months. In addition, striatal infusion of CERE-120 was found to have a safety and tolerability profile devoid of side effects or toxicological responses, for at least 12 months post-treatment, even at dose multiples 125 times that of the lowest efficacious dose tested. These results support the ongoing CERE-120 clinical program in PD patients.

Striatal delivery of CERE‐120, an AAV2 vector encoding human neurturin, enhances activity of the dopaminergic nigrostriatal system in aged monkeys

Neurturin (NTN) is a potent survival factor for midbrain dopaminergic neurons. CERE‐120, an adeno‐associated virus type 2 (AAV2) vector encoding human NTN (AAV2‐NTN), is currently being developed as a potential therapy for Parkinsons disease. This study examined the bioactivity and safety/tolerability of AAV2‐NTN in the aged monkey model of nigrostriatal dopamine insufficiency. Aged rhesus monkeys received unilateral injections of AAV2‐NTN into the caudate and putamen, with each animal therefore serving as its own control. Robust expression of NTN within the nigrostriatal system was observed 8 months postadministration. 18F‐fluorodopa imaging using positron emission tomography revealed statistically significant increases in 18F‐fluorodopa uptake in the injected striatum compared with the uninjected side at 4 and 8 months. In addition, at 8 months postadministration, a significant enhancement in tyrosine hydroxylase immunoreactive fibers and an increase in the number of tyrosine hydroxylase immunoreactive cells was observed in the AAV2‐NTN injected striatum compared with the uninjected side. Robust activation of phosphorylated extracellular signal‐regulated kinase immunoreactivity in the substantia nigra was also observed. Histopathological analyses revealed no adverse effects of AAV2‐NTN in the brain. Collectively, these results are consistent with the neurotrophic effects of NTN on the dopaminergic nigrostriatal system and extend the growing body of evidence supporting the concept that AAV2‐NTN may have therapeutic benefit for Parkinsons disease.

Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS

The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1 and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1(G93A) mice. We observed robust and long-term intraspinal IGF-1 expression and partial rescue of lumbar spinal cord motor neurons, as well as sex-specific delayed disease onset, weight loss, decline in hindlimb grip strength and increased animal survival.

Insulin Growth Factor-1 Gene Transfer Enhances Neurovascular Remodeling and Improves Long-Term Stroke Outcome in Mice

Background and Purpose— Insulin-like growth factor I (IGF-1) is a pleiotropic growth factor that has been demonstrated to protect against acute ischemic brain injury. Whether IGF-1 improves long-term functional outcome after ischemic stroke is not known. The aim of this study is to examine whether IGF-1 overexpression through adeno-associated virus (AAV) -mediated gene transfer enhances neurovascular remodeling and improves functional outcome in a mouse model of focal cerebral ischemia. Methods— Long-term cerebral IGF-1 overexpression was achieved with the AAV transduction system through stereotaxic injection. Control mice were injected with AAV–green fluorescent protein or saline. Three weeks after gene transfer, the mice underwent permanent distal middle cerebral artery occlusion. Histological and behavioral analyses were performed at day 21 after middle cerebral artery occlusion. Results— IGF-1 gene transfer compared with control treatment significantly improved motor performance assessed by sensorimotor tests. The functional recovery was accompanied by reduced volume of cerebral infarction. Immunohistochemical analysis with endothelial cell marker CD31 revealed that IGF-1 gene transfer potently increased neovessel formation in the periinfarct and injection needle tract area compared with AAV–green fluorescent protein transduction. Increased vascular density was associated with increased local vascular perfusion. Additionally, AAV-IGF-1 treatment enhanced neurogenesis in the subventricular zone compared with AAV–green fluorescent protein treatment. Conclusions— These data demonstrate that IGF-1 overexpression promoted long-lasting functional recovery after cerebral infarction. The improved functional performance was paralleled by enhanced neovascularization and neurogenesis.

Expression, bioactivity, and safety 1 year after adeno-associated viral vector type 2-mediated delivery of neurturin to the monkey nigrostriatal system support cere-120 for Parkinson's disease.

OBJECTIVEParkinsons disease is characterized by profound motor deficits that result mainly as a consequence of degeneration of midbrain dopaminergic neurons. No current therapy slows or halts disease progression. Neurturin (NTN) and glial cell line–derived neurotrophic factor have potent neuroprotective and neurorestorative effects on dopaminergic neurons, but their use in treating Parkinsons disease has been limited by significant delivery obstacles. In this study, we examined the long-term expression, bioactivity, and safety/tolerability of CERE-120, an adeno-associated virus type 2 vector encoding human NTN, after bilateral stereotactic delivery to the striatum of nonhuman primates. METHODSTwelve naïve rhesus macaques received bilateral stereotactic injections of 1 of 2 CERE-120 doses or vehicle to the caudate and putamen. Neurological and clinical parameters were monitored for up to 1 year postadministration, after which animals were sacrificed for histological analyses. RESULTSDose-related NTN expression was observed at 1 year and was associated with enhanced tyrosine hydroxylase immunolabeling in the striatum, hypertrophy of tyrosine hydroxylase–positive cells in the substantia nigra, and induction of extracellular signal–regulated kinase signaling in the substantia nigra. Extensive, formal analyses, conducted in accordance with Good Laboratory Practice Regulations, across multiple time points revealed no evidence of clinical, neurological, or systemic toxicity. CONCLUSIONThe present study provides evidence of long-term expression and bioactivity of NTN on the dopaminergic nigrostriatal system after bilateral stereotactic delivery of CERE-120 to the striatum. Furthermore, no evidence of any adverse effects for up to 1 year postadministration was observed. These findings reveal a wide safety margin for CERE-120 and collectively support the ongoing clinical testing of the efficacy and safety of CERE-120 in patients with Parkinsons disease.

Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Intraspinal cord delivery of IGF-I mediated by adeno-associated virus 2 is neuroprotective in a rat model of familial ALS.

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1(G93A) rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1(G93A) rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.

Transgene Expression, Bioactivity, and Safety of CERE-120 (AAV2-Neurturin) Following Delivery to the Monkey Striatum

Neurturin (NTN) is a neurotrophic factor for dopaminergic neurons that may be therapeutic for patients with Parkinsons disease (PD). As a crucial component in a series of nonclinical translational studies aimed at testing whether CERE-120 should advance into clinical trials in PD subjects, we characterized the expression, bioactivity and safety of CERE-120, an adeno-associated virus type-2 (AAV2) vector encoding NTN, following delivery to the striatum of nonhuman primates. Monkeys received bilateral injections of CERE-120 across a tenfold range of doses (6 x 10(10) to 6 x 10(11) vector genomes per animal) or formulation buffer (FB) control. We report here, for the first time, a dose-related: increase in NTN protein expression within the striatum and substantia nigra (SN) pars compacta of nonhuman primates; increase in nigrostriatal tyrosine hydroxylase (TH), (the rate-limited enzyme for dopamine); and activation of phosphorylated signal-regulated kinase (a common neurotrophic signaling event). Additionally, extensive toxicology testing revealed no adverse effects of CERE-120 on in-life measures, neurotoxicity (in any site throughout the brain) or systemic pathology (in any organ or tissue) across the tenfold range of doses. Collectively, these data provide substantial novel evidence for the potential utility of CERE-120 as a novel treatment for PD and support ongoing clinical trials testing CERE-120 in PD patients.

Long-Term Reversal of Cholinergic Neuronal Decline in Aged Non- Human Primates by Lentiviral NGF Gene Delivery

Spontaneous atrophy of basal forebrain cholinergic neurons occurs with aging in the non-human primate brain. Short-term reversal of this atrophy has been reported following ex vivo nerve growth factor (NGF) gene delivery, but long-term effects of in vivo NGF gene delivery in the aged primate brain have not to date been examined. We tested the hypothesis that long-term lentiviral NGF intraparenchymal gene delivery would reverse age-related cholinergic decline, without induction of adverse effects previously observed following sustained intracerebroventricular growth factor protein exposure. Three aged rhesus monkeys underwent intraparenchymal lentiviral NGF gene delivery to the cholinergic basal forebrain. 1 year later, cholinergic neuronal numbers were quantified stereologically and compared to findings in four controls, non-treated aged monkeys and four young adult monkeys. Safety was assessed on several variables related to growth factor exposure. We now report that lentiviral gene delivery of NGF to the aged primate basal forebrain sustains gene expression for at least 1 year, and significantly restores cholinergic neuronal markers to levels of young monkeys. Aging resulted in a significant 17% reduction (p<0.05) in the number of neurons labeled for the cholinergic marker p75 among basal forebrain neurons. Lentiviral NGF gene delivery induced significant (p<0.05) and nearly complete recovery of p75-labeled neuronal numbers in aged subjects to levels observed in young monkeys. Similarly, the size of cholinergic neurons in aged monkeys was significantly reduced by 16% compared to young subjects (p<0.05), and lentiviral NGF delivery to aged subjects induced complete recovery of neuronal size. Intraparenchymal NGF gene delivery over a one-year period did not result in systemic leakage of NGF, activation of inflammatory markers in the brain, pain, weight loss, Schwann cell migration, or formation of anti-NGF antibodies. These findings indicate that extended trophic support to neurons in the non-human primate brain reverses age-related neuronal atrophy. These findings also support the safety and feasibility of lentiviral NGF gene transfer for potential testing in human clinical trials to protect degenerating cholinergic neurons in Alzheimers disease.