Effect of Hypocretin Restoration on Wakefulness in Narcoleptic Patients

Effect of Hypocretin Restoration on Wakefulness in Narcoleptic Patients

Gayathri Shastri

Monash University

Effect of Hypocretin Restoration on Wakefulness in Narcoleptic Patients

        Narcolepsy is a neurological disorder that is caused by a selective loss of hypothalamic neurons, which produce hypocretin (i.e. also known as orexin) neuropeptides (Naumann & Daum, 2003; Peacock & Benca, 2010; Nishino, 2007; Scammell, 2003). The diagnosis of narcolepsy is established by four underlying symptoms consisting of excessive daytime sleep, cataplexy, sleep paralysis and hypnagogic hallucinations. The onset of narcolepsy begins at adolescence and patients usually require lifelong treatment to manage and control narcolepsy.

        Past research have discovered that narcolepsy is caused by selective loss of hypocretin producing neurons in the hypothalamus, leading to great progression in understanding the neuropathology of narcolepsy (Peyron et al. 1998; Thannickal et al. 2000; Crocker et al. 2005). Hypocretin peptides, known as Hcrt-1 and Hcrt-2 regulate REM sleep and promote wakefulness by increasing the activity of neurons during the active period (Bourgin et al. 2000; Adamantidis, Zhang, Aravanis, Deisseroth & de Lecea, 2007; Mochizuki et al. 2011). Several studies have used narcoleptic animal models (murine and canine) to show a lack of hypocretin signalling, resulting in decreased arousal levels during active period with much shorter spans of wakefulness, cataplexy and reduced locomotor activity (Chemelli et al. 1999; Hara et al. 2001; Espana, McCormack, Mochizuki & Scammell, 2007). This loss in orexins may possibly account for unstable brain regions that are responsible for regulating wake-promoting activities and REM-sleep, leading to frequent sleep attacks and intrusive cataplexic episodes.

        Previous findings have shed light on the cause of narcolepsy, but they have not been translated into clinical treatments. For example, chronic daytime sleep is the obvious underlying symptom in narcoleptic patients, however, even with optimal diagnostic tests and treatment, only a minority group of patients demonstrate normal alertness level (Mitler & Hayduk, 2002). Improvements on clinical aspects of narcolepsy are suboptimal because medications address only the symptoms, but not the fundamental hypocretin deficiency. The ideal treatment for narcolepsy would be to restore hypocretin levels in brain regions promoting wakefulness during active periods using gene therapy.

        The proposed study focuses on using gene therapy to restore hypocretin levels in the brain to promote longer wake periods by coaxing other brain cells to produce hypocretins to constitute and replenish the loss of normal hypocretin-producing neurons. Gene therapy has been used in various animal models and has earned its name for aiding in finding a cure for Parkinson’s disease (Coune, Schneider & Aebischer, 2012; LeWitt et al. 2011; Bartus, Weinberg & Samulski, 2014). Using a mouse model gene therapy study as reference for creating a viral vector (Kantor et al. 2013), narcoleptic patients with hypocretin deficiency will be microinjected with an adeno-associated viral vector coding for hypocretin (AAV-hypocretin).  The main purpose behind the proposed study is to investigate if restoration of hypocretin neurons using a viral vector (AAV-hypocretin) in narcoleptic patients can promote longer duration in wake period. The study hypothesises that excessive sleep in narcoleptic patients would significantly improve with hypocretin gene therapy.

Method

Participants

        Twenty participants (10 narcoleptic participants and 10 healthy participants). Narcoleptic participants will be recruited from sleep clinics based on the inclusion diagnostic criteria. . Inclusion criteria consisted of clinical record of excessive sleep, mean latency of sleep less than five minutes and two or more sleep-onset REM periods on Multiple sleep latency test, history of vivid dreams or hallucinations and/or cataplexy (not too severe to require treatment during experiment or testing), other sleep disorders ruled out with polysomnography. Once a suitable narcoleptic participant is identified, a suitable control participant will be recruited by word of mouth or advertisement. Each sample group of 10 will consists of 5 control and 5 treatment participants according to their matched pairs. All participants will be provided with informed consent and can withdraw at any time. Participants will be paid an honorarium for their time.

Vector

        The vector and the construction of vector will be based and modified as per Kantor et al. (2013) study. A recombinant adeno-associated virus (AAV) will be used due to their high transduction efficiency in neurons, which yield long-lasting gene expression. AAV vectors are in popular demand in genetic experiments in humans and animal models due to their desirable properties (High, Aubourg, 2011; Griffey, Macauley, Ogilvie & Sands, 2005). The experimental AAV will contain genes coding for prepro-hypocretin and GFP (a green fluorescence protein), which will be linked by an internal ribosome entry site. The control vector will only contain the GFP sequence. The vectors will be created using a tripartite transfection which will consist of the AAV vector plasmid, AAV-rep/cap expression plasmid and adenovirus mini-plasmid (Lu, Qu, Yang, Xu & Xiao, 2011; Xiao, Li & Samulski, 1998)

Design

        The proposed study is a randomised matched pair design. A randomised design ensures that the study treatment causes the expected result on human health where each participant will be randomly assigned to receive either the AAV-hypocretin vector or AAV-GFP control vector. The independent variable is level of hypocretin and the dependant variable is period of wakefulness.         

Materials

        Vectors, Electroencephalogram (EEG) and Electromyogram (EMG) electrodes, Polysomnography and MSLT.  

Procedure

Polysomnographic recordings and multiple sleep latency test will be required of all participants to ensure no other sleep disorders are present in narcoleptic and control groups prior to being microinjected with the respective vectors, AAV-hypocretin or AAV-GFP. Participants will be anaesthetised and microinjected with the respective vectors into the dorsomedial nucleus of the hypothalamus. This region was chosen targeted because past research have proven that when driven by a nonspecific promoter gene, various neurons in the ventral hypothalamus produce Hcrt-1 (Mieda, Willie, Hara, Sinton, Sukurai & Yanagisawa, 2004). As described in Kantor et al. (2013) study, participants will be implanted with EEG electrodes which are stainless steel screw electrodes inserted epidurally over the frontal and parietal cortex. EMG signals use a pair of stainless steel wire for acquired signal and inserted into the neck extensor muscles.