Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease.

Mutations in the CLN5 gene cause the fatal, pediatric, neurodegenerative disease CLN5 neuronal ceroid lipofuscinosis. Affected children suffer progressive neuronal loss, visual failure and premature death. Presently there is no treatment. This study evaluated dual intracerebroventricular (ICV) and intravitreal (IVT) administration of a self-complementary adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) into CLN5 affected sheep (CLN5-/-) at various disease stages. CLN5 disease progression was slowed in pre-symptomatic sheep who received a moderate dose of scAAV9/oCLN5, whilst a higher ICV dose treatment in early and advanced symptomatic animals delayed or halted disease progression. Intracranial (brain) volume loss was attenuated in all treatment cohorts, and visual function was also sustained in both the early and advanced symptomatic treated sheep over the 24-month duration of the study. Robust CLN5 protein expression was detected throughout the brain and spinal cord, and improvements in central nervous system and retinal disease correlates were observed. These findings hold translational promise for extending and improving the quality of life in both pre-symptomatic and symptomatic CLN5 patients, and prompted the initiation of the first in-human Phase I/II clinical trial testing ICV/IVT administration of scAAV9 encoding human CLN5 (https://clinicaltrials.gov/; NCT05228145).

Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease.

CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is characterised by progressive neuronal loss, blindness, and premature death. There is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected (CLN5-/-) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three disease stages. The treatment delayed disease progression, extended survival and slowed stereotypical brain atrophy in most animals. Of note, one high dose treated animal only developed mild disease symptomology and survived to 60.1 months of age, triple the natural life expectancy of an untreated CLN5-/- sheep. Eyesight was not preserved at any administration age or dosage. Histopathologic examination revealed that greater transduction efficiency was achieved through higher ICV doses, and this resulted in greater amelioration of disease pathology. Together with other pre-clinical data from CLN5-/- sheep, the safety and efficacy data from these investigational new drug (IND)-enabling studies supported the initiation of the first in-human CLN5 gene therapy clinical study using the ICV delivery route for the treatment of CLN5 NCL. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05228145.

Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.

Progressive MRI brain volume changes in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited lysosomal storage disorders characterized by progressive neurodegeneration leading to motor and cognitive dysfunction, seizure activity and blindness. The disease can be caused by mutations in 1 of 13 ceroid lipofuscinosis neuronal (CLN) genes. Naturally occurring sheep models of the CLN5 and CLN6 neuronal ceroid lipofuscinoses recapitulate the clinical disease progression and post-mortem pathology of the human disease. We used longitudinal MRI to assess global and regional brain volume changes in CLN5 and CLN6 affected sheep compared to age-matched controls over 18 months. In both models, grey matter volume progressively decreased over time, while cerebrospinal fluid volume increased in affected sheep compared with controls. Total grey matter volume showed a strong positive correlation with clinical scores, while cerebrospinal fluid volume was negatively correlated with clinical scores. Cortical regions in affected animals showed significant atrophy at baseline (5 months of age) and progressively declined over the disease course. Subcortical regions were relatively spared with the exception of the caudate nucleus in CLN5 affected animals that degenerated rapidly at end-stage disease. Our results, which indicate selective vulnerability and provide a timeline of degeneration of specific brain regions in two sheep models of neuronal ceroid lipofuscinoses, will provide a clinically relevant benchmark for assessing therapeutic efficacy in subsequent trials of gene therapy for CLN5 and CLN6 disease.

Intravitreal Injections in the Ovine Eye.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

The Translational Benefits of Sheep as Large Animal Models of Human Neurological Disorders.

The past two decades have seen a considerable rise in the use of sheep to model human neurological disorders. While each animal model has its merits, sheep have many advantages over small animal models when it comes to studies on the brain. In particular, sheep have brains more comparable in size and structure to the human brain. They also have much longer life spans and are docile animals, making them useful for a wide range of in vivo studies. Sheep are amenable to regular blood and cerebrospinal fluid sampling which aids in biomarker discovery and monitoring of treatment efficacy. Several neurological diseases have been found to occur naturally in sheep, however sheep can also be genetically engineered or experimentally manipulated to recapitulate disease or injury. Many of these types of sheep models are currently being used for pre-clinical therapeutic trials, particularly gene therapy, with studies from several models culminating in potential treatments moving into clinical trials. This review will provide an overview of the benefits of using sheep to model neurological conditions, and highlight naturally occurring and experimentally induced sheep models that have demonstrated translational validity.

Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5-/- or CLN6-/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5-/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6-/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5-/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5-/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.

Chemical neuroanatomy of the substantia nigra in the ovine brain.

The substantia nigra is an integral component of the basal ganglia circuitry for limbic and motor functions. Dysfunction and degeneration of the basal ganglia are fundamental aspects of neurodegenerative diseases such as Parkinson's disease and Huntington's disease. With the increasing use of sheep to model neurological diseases, it is crucial to understand the anatomy and neurochemistry of these key basal ganglia nuclei in the normal sheep brain and how they compare to the human brain. Therefore, studies of the gross anatomy, cellular morphology, and neurochemical expression patterns within the sheep substantia nigra were performed. We show that the sheep substantia nigra reflects all important aspects of the anatomy and neurochemistry of the human substantia nigra, with only minor inter-species differences evident. Many neurochemicals that are central to the functioning of the SN, and wider basal ganglia circuitry, are present throughout the sheep SN. In a wider context, the results of this study provide evidence that the sheep substantia nigra accurately reflects the anatomy of the human substantia nigra, which validates the use of sheep models of basal ganglia neurological disorders.