2.1.3.1 EFECTOS DE LA DOPAMINA

Determining the natural progression of disease was essential for evaluating therapeutic success in the gene therapy study. As in the CLN5 gene therapeutic study, sheep in the treatment and control cohorts were assessed for neurological, behavioural, motor and visual function based on an ovine- specific clinical rating scale (Table 3.1, see Section 3.3). Clinical rating scores (CRS) decreased from 5 (normal function) to 0 (progressive disease with spontaneous tetanic seizures, defined as the humane end-point) as animals became more debilitated from the disease. Routine monthly physical and neurological examinations were performed, with concurrent open- and closed-field behavioural observation, beginning on average at 4.3 months of age.

CLN6 heterozygous sheep were clinically normal throughout the study period, consistently scoring 5 on the rating scale (Figure 7.3) and exhibited no functional deficits. In comparison, clinical signs of CLN6 disease in homozygous affected (CLN6-/-) sheep developed slowly and progressively from 7  0.9 months of age. They initially presented with low head carriage, which was especially apparent when they were passing through gates, races, a maze (see Section 7.3.2.4) and in shadows (CRS 4). From 9 months of age, individual sheep began to show visual deficits with an overt head tilt, thereby extending their visual field, and depressed visual reflexes, namely the menace (eye preservation), pupillary light and dazzle responses. By 10 to 12 months CLN6-/- sheep were smaller and had less condition than their heterozygous counterparts; this coincided with the onset of winter, when grazing was limited. Despite the difference in live weights at this timepoint, the subsequent growth rate of the CLN6-/- cohort mirrored that of the CLN6+/- cohort (Figure 7.2). From 12.6  1.0 months, all untreated CLN6 -/- sheep were functionally blind (CRS 3).

An overt neurological disease phenotype (CRS 2) was detected in the remainder of the affected South Hampshire sheep from 15.4  1.0 months of age, with reduced mentation and spontaneous episodes of head nodding and facial twitches, particularly of the eyelids, ears and lips. There was however significant variation in the disease onset and progression in the CLN6-/- cohort. Three untreated CLN6-/- sheep died naturally, but prematurely with advanced disease symptoms, during the study (Table 7.1). 1013/12 died at 12.6 months, with disease-related pathology observed post

mortem. Another animal (1028/13) developed stress-induced tremors at 12.7 months and died of

misadventure (falling into a ditch) at 13.4 months. A second sheep (1041/13) also developed severe inducible tremors, with a circling tendency resembling encephalitic listeriosis, and was euthanised at 13.4 months. Thus the sample size of the untreated CLN6-/- cohort decreased with age prior to trial completion.

138 After 17.2  0.8 months, CLN6-/- sheep exhibited progressively more obvious changes in sensation (blindness), mental state (behavioural changes), and posture or movement (motor deficits), termed the advanced disease state (CRS 1). Menace and dazzle responses were absent and pupillary reflexes were diminished. Affected sheep self-segregated from the flock, showed a reduced awareness of their surroundings and performed repetitive activities, such as inefficient or sham eating, aimless walking and compulsive circling in a confined space. Sheep showed a higher propensity to stumble, and when they stood still often adopted a wide stance with more manifest hindlimb paresis and localised tremors. Spontaneous seizure activity was not evident in the CLN6-/- cohort during this study, however this manifestation of the disease usually presents from 18 months of age and only occurs in a minority of the affected sheep (Palmer and Mitchell, personal observation). Despite affected South Hampshire sheep having lived up to 29 months with extensive nursing, all CLN6-/- sheep in this study were euthanised by 19.2 months for humane reasons with varying degrees of clinical disease (Table 7.1).

A.

Score Clinical phenotype Clinical status * Age (months)

5 Normal Normal <7.0  0.9

4 Pre-clinical Low head carriage

Propensity to crouch, baulk and stumble

7.0  0.9 3 Blind Head tilt/stargazing

Visual deficits

Decreased or lost menace (blink) response Reduced herding

12.6 ± 1.0

2 Overt/neurological Motor, cognitive and proprioceptive deficits Decreased startle to auditory stimuli

Wide stance

15.4 ± 1.0

1 Advanced Progressive disease

+/- induced tetanic seizures

18.1 ± 0.9 0 Terminal Spontaneous tetanic seizures Not observed * Onset of inducible mild tremors in individual sheep began as early as 12.7 months. Spontaneous convulsive seizures were not reported for the CLN6-/- _{sheep in this study but have been observed previously in the field in}

animals approaching or into their second year of life.

Figure 7.3 Clinical progression of CLN6 deficient sheep following viral-mediated CLN6 gene transfer Treated sheep were followed for 24 months after treatment ( LV-CLN6,AAV9-CLN6) and monitored against disease progression in untreated CLN6-/- sheep. A. Ages of symptom onset are shown for untreated CLN6-/- sheep (mean  SEM, n = 9). The scale is based on multi-focal neurological and motor defects, with initial behavioural deficits at 7  0.9 months of age. B. One animal (1027/13) responded robustly to the treatment showing clinical scores consistent with untreated CLN6+/- controls, whereas the remaining five treated animals responded less favourably, developing behavioural deficits from 12.7  0.6 months and loss of vision and other clinical symptoms from 14  0.8 months of age.

1 2 3 4 5 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Se ve ri ty o f dis e as e Cli n ical rating sco re Age (months) CLN6+/- CLN6-/- CLN6-/- (LV or AAV treated) 1027/13 (AAV treated) B.  

140

7.3.2.2 Clinical status in treated CLN6-/- sheep

At baseline (4.3 months), no neurological abnormalities were detected in the study animals with all cohorts scoring 5 (Figure 7.3). The treated cohort could be divided into two groups: one animal (1027/13) responded so favourably to AAV9-CLN6 treatment that it was indistinguishable clinically from the untreated CLN6+/- cohort over the duration of the study whereas the same level of therapeutic efficacy was not achieved by the other five treated animals. At the pre-determined study endpoint of 18 months of age, sheep 1027/13 showed no clinical evidence of ovine NCL disease symptoms and, with a CRS of 5, the trial was extended to gain an insight into the persistence of efficacy.

At 26.2 months of age (23.9 months post-treatment), 1027/13 was euthanised. The only clinicopathological indication of NCL detected in this animal was low level retinal damage, described below, which did not impact her visual functionality. Bilateral funduscopic examination at 18 months of age, conducted by an independent veterinary ophthalmologist, revealed a degree of tapetal hyperreflectivity and mild attenuation of the retinal vasculature in 1027/13 compared with untreated

CLN6+/- controls. Electroretinography (ERG) and funduscopy performed on 1027/13 just prior to

euthanasia corroborated this finding. ERG waveforms in both eyes of untreated CLN6-/- animals began to decrease from 12 - 15 months of age, becoming extinguished (flatline) in some individuals from 18 months (Katharina Russell, personal communication), confirming retinal dysfunction in the ovine model. However a- and b-waveforms were detected for 1027/13 at 26 months of age, although the amplitude of the a-wave was slightly reduced compared with fully sighted heterozygous controls (Figure 7.4). Only a single ERG episode was recorded for 1027/13, hence no longitudinal data are available. At this age, the menace response and pupillary light reflexes were still intact. Mild vascular attenuation was confirmed in the tapetal and non-tapetal fundus, but this was much less marked than in younger untreated CLN6-/- animals (Katharina Russell, personal communication).

Although the other five treated CLN6-/- animals did not show the same degree of functional correction, disease onset was delayed in them by a few months on average (Figure 7.3). These sheep were clinically normal until 12.7  0.6 months of age, when they began to baulk at obstacles, shadows and their cohorts (CRS4). The presentation of a head tilt in some animals and reduced menace reflexes indicated the onset of visual deficits, with functional blindness reported at 14  0.8 months (CRS 3). During this early-clinical stage, animals 1004/13 and 1002/13 were euthanised due to the cumulative effects of copper toxicity. A third sheep (1025/13) was also euthanised early, at 13.8 months, because of overt clinical signs of NCL, which included blindness, confusion and circling. Animals 1001/13 and 1026/13 saw the trial to completion, but were functionally blind from 15.6 and 12.6 months respectively, and developed an overt clinical phenotype prior to euthanasia.

Figure 7.4 Electroretinography (ERG) as an indicator of retinal damage

A. Scotopic electroretinogram from the left fundus of an 18.8 month old CLN6+/- sheep with normal