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.

Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

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.

Ecotoxicological assessment of acid mine drainage: electrophysiological changes in earthworm (Aporrectodea caliginosa) and aquatic oligochaete (Lumbriculus variegatus).

Ecological health of 15 sites in two mining areas on the West Coast of the South Island, New Zealand, was assessed using a non-invasive electrophysiological technique. The conduction velocity (CV) changes in the medial giant fibres (MGF) of the terrestrial earthworm Aporrectodea caliginosa were measured on days 0, 1, 2, 4, 7, 14, and 21 following exposure to soil and/or sediment from six acid mine drainage (AMD) sites, and aquatic oligochaete Lumbriculus variegatus at 0, 3, 6, and 24 h following exposure to water from 14 AMD sites. The colour of the soil/sediments varied from red-brown to black with pH ranging from 4.46 to 7.37. The colour of AMD water samples varied from clear, black, brown to orange, and the pH ranged from 2.99 to 7.66. The CV decreased progressively in A. caliginosa exposed to most soil and sediment samples from the AMD sites (compared with controls exposed to soil from an organic farm) and this was most evident in measurements taken at 7 days. Based on the CV measurements taken on day 7, sites 3 > 2 > 1 were significantly (P < 0.05) the most toxic to earthworms. The CV of L. variegatus exposed to AMD water sampled from many sites also decreased progressively and this was significantly lower than the controls in the measurements taken at 24 h from sites 3 > 9 > 7 > 11. It is proposed that MGF CV in A. caliginosa and L. variegatus worms can be used as a non-invasive, sensitive, biomarker to monitor the toxicity of AMD sites.

Electroretinography data from ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.

This article presents datasets associated with the research article entitled "Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease" (Murray et al., [1]). The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of fatal inherited diseases caused by mutations in a number of CLN genes that lead to degenerative and fatal encephalopathies in children. Naturally-occuring sheep models of NCL exist. Affected sheep share the clinical and pathological features of the human disease, including retinal degeneration. Electroretinography (ERG) was employed to characterise the physiological changes in the degenerating retina of CLN5 and CLN6 forms of ovine NCL. ERGs were performed every two months from 3 to 17 months of age in 11 NCL affected (6 CLN5-/ - and 5 CLN6-/- ) sheep and 12 clinically normal heterozygous controls (6 CLN5+/ - and 6 CLN6 +/-) under three different adaptation conditions. A-wave and b-wave amplitudes were collected from each eye using the Eickemeyer Veterinary ERG system. These are the first longitudinal datasets assessing the progression of retinal degeneration in ovine NCL, aiding in characterisation of the disease process and providing insight into optimal therapeutic windows for subsequent studies.

Nerve conduction velocity as a non-destructive biomarker in the earthworm Aporrectodea caliginosa exposed to insecticides.

Earthworms are important and useful soil organisms, but in agricultural soils, they are potentially exposed to a wide variety of pesticides. Insecticides represent the highest threat to earthworms and many are neurotoxic. There is a need for a reliable, relevant, simple biomarker to assess the sub-lethal effects of neurotoxic insecticides on earthworms under laboratory or field conditions. The Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.5×, 1× (normal field application rate), and 5× concentrations of a carbamate (Pirimor®) and an organophosphate (Lorsban®) insecticides. The nerve conduction velocity (NCV) of the medial giant fibers of A. caliginosa earthworm was recorded on days 0, 1, 2, 3, 4, and 7 to quantify sub-lethal neurotoxic effects. Acetylcholinesterase (AChE) enzyme activity of A. caliginosa homogenates was measured at the conclusion of the experiment. Pirimor® but not Lorsban® induced a significant decrease in NCV on days 3, 4, and 7 at 1× and 5× doses. A significant dose-dependent decrease was observed on AChE activity to Pirimor® at the doses used but not Lorsban®. A clear relationship is observed between AChE activity and NCV in the case of Pirimor®. This study showed that NCV is a sensitive biomarker that correlates well with classical biomarker measurements such as AChE enzyme activity. This technique could be used to study the impact of insecticides on earthworms and also their recovery.

Computed tomography provides enhanced techniques for longitudinal monitoring of progressive intracranial volume loss associated with regional neurodegeneration in ovine neuronal ceroid lipofuscinoses.

INTRODUCTION: The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well-established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral-mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans. METHODS: Computed tomography (CT)-based three-dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL. RESULTS: ICVs of NCL-affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5-/- sheep and 11-15 months in CLN6-/- sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (-12; 23). CONCLUSIONS: Computed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.

Differential response of C-type natriuretic peptide to estrogen and dexamethasone in adult bone.

C-type natriuretic peptide (CNP) is crucial in promoting endochondral bone growth in mammals including humans but whether this paracrine hormone participates in maintaining bone integrity in the mature skeleton is unknown. Accordingly we studied changes in plasma and bone tissue CNP in anoestrus adult ewes receiving short term anabolic (estrogen) or catabolic (dexamethasone) treatment for 7days. CNP and the aminoterminal fragment of the CNP prohormone (NTproCNP) were measured in plasma and extracts of cancellous bone excised from vertebral, iliac, tibial and marrow tissues. Concentrations of CNP peptides were much higher in vertebral and iliac extracts than those of tibial or marrow. Both plasma CNP and NTproCNP increased rapidly after estrogen followed by a later rise in bone alkaline phosphatase. Vertebral and iliac (but not tibial or marrow) CNP peptide content were significantly increased by estrogen. Consistent with a skeletal source, plasma NTproCNP was significantly associated with vertebral tissue CNP. In contrast, bone tissue CNP peptide content was unaffected by dexamethasone despite suppression of plasma CNP peptides and bone alkaline phosphatase. We postulate that increases in trabecular bone CNP reflect new endosteal bone formation in these estrogen responsive tissues whereas reduced plasma CNP peptides after dexamethasone, without change in cancellous bone content, reflects reductions in cortical bone turnover.

Effect of nutrition on plasma C-type natriuretic peptide forms in adult sheep: evidence for enhanced C-type natriuretic peptide degradation during caloric restriction.

Previous studies in lambs and children show that the plasma concentration of amino terminal pro-C-type natriuretic peptide (NTproCNP), a stable product of proCNP, is strongly correlated with skeletal growth and markers of bone formation. Consistent with these findings, CNP expression is sensitive to nutritional status and is reduced by caloric restriction (CR) in both the fetus and the postnatal lamb. However, the effect of nutritional status on CNP in the adult, once linear growth is complete, is unknown. Hypothesizing that reduced CNP synthesis during CR is contingent on the presence of active growth plates, we studied the effect of CR ( 25% of maintenance) or loading (CL, 200% of maintenance) on CNP forms and alkaline phosphatase (ALP) in adult ewes and compared the findings to responses in a control group (C) fed a maintenance diet of 10.6 MJ of metabolizable energy. Live body weight was reduced (17%) in the CR group and increased (10%) in the CL group after 16 days of intervention. Plasma CNP concentration and ALP both fell in CR sheep and were significantly lower than C (P < .05 for both), returning toward basal levels 1 week after refeeding. In contrast, plasma NTproCNP did not differ (CR vs C). There were no significant changes in CNP forms and ALP in CL sheep compared with C. Fall in plasma CNP but not in NTproCNP in CR adult sheep suggests that CNP degradation (not synthesis) is altered, and contrasts with previous findings in growing lambs where CR reduces both CNP forms.

C-type natriuretic peptide forms in pregnancy: maternal plasma profiles during ovine gestation correlate with placental and fetal maturation.

Circulating concentrations of C-type natriuretic peptide (CNP) and a related amino terminal fragment (NTproCNP) were measured at weekly intervals from preconception to 3 wk postpartum in ewes with twins (n = 8) and nonpregnant ewes (n = 8). In contrast to low and stable values in nonpregnant ewes (CNP, 0.75 +/- 0.08; NTproCNP, 22 +/- 2 pmol/liter), CNP forms increased abruptly at 40-50 d of gestation and rose to peak values (CNP, 31 +/- 5, NTproCNP, 270 +/- 16 pmol/liter) at about d 120. Approximately 7 d prepartum, the concentration of both CNP forms fell precipitously to preconception values immediately postpartum. In separate studies, circulating maternal CNP forms were positively related to fetal number at d 120. Consistent with a major contribution from the placenta to circulating levels, the concentrations of CNP forms were elevated in the placentome (cotyledon: CNP, 18 +/- 4, NTproCNP, 52 +/- 10 pmol/g; caruncle: CNP, 13 +/- 3, NTproCNP, 31 +/- 6 pmol/g) and much higher than those of intercaruncular uterine tissue (CNP, 0.19 +/- 0.05, NTproCNP, 0.98 +/- 0.2 pmol/g) in late-gestation ewes (P < 0.001, n = 4). These distinctive patterns of maternal plasma CNP forms, positive relation with fetal number, and greatly elevated protein concentrations in the placentome demonstrate the hormone's strong relation to placental and fetal maturation. The findings provide a firm basis for future studies of the functional role of CNP in fetal-maternal welfare.

Effect of sex steroids on plasma C-type natriuretic peptide forms: stimulation by oestradiol in lambs and adult sheep.

Although C-type natriuretic peptide (CNP) is crucial to post-natal endochondral growth, roles for the hormone in pubertal bone growth and the physiological effects of sex steroid substitution on CNP synthesis are not known. Using a plasma marker of CNP tissue synthesis (amino-terminal proCNP, NTproCNP), we have studied the effect of exogenous oestrogen (E(2)) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E(2) in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E(2) promptly increased plasma NTproCNP and CNP (P<0.001) to peak on day 2, and bALP (P<0.001 peaking on day 7), whereas no significant stimulation in response to T was observed in male lambs. Linear bone growth and live weight were unaffected. In adult anoestrous ewes, basal concentrations of CNP forms and bALP were lower than in ewe lambs, in keeping with skeletal maturity, but adults responded similarly to E(2). Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P<0.001), were induced by E(2). Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E(2)-induced CNP expression) were excluded by showing similar E(2)-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E(2) stimulates plasma CNP forms and bALP in lambs and adult sheep and raise the possibility that CNP also participates in bone formation in the mature skeleton.

Response of plasma CNP forms to acute anabolic and catabolic interventions in growing lambs.

Using a novel marker of C-type natriuretic peptide (CNP) synthesis [amino-terminal pro-CNP (NT-proCNP)], we have recently shown that plasma NT-proCNP is strongly correlated with skeletal growth and markers of bone formation and is reversibly reduced by glucocorticoids. The effects on CNP of other catabolic or anabolic factors, known to affect skeletal growth, are unknown. Accordingly, we have studied the response of plasma CNP forms to acute catabolic (caloric restriction) and anabolic [growth hormone (GH) stimulation] interventions in lambs and related the findings to circulating IGF-I levels, growth velocity, and markers of bone formation. Lambs fed a reduced caloric intake (25% of normal) for 6 days exhibited reduced live weight, plasma urea, and IGF-I (P < 0.001 for all) compared with control lambs. Basal levels of NT-proCNP (40.1 +/- 0.9 pmol/l) fell promptly to a nadir (28.1 +/- 0.8 pmol/l, P < 0.001) on day 6, returning rapidly to basal levels upon refeeding. Although plasma alkaline phosphatase (ALP) fell (P < 0.001), reductions in metacarpal growth velocity were not significant within the 12-day period of study. In contrast to caloric restriction, long-acting bovine recombinant GH (2.5 mg/kg on days 0 and 6), as expected, increased plasma IGF-I more than twofold above control for 12 days (P < 0.001). Growth velocity did not differ during the 30 days of observation, and, consistent with unchanged growth velocity, plasma NT-proCNP and ALP were also unaffected. In conclusion, CNP synthesis and markers of bone formation are acutely sensitive to catabolism but unaffected by doses of GH that fail to stimulate skeletal growth.