Gene Therapy Batten Disease Sheep PDF 2023

TYPE Original Research PUBLISHED 08 August 2023 DOI 10.3389/fg...

TYPE Original Research PUBLISHED 08 August 2023 DOI 10.3389/fgene.2023.1212228 Long-term safety and dose OPEN ACCESS escalation of EDITED BY Paschalis Nicolaou, The Cyprus Institute of Neurology and intracerebroventricular Genetics, Cyprus REVIEWED BY CLN5 gene therapy in sheep Hemanth Ramesh Nelvagal, University College London, United Kingdom supports clinical translation for Bruce Frederic Smith, Auburn University, United States CLN5 Batten disease Nicolina Cristina Sorrentino, Telethon Institute of Genetics and Medicine (TIGEM), Italy Nadia L. Mitchell 1,2*, Samantha J. Murray 1, Martin P. Wellby 1, *CORRESPONDENCE Graham K. Barrell 1, Katharina N. Russell 1, Ashley R. Deane 1, Nadia L. Mitchell, [email protected] John R. Wynyard 1, Madeleine J. Palmer 1, Anila Pulickan 1, RECEIVED 07 June 2023 Phillipa M. Prendergast 1, Widler Casy 3, Steven J. Gray 3 and ACCEPTED 24 July 2023 PUBLISHED 08 August 2023 David N. Palmer 1,2 1 CITATION Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, New Zealand, 2Department of Mitchell NL, Murray SJ, Wellby MP, Radiology, University of Otago, Christchurch, New Zealand, 3Department of Pediatrics, University of Texas Barrell GK, Russell KN, Deane AR, Southwestern Medical Center, Dallas, TX, United States Wynyard JR, Palmer MJ, Pulickan A, Prendergast PM, Casy W, Gray SJ and Palmer DN (2023), Long-term safety and dose escalation of intracerebroventricular CLN5 gene CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal therapy in sheep supports clinical neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is translation for CLN5 Batten disease. Front. Genet. 14:1212228. characterised by progressive neuronal loss, blindness, and premature death. There doi: 10.3389/fgene.2023.1212228 is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) COPYRIGHT delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/ © 2023 Mitchell, Murray, Wellby, Barrell, oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected Russell, Deane, Wynyard, Palmer, (CLN5−/−) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three Pulickan, Prendergast, Casy, Gray and Palmer. This is an open-access article disease stages. The treatment delayed disease progression, extended survival and distributed under the terms of the slowed stereotypical brain atrophy in most animals. Of note, one high dose treated Creative Commons Attribution License animal only developed mild disease symptomology and survived to 60.1 months of (CC BY). The use, distribution or reproduction in other forums is age, triple the natural life expectancy of an untreated CLN5−/− sheep. Eyesight was permitted, provided the original author(s) not preserved at any administration age or dosage. Histopathologic examination and the copyright owner(s) are credited revealed that greater transduction efficiency was achieved through higher ICV and that the original publication in this journal is cited, in accordance with doses, and this resulted in greater amelioration of disease pathology. Together accepted academic practice. No use, with other pre-clinical data from CLN5−/− sheep, the safety and efficacy data from distribution or reproduction is permitted these investigational new drug (IND)-enabling studies supported the initiation of which does not comply with these terms. 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 KEYWORDS neuronal ceroid lipofuscinosis, neurodegenerative disease, gene therapy, adeno- associated virus, intracerebroventricular, sheep Frontiers in Genetics 01 frontiersin.org Mitchell et al. 10.3389/fgene.2023.1212228 1 Introduction 2 Materials and methods The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a 2.1 Animals group of monogenic inherited lysosomal storage diseases characterized by progressive neurodegeneration, cortical atrophy, Supplementary Table S1 summarizes the study design. Clinically and blindness. Few effective treatments exist; the disease is presently healthy CLN5 heterozygous (CLN5+/−) and CLN5 affected fatal. Variant late-infantile NCL or CLN5 disease is caused by (CLN5−/−) Borderdale sheep were diagnosed at birth (Frugier autosomal-recessive mutations in the CLN5 gene. Whilst the et al., 2008) and maintained at Lincoln University under NIH function of this gene is unclear, it is known to encode a guidelines for the Care and Use of Animals in Research and the glycosylated, soluble non-enzymatic lysosomal protein NZ Animal Welfare Act (1999). The animal studies were reviewed (Isosomppi et al., 2002). Human CLN5 disease usually presents and approved by the Lincoln University Animal Ethics and with motor dysfunction and impaired cognition between 1.5 and Institutional Biosafety Committees. 8 years of age (Xin et al., 2010; Mole and Williams, 2013; Simonati Sheep were randomized into treatment groups based et al., 2017), followed by progressive visual decline, seizures and on identification codes assigned to them at birth. Over dementia leading to premature death between the ages of a period of 3 years, nineteen CLN5 −/− ewes received 14–36 years (Nita et al., 2016; Simonati et al., 2017). bilateral intracerebroventricular (ICV) administration of a A naturally occurring sheep model of CLN5 NCL exists in self-complementary adeno-associated virus serotype 9 vector New Zealand Borderdale sheep (Jolly et al., 2002; Frugier et al., expressing ovine CLN5 (scAAV9/oCLN5) at various ages and 2008). Affected animals exhibit a human CLN5 disease-like dosage levels. phenotype including progressive brain atrophy and loss of vision Treated sheep were euthanized by penetrating captive bolt to the from 11 to 12 months of age, and a shortened life span cervical spine and immediate exsanguination at predetermined of 0.01, *p < 0.05). Low dose (LD); moderate (MD); high (HD) dose. halt or slowing of further astrocytosis, brain macrophage formation was elevated in the thalamus (Figure 6; Supplementary Figure S4), it and recruitment. Although astrocytes were hypertrophied in the did not significantly exceed that of much younger untreated CLN5−/− treated brains, the strong meshwork of activated astrocytes was not sheep and was noticeably less than that observed in the cortical observed and total reactivity of both neuroinflammatory markers regions. There was no evidence of astrocytic or microglial activation was often significantly less than that in untreated CLN5−/− brains. In in either the untreated CLN5−/− or ICV treated cerebella some instances, GFAP immunoreactivity in the treated motor cortex (Supplementary Figure S5). approached healthy control CLN5+/− levels. Gliosis would have been pronounced in the three advanced symptomatic treated animals’ brains at their time of ICV delivery 3.9 Lysosomal storage (9 months). Treatment had no substantive effect in two animals of the cohort, but amelioration in disease-associated Punctate globular storage bodies were densely packed into the neuroinflammation was observed in the brain of animal 1163–16 cortical neurons and cerebellar Purkinje cells of untreated CLN5−/− (Supplementary Tables S3, S4). Glial activation within the brain of sheep brains (Figure 7; Supplementary Figure S6). There was less this animal was to a much lesser extent than without treatment. storage in one pre-symptomatic MD treated sheep (1104–16), but To verify if reduced CLN5 transduction in the subcortex resulted the LD or MD was insufficient to attenuate accumulation in the in heightened neuroinflammation, GFAP and GSB4 reactivities were remaining animals. also assessed in the thalamus and cerebellum of ICV treated Quantitative threshold analysis showed there was significantly sheep. Although expression of both neuroinflammatory markers less fluorescent material in the primary visual and parieto-occipital Frontiers in Genetics 09 frontiersin.org Frontiers in Genetics Mitchell et al. TABLE 1 Summary of in-life efficacy and neuropathological terminal endpoints following ICV scAAV9/oCLN5. Study Treatment ICV Sheep Tx End Clinical Rate of Loss Intracranial Terminal Cortical Neuroinfl Lysosomal CLN5 dose Age point Description Declineb of volume brain thicknessd ammation storage expression (vg)a (m) (m) vision: change weight (mL)c (g) Pre- ICV scAAV9/ LD/1 1102–16 3.2 53.8 Delayed clinical -0.4 Yes -0.7 65.7 45-67% High Extensive Mild to symptomatic oCLN5 8.0x1010 disease moderate in cortex, sparse remaining CNS 1111–16 3.2 47.3 Delayed clinical -0.5 Yes -3.0 66.0 38-58% High Extensive Moderate in disease M1, sparse remaining CNS 1128–16 2.9 53.5 Delayed clinical -0.4 Yes 1.2 73.5 44-67% Moderate Extensive Moderate to disease extensive across cortex LD/2 1110–18 3.5 30.4 Delayed clinical -0.9 Yes -1.0 72.4 59-70% High Extensive Sparse in 8.2x1010 disease cortex 1115–18 3.5 17.9 Euthanised -0.7 Yes -0.1 85.0 73-81% Moderate Extensive Mild in M1, with PEM sparse remaining CNS 1122–18 3.0 24.0 Moderate -1.1 Yes 1.5 74.8 53-67% Moderate Reduction clinical disease 10 MD 1104–16 3.2 27.4 Delayed clinical -0.8 Yes -7.0 65.4 70-79% Low Reduction Mild across 2.4x1011 disease cortex 1120–16 3.2 50.4 Delayed clinical -0.4 Yes 0.3 82.4 51-74% Moderate Extensive Moderate disease across cortex 1123–16 2.9 44.7 Delayed clinical -0.4 Yes -1.2 59.0 42-58% Moderate Extensive Moderate in disease M1 and V1 Early ICV scAAV9/ MD 1185–16 6.0 21.4 Moderate -0.8 Yes -5.5 73.5 59-82% Low Reduction Mild to symptomatic oCLN5 2.7x1011 clinical disease extensive across cortex 1186–16 6.0 49.6 Delayed clinical -0.4 Yes -6.0 74.0 50-64% Low Reduction and disease cerebellum, very little in 1187–16 6.0 49.6 Delayed clinical -0.3 Yes -15.4 75.1 42-60% Moderate Reduction subcortex disease 10.3389/fgene.2023.1212228 HD 1164–15 7.5 60.1 Mild decline -0.2 Yes -6.0 78.3 60-67% Moderate Slight reduction Mild to 2.8x1012 moderate across cortex and subcortex frontiersin.org 1165–15 7.5 22.5 Mild clinical -0.7 Yes -4.5 76.8 62-72% Moderate Reduction Mild to disease extensive across cortex (Continued on following page) Frontiers in Genetics Mitchell et al. TABLE 1 (Continued) Summary of in-life efficacy and neuropathological terminal endpoints following ICV scAAV9/oCLN5. Study Treatment ICV Sheep Tx End Clinical Rate of Loss Intracranial Terminal Cortical Neuroinfl Lysosomal CLN5 dose Age point Description Declineb of volume brain thicknessd ammation storage expression (vg)a (m) (m) vision: change weight (mL)c (g) 1170–15 7.3 42.3 Delayed clinical -0.5 Yes -7.0 73.9 54-77% Low Reduction and disease cerebellum, very little in 1172–15 7.3 22.3 Mild decline -0.3 Yes -0.6 73.5 73-77% Moderate Reduction subcortex Advanced ICV scAAV9/ MD 1143–16 9.0 18.5 Stereotypical -1.6 Yes -6.7 67.9 50-72% High Extensive Mild across symptomatic oCLN5 2.7x1011 clinical disease cortex 1163–16 8.8 30.9 Delayed clinical -0.6 Yes -5.4 71.8 56-80% Low Reduction Mild to disease extensive across cortex and cerebellum, very little in subcortex 1165–16 8.8 21.6 Moderate -1.1 Yes -7.8 69.8 42-72% High Extensive Mild across clinical disease cortex 11 Control Nil n = 12 N/A >24 Normal 0.0 No 3.3 106.1 ± 1.5 100% None No present Endogenous CLN5+/- throughout CNS Untreated Nil n = 15 N/A 18.6 Stereotypical -1.7 Yes -9.4 62.0 ± 1.0 37-42% Extensive Extensive Absent CLN5-/- clinical disease Abbreviations: CNS, central nervous system; HD, high dose; ICV, intracerebroventricular; m, months; M1 primary motor cortex; MD, moderate dose; N/A not applicable; oBDRS, ovine Batten disease rating scale; Tx Treatment. a Two low dose trials have been performed. The first was designated LD/1 and the second LD/2. b Rate of decline in oBDRS, units per month. For treated sheep this is from baseline (treatment age) to end point; for controls this is from 3 months to end point. c Total intracranial volume change from baseline (treatment age) to end point for treated sheep, or from 3 to 19 months of age for controls (Russell et al., 2018). d % cortical thickness of 24-month-old healthy control CLN5+/− 10.3389/fgene.2023.1212228 frontiersin.org Mitchell et al. 10.3389/fgene.2023.1212228 cortex of the early symptomatic treated brains compared with much clinical benefit. Three months of age is a pre-symptomatic disease younger CLN5−/− sheep (Figure 7). Of note, HD-treated sheep stage in CLN5 affected sheep, modelling patients being symptom- 1164–15 and 1170–15 who were euthanized at 60.1 and free, with maximum functional neuronal cells present, and before 42.3 months of age respectively, had less than or similar storage cumulative pathogenic processes, including neuroinflammation and levels to untreated CLN5−/− animals nearly 2 years younger lysosomal storage body accumulation, become manifest. Given that (Supplementary Table S5). a CLN5 diagnosis in humans can take years, the equivalent pre- Storage body accumulation in the advanced symptomatic symptomatic population in humans is normally restricted to treated sheep paralleled neuroinflammation. Storage burden was younger siblings or close relatives of already diagnosed significantly reduced in sheep 1163–16 compared with untreated symptomatic CLN5 patients. CLN5−/− sheep, particularly in the visual and motor cortices Nine CLN5−/− sheep have received pre-symptomatic ICV (Figure 7; Supplementary Table S5). scAAV9/oCLN5 therapy at 3 months of age at two doses Although fewer transduced cells were detected in the subcortex (8.0–8.2 × 1010 to 2.4 × 1011 vg). The treatment provided long- of ICV treated sheep than cortical structures, this did not have a term therapeutic benefit. Clinical disease was considerably delayed greater effect on subcortical lysosomal accumulation. Storage and five of the treated animals survived to more than double the burden was attenuated in the thalamus and cerebellum for the natural life expectancy of untreated CLN5−/− sheep. Longitudinal majority of ICV treated sheep, and significantly reduced in the neuroimaging indicated a halt or slowing in the stereotypical brain thalamus of MD treated sheep and the cerebellum and thalamus of atrophy of ovine CLN5 disease and four of these sheep maintained HD treated sheep when compared with untreated CLN5−/− sheep intracranial volumes in the range of healthy CLN5+/− controls before (Figure 7; Supplementary Figure S6). declining slowly over time. One pre-symptomatic treated sheep (1104–16) did not respond as positively to the moderate dose (2.4 × 1011 vg) ICV treatment and was euthanized at 27.4 months 3.10 In vitro vector analyses of age with developing clinical disease. Despite its 7.0 mL loss in intracranial volume, this was at a slower rate than untreated CLN5−/− Finally, in addition to an ovine CLN5 (oCLN5) construct sheep and post-mortem analyses revealed a significant reduction in generated for the in vivo studies in sheep, a parallel human disease-associated neuropathology. The low level of CLN5 (hCLN5) construct was made for possible downstream CLN5 transduction efficiency in the brain of this animal is likely human use. Both plasmid constructs were identical between the causative of the reduced therapeutic benefit compared with her inverted terminal repeats (ITRs), except for the species specificity of similarly treated cohorts. the CLN5 transgene. Both human and ovine genes underwent One of the pre-symptomatically treated sheep (LD 1115–18) was independent codon-optimizations. To confirm expression of the euthanized on welfare grounds at 17.9 months of age due to constructs and compare their relative strength of expression, both suspected acute polioencephalomalacia (PEM). This neurological plasmids were transfected into HEK293 cells and protein levels disease, also known as cerebrocortical necrosis, is seen worldwide in assessed by Western blotting. While minimal endogenous ruminants and presents sub-acutely with similar clinical signs to CLN5 protein could be detected by Western blot under the ovine Batten disease hence diagnosis was not detected. PEM is experimental conditions, expression was readily apparent in a primarily associated with thiamine or dietary deficiencies (Lévy, dose-responsive manner 48 h following transfection of either the 2020) but no cause was identified in this case and no other animals in hCLN5 or oCLN5 plasmid (Supplementary Table S7). When 2 ug of the flock developed the condition. The brain of sheep LD plasmid was transfected, the hCLN5 construct showed a 1.26-fold 1115–18 lacked the definitive post-mortem tests for PEM, faint higher expression compared to oCLN5 (p = 0.002), whereas when yellow discoloration and/or fluorescence of the grey matter under 1 ug was transfected the hCLN5 showed 2.82-fold higher expression ultraviolet light, so diagnosis could not be confirmed. However, it that was not statistically significant (p = 0.225). occurred 15 months after treatment, thus there was no evidence to attribute this condition to the gene therapy vector, anaesthesia, surgical or administration procedure. 4 Discussion These results in pre-symptomatic sheep correlate well with previous CLN5 gene therapy studies in the Borderdale sheep These studies provide pre-clinical proof-of-concept data which model. Both single-stranded AAV9 and lentiviral vectors supported the successful United States Food and Drug expressing ovine CLN5 have been shown to normalize Administration clearance of the CLN5 gene therapy product as intracranial volume and prevent disease onset and progression an investigational new drug for the treatment of human when delivered to CLN5−/− sheep at 3 months of age (Mitchell CLN5 Batten disease. New Zealand Borderdale sheep with et al., 2018). This study showed that the current central nervous naturally occurring CLN5 NCL were used as they represent an system (CNS) viral vector of choice, self-complementary AAV9, can ideal model in which to develop and test potential gene therapies for also be added to the suite of therapeutic vector platforms for this largely pediatric condition. Nineteen CLN5−/− sheep received CLN5 disease. ICV scAAV9/oCLN5 therapy at three different ages representing Most human cases of Batten disease only become diagnosed different stages of disease development and over a 35-fold range of following the development of overt disease, represented by the six- doses. A summary of the results is presented in Table 1. month-old early symptomatic CLN5−/− cohort. At this age, affected Theoretically the earlier gene therapy can be administered to sheep demonstrate mild movement dysfunction and early visual patients with neuropathic lysosomal storage diseases, the more the loss, and cortical atrophy and neurodegeneration is obvious. Seven Frontiers in Genetics 12 frontiersin.org Mitchell et al. 10.3389/fgene.2023.1212228 CLN5−/− sheep received moderate (2.7 × 1011 vg) or high dose (2.8 × A number of gene therapy clinical trials have been performed, 1012 vg) ICV scAAV9/oCLN5 therapy between six and 7 months of are underway, or are in preparation, for the different forms of NCL age. The treatment slowed disease progression in most treated (https://clinicaltrials.gov/), including the first in-human CLN5 gene animals. Live weight largely normalized, quality of life was therapy study based on these and other efficacy studies in CLN5−/− improved, and lifespan extended to more than double to triple sheep (Clinicaltrials.gov identifier: NCT05228145). Routes of the natural expectancy of CLN5−/− sheep. Three animals survived to administration have typically been intraparenchymal (CLN2) or be over 42 months of age, and a fourth (1164–15) until over intrathecal into the lumbar cistern (CLN3, CLN6, CLN7). Pilot 60 months when it died unexpectedly of other causes. Aside from studies in a small number of healthy sheep indicate that the ICV loss of vision, these sheep showed only mild phenotypic changes route resulted in the most robust widespread distribution of vector long after ICV injection. In particular, sheep 1164–15 had only mild throughout the entire CNS, compared to intracisternal magna, episodes of stereotypical behavior and ataxia at her untimely death lumbar intrathecal or intraparenchymal delivery. Delivery directly and did not show manifest ataxia, localized seizure activity or into the cerebrospinal fluid (CSF) was preferred for require extensive nursing. Intracranial volumes at injection were CLN5 therapeutics, as it allows for maximum transduction of the equivalent to those of untreated CLN5−/− sheep, and losses observed CNS and circumvents the blood-brain and blood-CSF barriers, over the duration of the study occurred much more slowly than they reducing the risk of an immune response or off-target side do in untreated CLN5−/− sheep. effects, seen with systemic (intravenous) delivery of Amelioration of all disease-related neuropathological markers AAV9 vectors and other biologics (Zincarelli et al., 2008; Xie indicated long-term treatment efficacy, albeit not complete. The et al., 2011; Sadekar et al., 2022). Increasingly the ICV delivery treated sheep with the least therapeutic efficacy (MD 1185–6) was route is being recognized as a powerful way to transduce widespread the animal with the lowest number of transduced cells, whilst there areas of the brain in CNS disorders (Hughes et al., 2018; Bey et al., were many CLN5-positive cells in the motor, visual and parieto- 2020; Hinderer et al., 2020; Galvan et al., 2021). It is the infusion occipital cortices, hippocampus and cerebellum of the HD early route for the only current approved NCL enzyme replacement symptomatic treated sheep who survived to 60.1 months of age, therapy, cerliponase alfa, for CLN2 disease (Schulz et al., 2018) indicating some linkage between transduction efficiency and and is the delivery route of choice to target the brain in the current therapeutic efficacy. CLN5 gene therapy clinical trial. Most human CLN5 cases are of a variant late infantile onset Consistent with similar studies in non-human primates (Naidoo form, presenting between 1.5 and 8 years of age (Xin et al., 2010; et al., 2018; Bey et al., 2020; Galvan et al., 2021), this sheep study Mole and Williams, 2013; Simonati et al., 2017), then progressing shows that the ICV delivery route did not result in high levels of to severe impairment in the learning/cognition, motor, language, transduction of deeper brain structures, such as the basal ganglia vision, and seizures domains and premature death. The advanced (striatum, putamen and caudate nucleus) or thalamus. However, symptomatic nine-month-old CLN5−/− cohort was studied to failure to greatly transduce these regions that are not as severely determine the effectiveness of a treatment at this stage. At this affected in ovine CLN5 disease (Mitchell et al., 2023) did not result age, untreated CLN5−/− sheep demonstrate clear phenotypic in increased subcortical disease pathology and seems unlikely to deficits, particularly related to visual loss, and the affected have contributed greatly to disease progression. Levels of brain has reduced to approximately 85% the weight of a neuroinflammation and storage burden in the thalamus and healthy sheep brain (Mitchell et al., 2023). Neuronal loss is cerebellum of ICV treated sheep aged 17.9–60.1 months of age advanced, particularly from the occipital cortex where cortical were not significantly different from much younger 24-month- thicknesses are reduced to half those of a healthy CLN5+/− sheep old untreated CLN5−/− sheep. In fact, the four early symptomatic (Mitchell et al., 2023). treated sheep who received the highest ICV dose had significantly Unfortunately, due to limited vector availability, advanced less thalamic gliosis and lysosomal storage accumulation in their symptomatic sheep could only be treated with a MD treatment thalamus and cerebellum than untreated CLN5−/− sheep, suggesting and not the HD which had proved most efficacious in the early higher doses might even attenuate subcortical pathology. Much symptomatic cohort. Nevertheless, of the three CLN5−/− sheep who discussion arises over the benefits of unilateral versus bilateral received treatment at 9 months of age, one responded favorably ICV delivery route. Whilst bilateral injections have been used in (MD 1163–16). Disease progression was stabilized until 19 months most pre-clinical animal studies, therapeutic benefit has been post-treatment when the animal began to decline clinically, and it demonstrated in a canine model of CLN2 NCL after a single was euthanized at 30.9 months of age following increasing episodes unilateral injection of AAV serotype 2 expressing the canine of inducible tremors and stereotypical circling behavior. Intracranial CLN2/TPP1 enzyme (Katz et al., 2015). Injections in this study volume loss was moderate, at 5.4 mL from 9 to 30.9 months, but were bilateral except for one (HD 1172-15) that received both doses significantly less than untreated CLN5−/− sheep that lost an average into the right ventricle following difficulty in identifying the left of 9.4 mL between 3 and 19 months of age. In-life assessments of ventricle. This had no apparent impact on the efficacy of the efficacy suggest that another MD ICV treated advanced treatment. The sheep was clinically stable, lost minimal symptomatic sheep (1143–16) was a non-responder, whilst the intracranial volume and was euthanized early at 22.3 months of treatment had only minimal positive benefit to the third age, whilst in excellent clinical health. Post-mortem sheep. These two animals had fewer transduced cells, but neuropathological analysis revealed no significant difference in neuropathological analyses also revealed amelioration of common cortical thickness, neuroinflammatory marker expression, disease markers (cortical thinning, astrocytosis, microgliosis, storage lysosomal storage burden or CLN5 expression between the two body accumulation) to a degree. hemispheres suggesting widespread CNS transduction could be Frontiers in Genetics 13 frontiersin.org Mitchell et al. 10.3389/fgene.2023.1212228 achieved by unilateral delivery. The only asymmetry was a larger show that sheep appear to be tolerant to native and non-native amount of transduced sensory and motor neurons in the right transgenes (Banin et al., 2015; Gray-Edwards et al., 2018; O’Leary column of the cervical spinal cord compared with the left column. et al., 2023). In the current study, there was no apparent In line with previous NCL gene therapy studies in sheep and upregulation in neuroinflammation in treated sheep that could dogs (Katz et al., 2015; Mitchell et al., 2018). ICV delivery alone was not be considered disease-associated, and there was long-term not sufficient to protect against vision loss in the current study. transgene expression and no other clinical indications of toxicity. Unilateral intravitreal (IVT) delivery of scAAV9/oCLN5 has However, as the sheep did not receive any immunosuppressive resulted in maintenance of retinal structure and function in the drugs, this study did not provide translational data on the need treated eye of CLN5−/− sheep for up to 15 months post-treatment, for immunomodulation in human CLN5 patients. while the untreated eye displayed progressive electroretinographic Finally, the ovine and human CLN5 transgenes used in this study decline and severe atrophy on post-mortem examination (Murray were codon optimized. There is concern that this is not necessary for et al., 2021). A second IND-enabling study in sheep has expression of a gene in its native host and the process itself might affect subsequently shown that combined ocular and brain-directed protein conformation and function, detrimentally increasing gene therapy simultaneously halt neurological and retinal disease immunogenicity or reducing efficacy (Mauro and Chappell, 2014). in ovine CLN5 Batten disease (Murray et al., 2023), prompting Here, codon optimization was performed to strengthen the expression clinical translation of dual ICV and IVT administration for this cassette, ensure robust protein expression, and minimize the potential disease. for cryptic splice sites and start codons which might produce non- Although the ICV delivery route is efficacious in the brain, one of specific or immunogenic peptides. Whilst the native sequence is the concerns with a non-systemic delivery route is that not all regions of preferred, it was evident from this study that codon optimization the body are targeted. Typically, in NCL, pathology outside of the brain did not adversely affect functionality and the first-in-human and eye have not been considered due to the profound effect of CLN5 gene therapy Phase I/II Clinical Trial (NCT05228145) neurodegeneration, vision loss and death before other pathologies supported by these sheep studies will also use a codon-optimised emerge. However, the CLN5 gene mutation is present in every cell human CLN5 transgene. of the body, lysosomal storage is largely ubiquitous and in humans Overall, these studies in CLN5−/− sheep demonstrate the long- endogenous CLN5 protein is widely expressed (https://www. term efficacy of ICV scAAV9/oCLN5 gene therapy when delivered proteinatlas.org/). Extra-neuronal pathologies have previously been at pre-symptomatic, early symptomatic, and more advanced documented in the heart, liver and muscle tissue of CLN2−/− dogs symptomatic disease stages at the doses administered. Treatment whose lives were significantly extended through ICV CLN2 gene was well tolerated, delayed disease progression, stereotypical brain therapy (Katz et al., 2017). Here, the HD treated sheep atrophy, and disease pathology in the majority of the treated sheep 1,164–15 died unexpectedly at 60.1 months of age from but did not preserve vision at any dose or disease state evaluated. In gastrointestinal stasis. Descriptions of constipation, abdominal pain preparation for clinical translation, in vitro studies indicated higher and bloating, vomiting, gastric reflux, changes in gut microbiota, loss of expression levels of the proposed investigational drug, enteric neurons and impaired gastrointestinal motility have been AAV9 expressing the human CLN5 gene, than the ovine gene published and anecdotally reported for NCL patients and animal therapy product delivered in vivo. Typically for lysosomal storage models (Mole et al., 2021; Parker et al., 2021; Wang et al., 2021) so diseases like CLN5 NCL, the earlier the treatment the better (Sands long-term future therapeutic strategies may be required for other and Davidson, 2006), yet the findings from early and advanced pathologies, such as those in the enteric nervous system. symptomatic treated sheep demonstrate dose-dependent There were other limitations to this study. Although transduction efficiency is important. Here, the MD and HD histochemical analyses suggested the co-existence of vector- treatments were the most efficacious, prompting further studies transduced cells and cross-corrected cells within the treated in sheep with even higher doses and the inclusion of an ocular brain, it was not categorically possible to confirm that cross- delivery route for retinal targeting (Murray et al., 2023). This study correction was occurring in the ICV treated sheep brains. Strong indicates great translational promise for extending and improving CLN5-positive transduced cells were easily distinguished from the quality of life in post-symptomatic CLN5 patients, so long as diffuse weak CLN5-positive neighboring cells which were they have sufficient remaining cortical and cerebellar neurons to hypothesized to have taken up secreted CLN5 protein. However, benefit from CLN5 expression and that higher doses are likely to be as treated sheep brains were perfusion-fixed in situ, no fresh frozen more effective. brain tissue was available for vector copy number analyses for a direct comparison of CLN5 protein to AAV9 distribution. The CLN5 mutation in Borderdale sheep is caused by a splice site Data availability statement nucleotide substitution, resulting in the excision of exon 3 and a truncated protein (Frugier et al., 2008). Like sheep, many of the The raw data supporting the conclusion of this article will be disease-causing mutations in humans result in a putative shorter made available by the authors, without undue reservation. CLN5 protein (Basak et al., 2021), however some patients will have mutations that preclude any CLN5 protein synthesis. Therefore, there is a risk that CLN5−/− individuals, who lack expression of some Ethics statement or all natural CLN5 epitopes, could mount an immune response when they receive the CLN5 transgene, however this does not appear The animal study was reviewed and approved by Lincoln to be an issue in sheep. Indeed, CNS and ocular gene therapy studies University Animal Ethics Committee. Frontiers in Genetics 14 frontiersin.org Mitchell et al. 10.3389/fgene.2023.1212228 Author contributions Conflict of interest NM, GB, SG, and DP contributed to the conceptualisation and DP and NM received research funding from Neurogene Inc. for design of the study. NM, SG, and DP acquired the funding, provided this study. SG has received royalty income from Asklepios the resources and oversaw the project supervision. NM, SM, MW, Biopharmaceutical Inc. for intellectual property (I.P.) that was KR, AD, JW, MP, AP, PP, and WC performed the investigation, not used in this study, and he received royalty income from curated the data and performed the formal analyses. NM wrote the Neurogene Inc. for I.P. that was used in this study. first draft of the manuscript. All authors contributed to the article The remaining authors declare that the research was conducted and approved the submitted version. in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Publisher’s note This work was funded by CureKids NZ (6501, 3607), the Canterbury Medical Research Foundation (01/2019), the Batten All claims expressed in this article are solely those of the authors disease Support and Research Association (United States), Batten and do not necessarily represent those of their affiliated Disease New Zealand, Neurogene Inc. (all to DP and NM), and organizations, or those of the publisher, the editors and the the Maurice and Phyllis Paykel Trust (to NM). A University of reviewers. Any product that may be evaluated in this article, or Otago Health Sciences post-doctoral fellowship and Lincoln claim that may be made by its manufacturer, is not guaranteed or University post-doctoral fellowship were awarded by NM and endorsed by the publisher. SM respectively. Supplementary material Acknowledgments The Supplementary Material for this article can be found online The authors would like to thank the staff of the Johnstone at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1212228/ Memorial Laboratory at Lincoln University for animal care. full#supplementary-material References Banin, E., Gootwine, E., Obolensky, A., Ezra-Elia, R., Ejzenberg, A., Zelinger, L., et al. (2015). Isosomppi, J., Vesa, J., Jalanko, A., and Peltonen, L. (2002). 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