Protracted CLN3 Batten disease in mice that genetically model an exon-skipping therapeutic approach

Jessica L Centa; Matthew P Stratton; Melissa A Pratt; Jenna R Osterlund Oltmanns; Douglas G Wallace; Steven A Miller; Jill M Weimer; Michelle L Hastings

doi:10.1016/j.omtn.2023.05.025

. 2023 Jun 3;33:15–27. doi: 10.1016/j.omtn.2023.05.025

Protracted CLN3 Batten disease in mice that genetically model an exon-skipping therapeutic approach

Jessica L Centa ^1,⁸, Matthew P Stratton ^1,^2,⁸, Melissa A Pratt ³, Jenna R Osterlund Oltmanns ⁴, Douglas G Wallace ⁴, Steven A Miller ⁵, Jill M Weimer ^3,⁶, Michelle L Hastings ^1,^7,^∗

PMCID: PMC10285469 PMID: 37359347

Abstract

Genetic mutations that disrupt open reading frames and cause translation termination are frequent causes of human disease and are difficult to treat due to protein truncation and mRNA degradation by nonsense-mediated decay, leaving few options for traditional drug targeting. Splice-switching antisense oligonucleotides offer a potential therapeutic solution for diseases caused by disrupted open reading frames by inducing exon skipping to correct the open reading frame. We have recently reported on an exon-skipping antisense oligonucleotide that has a therapeutic effect in a mouse model of CLN3 Batten disease, a fatal pediatric lysosomal storage disease. To validate this therapeutic approach, we generated a mouse model that constitutively expresses the Cln3 spliced isoform induced by the antisense molecule. Behavioral and pathological analyses of these mice demonstrate a less severe phenotype compared with the CLN3 disease mouse model, providing evidence that antisense oligonucleotide-induced exon skipping can have therapeutic efficacy in treating CLN3 Batten disease. This model highlights how protein engineering through RNA splicing modulation can be an effective therapeutic approach.

Keywords: MT: Oligonucleotides: Therapies and Applications, antisense oligonucleotides/CLN3 Batten disease/exon skipping/lysosomal storage disease/pre-mRNA splicing

Graphical abstract

Hastings and colleagues generated a therapeutic mouse model of CLN3 Batten disease to show that correcting the open reading frame by exon skipping results in reduced disease burden. These findings validate their prior work, supporting the use of exon-skipping antisense oligonucleotides to treat patients with a CLN3^Δex7/8 mutation.

Introduction

CLN3 Batten disease, also known as CLN3 disease or juvenile neuronal ceroid lipofuscinosis (JNCL), is an autosomal recessive, lysosomal storage disease that affects young children. There are currently no treatments for this fatal disease that impacts as many as 1 in 25,000 live births in some populations.¹^,² Children with mutations in CLN3 develop normally for the first few years of their life and then progressively decline, with initial symptoms including visual deficits and seizures followed by cognitive decline, behavioral changes, and motor decline with loss of ambulation.³ Cardiac arrhythmias and difficulty feeding are common later in disease with death typically in the late teens or twenties.⁴^,⁵^,⁶^,⁷

CLN3 Batten disease is most commonly caused by a 966 base-pair deletion encompassing exons 7 and 8 of the gene (CLN3^Δex7/8), causing disruption of the open reading frame and a premature termination codon in exon 9. This disruption results in either nonsense-mediated decay of the mRNA or translation of a truncated protein lacking 257 amino acids at the C-terminus; however, the lack of commercially available antibodies has limited researchers’ ability to study the effects of CLN3 mutations at the protein level.⁸^,⁹ Though the function of CLN3 is not fully understood, the C-terminal tail of CLN3 contains a lysosomal targeting sequence and has been shown to have an important role in folding and exit from the endoplasmic reticulum.¹⁰ Additionally, most cases of CLN3 Batten disease not resulting from deletion of exons 7 and 8 are frame-shifting deletions and nonsense mutations that would lack this C-terminal domain.¹¹ Mutations that do not result in a C-terminal truncation are associated with protracted disease¹¹^,¹² (NCL Mutation Database: http://www.ucl.ac.uk/ncl), suggesting that mutations that do not result in the complete loss of CLN3 protein function may be better tolerated than frameshift mutations, which invariably cause severe forms of the disease. Based on this evidence, we reasoned that recovery of the protein coding open reading frame of CLN3^Δex7/8 could produce a protein with at least partial function that might result in a less severe, protracted form of the disease.

One approach to restore open reading frames caused by frame-shifting mutations that has shown promise in the clinic for other diseases, is to target the endogenous pre-mRNA splicing and splice out (skip) the exon or exons containing the premature termination codon using splice-switching antisense oligonucleotides (SSOs). If the exons flanking the spliced-out exon are in the same reading frame (symmetric with each other), then the resulting mRNA will encode the correct protein product, albeit with an internal deletion of the amino acids encoded by the spliced-out exon.

SSOs are a type of antisense oligonucleotide (ASO) that work specifically by base-pairing to their target RNA, thereby masking the target RNA from recognition by RNA processing machinery. To date, nine ASO drugs have received approval for clinical use, five of which are based on splice-switching principles, demonstrating the utility of this ASO-mediated splice modulation for treating disease.¹³ SSOs are frequently utilized to block binding of the spliceosome or splicing factors and, in so doing, modulate splicing. In this way, SSOs induce alternative pre-mRNA splicing, a process whereby different mRNA-spliced isoforms are generated from the same gene. Alternative splicing is a common occurrence in most human gene transcripts and is thought to be important for achieving organismal complexity from a relatively small genome.¹⁴^,¹⁵ Alternative protein isoforms encoded by a single gene may have subtle or gross differences in function and regulation of alternative splicing can fine-tune protein activity.¹⁶

The alternative splicing repertoire of CLN3 RNA transcripts appears to be extensive, though few studies have validated these isoforms or their function.¹⁷^,¹⁸ The splicing diversity may indicate a functionality or tolerance for alternative spliced protein isoforms. Considering this possibility, we devised a therapeutic approach that induces a CLN3 mRNA isoform lacking exons 5, 7, and 8, and examined whether the protein encoded by this mRNA has activity that can compensate for the aberrant function resulting from the disease-causing CLN3^Δex7/8 mutation. This CLN3^Δex5/7/8 isoform has been documented in humans, non-human primates, and dogs, suggesting possible functionality due to its evolutionary conservation across species.¹⁷ Here we examine whether this alternatively spliced isoform of CLN3, which in addition to lacking exons 7 and 8, also lacks exon 5, can provide therapeutic value. This CLN3^Δex5/7/8 isoform, unlike the disease-associated mutation, has an intact open reading frame and encodes a protein with an internal deletion.¹⁷

Our group has previously demonstrated that an SSO that induces exon 5 skipping of Cln3^Δex7/8 transcripts resulted in a reduced disease burden in mice.⁹ To further demonstrate that exon 5 skipping and recovery of the Cln3^Δex7/8 open reading frame provides disease rescue, we generated a mouse model that has a genetic deletion of exons 5, 7, and 8 and thus naturally expresses the SSO-induced Cln3^Δex5/7/8 isoform. We found that Cln3^Δex5/7/8 mice have a lower disease burden both behaviorally and pathologically compared with Cln3^Δex7/8 mice. Furthermore, this improvement was greater in mice homozygous for Cln3^Δex5/7/8 compared with mice with only one copy of Cln3^Δex5/7/8, suggesting a correlation between the amount of Cln3^Δex5/7/8 and therapeutic effect. These results provide critical validation of our prior findings that an SSO that induces exon 5 skipping ameliorates the effects of the disease in mice, and support the use of this technology to treat patients that have a CLN3^Δex7/8 mutation.

Results

Generation of Cln3^Δex5/7/8 mice

The concept that correction of the CLN3^Δex7/8 open reading frame by skipping of exon 5 will be therapeutic in CLN3 disease is based on the idea that the resulting CLN3^Δex5/7/8 mRNA isoform will produce a protein product. There are no validated, commercially available antibodies that can be used to detect CLN3 or its isoforms. However, expression of an epitope-tagged version of the full-length CLN3 (wild type [WT]) or the mutant CLN3^Δex7/8 or reading frame corrected CLN3^Δex5/7/8 demonstrate the ability of the CLN3^Δex5/7/8 mRNA to produce a protein product (Figure S1). With this proof-of-concept result, we moved forward with testing the therapeutic effect of replacing Cln3^Δex7/8 with a version lacking exon 5 that restores the open reading frame of the gene, we deleted exon 5 from the mouse Cln3^Δex7/8 allele using CRISPR-Cas9 gene editing. Guide RNAs flanking the intron 4-exon 5 junction and exon 5-intron 5 junction were used for germ-line, constitutive removal of exon 5 from Cln3^Δex7/8 (Figure 1A). PCR analysis of genomic DNA from the gene-edited mice produced the correctly sized product resulting from excision of exon 5 from the Cln3^Δex7/8 allele (Figures 1B and 1C). RT-PCR analysis and sequencing of the resulting cDNA confirmed that deletion of exon 5 results in a Cln3^Δex5/7/8 mRNA transcript (Figures 1B and 1D).

Sensorimotor coordination deficits associated with murine CLN3 disease are absent in a Cln3^Δex5/7/8 therapeutic model

To assess the effects of open reading frame correction on disease progression, behavioral assessments were performed on mice that were heterozygous and homozygous for the exon 5 deletion. Cln3^Δex7/8 mice have measurable motor deficits as early as 2 months of age.¹⁹^,²⁰ As expected, 3-month-old Cln3^Δex7/8 mice had significant deficits in sensorimotor coordination as assessed by latency to passive rotation and fall on an accelerating rotarod (Figure 2). Cln3^Δex7/8 mice slipped (Figure 2A) and fell (Figure 2B) from the rotarod significantly earlier than Cln3^+/Δex7/8 mice. Cln3^Δex5/7/8 mice performed significantly better on these tasks than Cln3^Δex7/8 mice, and neither heterozygous Cln3^{Δex7/8/Δex5/7/8} nor homozygous Cln3^Δex5/7/8 mice were significantly different from Cln3^+/Δex7/8 control mice in either measure (Figures 2A and 2B). These data show that Cln3^Δex5/7/8, Cln3^{Δex7/8/Δex5/7/8}, and Cln3^+/Δex7/8 mice do not exhibit the sensorimotor deficit phenotype that is present in Cln3^Δex7/8 mice, suggesting that the Cln3^Δex5/7/8 isoform can compensate for loss of Cln3 that occurs in Cln3^Δex7/8 mice.

Fine motor control is intact in Cln3^Δex5/7/8 therapeutic model compared with CLN3 disease mice

Sensorimotor coordination is a known deficit in mouse models of Batten disease; however, fine motor skills have not been extensively assessed. String-pulling behavior to retrieve a food reward has previously revealed disruptions in both topographic and kinematic organization of fine motor skills in rats suffering focal cortical damage as well as those exposed to simulated space radiation.²¹^,²²^,²³ To test whether fine motor skills are affected in Cln3^Δex5/7/8 and Cln3^Δex7/8 mice, we implemented a string-pulling task modified from previous studies in rats (Video S1). Across all genotypes, 4-month-old mice had no significant differences in the time required to successfully retrieve the food reward at the end of the string (Figure S2). However, male Cln3^Δex7/8 disease mice required significantly more contacts with the string in order to retrieve the reward compared with all other groups (Figures 3A and 3B). This deficit was not seen in female mice. Irrespective of sex, Cln3^Δex7/8 mice also made significantly more misses when grabbing the string compared with all other groups (Figure 3C, Video S2). Though both sides were significantly affected, Cln3^Δex7/8 mice made more missed attempts when grabbing for the string with their right paw than their left (Figure 3C). Cln3^Δex7/8 mice also used their mouth or both paws simultaneously to pull on the string significantly more than all other groups, a behavior interpreted as compensatory for the aforementioned deficits (Figure 3D, Video S3). Across all three measures, Cln3^{Δex7/8/Δex5/7/8} and Cln3^Δex5/7/8 mice had fine motor skills similar to control mice, making significantly fewer mistakes and compensatory grabs than Cln3^Δex7/8 mice. Taken together, these data show that Cln3^Δex5/7/8, Cln3^{Δex7/8/Δex5/7/8}, and Cln3^+/Δex7/8 mice do not exhibit the fine motor skills deficit that is present in Cln3^Δex7/8 mice.

Video S1. Representative video of a Cln3^+/∆ex7/8 control mouse completing the string pull task

Download video file^{(8MB, mp4)}

Video S2. Representative video of a Cln3^∆ex7/8 mouse frequently missing when attempting to grab the string

Download video file^{(11.2MB, mp4)}

Video S3. Representative video of a Cln3^∆ex7/8 mouse using its mouth to pull the string with increased frequency

Download video file^{(7.8MB, mp4)}

Grip strength deficits seen in CLN3 disease mice are less severe in Cln3^Δex5/7/8 mice

Declines in handgrip strength are often considered a “vital sign” of aging in humans and it has been postulated that decreased grip strength is a sign of diminished neural and motor system capacity.²⁴^,²⁵^,²⁶^,²⁷ As such, the grip strength test has been used to evaluate motor function as well as cognitive deficits in rodent models of central nervous system (CNS) disorders.²⁸^,²⁹ To test for differences in strength between Cln3^Δex7/8 and Cln3^Δex5/7/8 mice, we measured forelimb grip strength at 4 months of age, using a sensitive, modified vertical grip test.³⁰ Across all genotypes, male mice had significantly greater average grip strength than female mice, and thus, male and female mice were assessed separately. Interestingly, only female Cln3^Δex7/8 mice had a significantly lower forelimb grip strength compared with control mice (Figure 4A), whereas male Cln3^Δex7/8 mice performed similarly to Cln3^+/Δex7/8 mice (Figure 4B). Female Cln3^Δex5/7/8 mice had significantly elevated grip strength compared with homozygous Cln3^Δex7/8 mice (Figure 4A). Regardless of sex, Cln3^Δex5/7/8 and Cln3^{Δex7/8/Δex5/7/8} mice both performed similarly to Cln3^+/Δex7/8 mice (Figures 4A and 4B). These data suggest that female Cln3^Δex5/7/8, Cln3^{Δex7/8/Δex5/7/8}, and Cln3^+/Δex7/8 mice do not exhibit the grip strength deficit that is present in Cln3^Δex7/8 mice.

Cln3^Δex5/7/8 mice do not exhibit anxiety-like behavior seen in CLN3 disease mice

There have been reports of generalized alterations in activity and anxiety-like behaviors in patients with CLN3 Batten disease,³¹ as well as altered open field behavior in Cln3^Δex7/8 mice.³² To test for this phenotype in the mice, we analyzed behavior in an open field arena (Figure 5A). The open field test is a sensorimotor task used to determine general activity levels, gross locomotor activity, exploration habits, and anxiety-like behavior in rodent models of CNS disorders.³³ At 3 months of age, there was no difference in global path and time spent in the center between Cln3^+/Δex7/8 control mice and Cln3^Δex7/8 disease mice (Figures 5B and 5C). However, homozygous Cln3^Δex7/8 mice made significantly fewer visits to the center of the open field than control or homozygous Cln3^Δex5/7/8 mice and both homozygous Cln3^Δex5/7/8 mice and heterozygous Cln3^{Δex7/8/Δex5/7/8} mice showed no difference from control Cln3^+/Δex7/8 mice (Figure 5D). These data reveal that Cln3^Δex5/7/8, Cln3^{Δex7/8/Δex5/7/8}, and Cln3^+/Δex7/8 mice do not have the anxiety-like behavioral phenotype that is present in Cln3^Δex7/8 mice.

Cln3^Δex5/7/8 mice have delayed histopathological signs of CLN3 Batten disease compared with Cln3^Δex7/8 disease mice

Having identified several measures of behavioral improvement in Cln3^Δex5/7/8 mice compared with Cln3^Δex7/8 mice, we next explored whether these improvements correlate with histopathological changes. An early pathological hallmark of CLN3 Batten disease is the accumulation of storage material, mainly composed of subunit C of mitochondrial ATP synthase (SCMAS), in the brains of patients with the disease and in animal models including Cln3^Δex7/8 mice.³⁴^,³⁵^,³⁶ To assess whether correction of the Cln3^Δex7/8 reading frame in the Cln3^Δex5/7/8 mice results in less SCMAS storage material, we quantitated SCMAS in mouse brains at postnatal day 70, an age when storage material is evident in Cln3^Δex7/8 mice.⁹ Cln3^Δex7/8 mice had elevated levels of SCMAS accumulation in the somatosensory (SS) cortex, thalamus, hippocampus, and visual cortex compared with control Cln3^+/Δex7/8 mice, whereas levels in homozygous Cln3^Δex5/7/8 mice were not significantly different from the control, non-disease mice (Figures 6A and S3A). Male homozygous Cln3^Δex5/7/8 mice had significantly lower SCMAS accumulation than homozygous Cln3^Δex7/8 mice in the somatosensory cortex and hippocampus, with an 81% and 89% reduction and large effect (1.75 and 1.32 standard deviations), respectively (Figure 6A, Table S1).

*Cln3*^Δex5/7/8 mice have less storage material accumulation early in disease compared with CLN3 disease model mice

Quantitative analysis of SCMAS in the somatosensory cortex (SS cortex), thalamus, hippocampus, visual cortex, and striatum of mice at age (A) p70 (B) p140, and (C) p360. For box and whisker plot: center line is the group median; limits 25th-75th percentile; whisker, Tukey. For separated scatterplot: center line is the individual mouse mean ± SEM; n = image fields per genotype. Mixed linear effects with Tukey’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, or non-significant (ns).

By postnatal day 140, SCMAS accumulation was significantly elevated in the somatosensory cortex, thalamus, and hippocampus of Cln3^Δex7/8 mice compared with Cln3^+/Δex7/8 (70%, 72%, and 83% higher, respectively; Figures 6B and S3B). In these same regions, Cln3^Δex5/7/8 mice did not have lower SCMAS compared with Cln3^Δex7/8 mice (Figures 6B and S3B, Table S1). By 12 months of age, SCMAS accumulation was significantly elevated across all five brain regions in Cln3^Δex7/8, Cln3^{Δex7/8/Δex5/7/8}, and Cln3^Δex5/7/8 mice (Figures 6C and S3C, Table S1). These data suggest that Cln3^Δex5/7/8 mice have less SCMAS accumulation in the CNS, in some brain regions at younger ages.

Pathology later in disease progression involves activated microglia and astrocytes in the CNS, which can be measured via immunoreactivity of cluster of differentiation protein 68 (CD68) and glial fibrillary acidic protein (GFAP), respectively. CD68 was significantly elevated in Cln3^Δex7/8 disease mice compared with controls. However, homozygous Cln3^Δex5/7/8 mice did not have significant reductions in CD68 compared with Cln3^Δex7/8 disease mice at any of the timepoints examined (Figures S4 and S5). Likewise, samples from postnatal day 70 mice did not have differences in GFAP levels, except in the thalamus, where female Cln3^Δex5/7/8 and Cln3^{Δex7/8/Δex5/7/8} mice both had GFAP levels indistinguishable from controls, but 76% and 82% lower than Cln3^Δex7/8 mice, respectively (Figures S6A and S7A). Similarly, in the visual cortex of postnatal day 140 Cln3^Δex5/7/8 mice, GFAP did not differ from control levels; however, it was significantly elevated in Cln3^Δex7/8 mice compared with Cln3^+/Δex7/8 mice (Figures S6B and S7B, Table S1). By 12 months of age GFAP was significantly elevated in the somatosensory cortex, thalamus, and visual cortex of Cln3^Δex7/8 mice as well as within the striatum of female Cln3^Δex7/8 mice (Figures S6C and S7C). GFAP was significantly lower in the striatum of female Cln3^Δex5/7/8 mice than Cln3^Δex7/8 mice and, regardless of sex, GFAP levels did not differ significantly from control levels in the striatum (Figure S6C).

Discussion

Here we report on in vivo genetic confirmation of a strategy to treat CLN3 Batten disease caused by a large frame-shifting deletion of exons 7 and 8 in the CLN3 gene (CLN3^Δex7/8), which is the most common disease-causing mutation. We created a mouse line that models the therapeutic strategy of deleting exon 5 in the context of the Cln3^Δex7/8 mutation. This Cln3^Δex5/7/8 mouse expresses a Cln3 gene lacking exons 5, 7, and 8, which has an intact open reading frame to the natural stop codon in exon 15. We have previously shown that inducing exon 5 skipping using a splice-switching ASO is therapeutic in mice lacking exons 7 and 8.⁹ These Cln3^Δex5/7/8 mice demonstrate an important proof-of-concept of the disease-sparing effects resulting from correcting the CLN3 open reading frame by skipping exon 5, supporting the clinical development of splice-switching ASOs to achieve this goal. Indeed, we find that the Cln3^Δex5/7/8 isoform ameliorates the effects of CLN3 Batten disease compared with Cln3^Δex7/8 mice, validating our previous findings that SSO-mediated reading frame correction can treat features of this lysosomal storage disease.

Reading frame corrected Cln3^Δex5/7/8 mice show significant improvements compared with the disease model Cln3^Δex7/8 mice across multiple behavioral and histological measures, though most improvements were observed at younger ages. Since the Cln3^Δex7/8 mice do not have a severely shortened lifespan that is seen in patients with the disease, survival was not assessed.³⁶ At 3 to 4 months of age, Cln3^Δex5/7/8 mice showed improvements across all behavioral measures compared with Cln3^Δex7/8 mice (Figure 2, Figure 3, Figure 4, Figure 5). Similarly, at 70 days of age, SCMAS accumulation in Cln3^Δex5/7/8 male mice was significantly lower than that of Cln3^Δex7/8 in the SS cortex and hippocampus (Figure 6). However, by 140 days, Cln3^Δex5/7/8 mice had elevated SCMAS accumulation, more similar to Cln3^Δex7/8 mice. Though it remains possible that these improvements stem from a loss of Cln3^Δex7/8 as a possible dominant negative isoform, rather than a rescue of function by correcting the Cln3 open reading frame, this is unlikely given that Cln3-KO mice exhibit similar deficits to Cln3^Δex7/8 mice.³⁷^,³⁸^,³⁹ Nonetheless, this therapeutic approach would be effective in either situation, by both decreasing the CLN3^Δex7/8 isoform and increasing the reading frame corrected isoform. These findings suggest that skipping of exon 5 and correcting the Cln3^Δex7/8 open reading frame may result in a protracted disease course, similar to that seen in patients with missense mutations.¹¹^,¹²

Though the elevated SCMAS reactivity at later ages suggests that the deletion of exon 5 and correction of the open reading frame may only slow and not completely prevent accumulation of SCMAS, this pathology may not be an accurate indicator of disease. Recent work has shown that prevention of storage material accumulation is not required for correcting neuronal network physiology in CLN3 Batten disease.⁴⁰ Thus, SCMAS accumulation may not be a conclusive metric for assessing disease progression and therapeutic efficacy. Our results support this possibility in that our Cln3^Δex5/7/8 mice showed significant phenotypic improvement on the grip strength and string-pull behavioral assessments despite exhibiting elevated SCMAS just 2 weeks later (Figures 3, 4, and 6).

In addition to validating the therapeutic benefit of exon 5 skipping, this study reveals a disease phenotype in Cln3^Δex7/8 mice related string-pulling behavior that has not been previously reported (Figure 3). The results of the string-pull test and grip strength test may be indicative of proprioceptive deficits in Cln3^Δex7/8 mice. Kinematic analysis of the string-pulling behavior did not reveal overt differences in reach and withdrawal movements or in the average peak speed and average distance traveled of the paws and nose (data not shown). However, analysis of pulling accuracy and efficiency revealed that the number of misses and engagement of mouth and double-paw grabs were significantly different across genotypes.

Proprioceptive deficits have been seen in neurodegeneration models of CLN5 Batten disease,⁴¹ multiple sclerosis,⁴² Friedrich ataxia,⁴³ and Charcot-Marie-Tooth disease,⁴⁴ but to our knowledge have not yet been documented in CLN3 Batten disease. However, postmortem investigations of patients with CLN3 Batten have shown atrophy of the cerebral hemispheres, cerebellum, and brain stem along with demyelination and axonal loss within peripheral nerves.⁴⁵ If Cln3^Δex7/8 mice experience a similar peripheral neuropathy to that observed in human patients, it could result in numbness, which could contribute to proprioceptive deficits like those observed here. Further investigation into sensory nerve function and gait analysis would help determine if these deficits are in fact proprioceptive in nature or if they are being caused by another neuropathy.⁴⁶

Our results also provide insights into the amount of exon 5 skipping that might be necessary to see a therapeutic effect with an SSO, such as the one we have previously described.⁹ While Cln3^{Δex7/8/Δex5/7/8} and homozygous Cln3^Δex5/7/8 mice both showed improvements in their overall average for rotarod, grip strength, and open field test assessments, these improvements were only statistically significant in homozygote Cln3^Δex5/7/8 mice (Figures 2,4, and 5). The only exception was the string pull, in which both Cln3^{Δex7/8/Δex5/7/8} and Cln3^Δex5/7/8 mice were significantly better at the task than the Cln3^Δex7/8 mice, though having two copies of Cln3^Δex5/7/8 was associated with better performance. In our previous study, Cln3^Δex7/8 mice treated with an exon 5 skipping SSO showed significant behavioral and histopathological improvements over control SSO-treated mice, and splicing analysis indicated 60%–80% exon 5 skipping at the time of analysis.⁹ Taken together, these data suggest that the threshold for therapeutic efficacy may occur with ≥50% exon 5 skipping in mice.

In addition to these gene dosage differences, we also found differences in disease severity between sexes (Figures 3, 4, 6, S4, and S6). While both male and female Cln3^Δex7/8 mice had significant deficits in the string-pull behavioral assessment at 4 months of age, male Cln3^Δex7/8 mice showed more severe deficits, requiring a greater overall number of grabs to retrieve the food reward at the end of the string (Figure 3). This sexual dimorphism in fine motor skill deficit could be the result of earlier disease onset in male Cln3^Δex7/8 mice, as suggested by the more severe SCMAS accumulation compared with females in the somatosensory cortex and hippocampus at p70, and is similar to reports of earlier disease onset seen in male patients.⁷ Conversely, at 3 months of age, female Cln3^Δex7/8 mice had a significant decrease in forelimb grip strength, whereas male Cln3^Δex7/8 mice showed no such deficit (Figure 4). These results are reminiscent of human disease, with reports that male patients lose physical function later than female patients, possibly due to a lower baseline muscle mass in females or differences in sex hormones, where testosterone has been shown to serve a protective role against muscle atrophy.⁷^,⁴⁷^,⁴⁸

The Cln3^Δex7/8 mice also exhibited sexual dimorphism in histopathology. At postnatal day 70, male Cln3^Δex7/8 mice exhibited significantly more SCMAS accumulation than female mice in the somatosensory cortex, hippocampus, and visual cortex (Figure 6). However, at postnatal day 140, there were no differences between the sexes. This result is consistent with disease onset and progression in patients with CLN3 Batten disease, where males typically present with disease symptoms earlier, but females eventually catch up in severity.⁶^,⁷ Conversely, at postnatal day 70, female Cln3^Δex7/8 mice had significantly higher microglial immunoreactivity than their male counterparts in the visual cortex and striatum (Figure S4).

Sex differences in autoimmune disorders are well-documented, with females being nine times more likely to be affected by a systemic immune disorder than males.⁴⁹ Indeed, even in healthy individuals, females have increased immunoreactivity, an increased number of circulating antibodies, and a higher number of T cells.⁴⁹ By postnatal day 140 and beyond, it is possible that these sex differences disappear due to the overall severity of the disease course, regardless of sex. Differences in histology and behavior based on sex are also reported in a CLN6 Batten disease mouse model where males have higher autofluorescent storage material at 2 months of age and increased reactive gliosis; however, female mice perform worse on behavioral tests and have a decreased lifespan compared with males.⁵⁰ Additionally, in the CLN8 Batten disease mouse model, females demonstrate more severe histopathology, behavioral deficits, and shorter lifespan compared with male mice.⁵¹ Sex differences have also been shown previously in Cln3^Δex7/8 mice.¹⁹^,⁵² These differences in disease progression and treatment response are consistent with the sex differences we observed in behavior and histopathology. Sex differences are not unique to Batten disease and are also found in other neurodegenerative disease, such as Alzheimer’s disease.⁵³ Given these documented differences in disease onset and progression, it will be important to consider such variables when evaluating individual patient responses to future therapies in the clinic.

When seeking to develop novel interventions for a debilitating disease, it is critical to explore multiple therapeutic avenues given that a single drug can rarely serve as the “silver bullet” intervention for all patients. Along these lines, a phase I/IIa clinical trial for adeno-associated virus (AAV)-mediated gene therapy in CLN3 Batten disease is ongoing (NCT03770572).⁵⁴ Comparing our treatment modality with the available preclinical AAV data, we note that the therapeutic outcomes reported in the current study are similar to those previously seen in Cln3^Δex7/8 mice treated with hCLN3 gene therapy. Bosch et al. demonstrated that systemic delivery of scAAV9/MeCP2-hCLN3 reverses motor deficits in Cln3^Δex7/8 mice, similar to our own rotarod and string-pull data.⁵⁵ When evaluating histopathology 5 months post-injection, they saw only trends toward lower SCMAS accumulation and astrocyte activation in the thalamus and somatosensory cortex.⁵⁵

As we have shown, skipping exon 5 to correct the Cln3 open reading frame has a therapeutic efficacy in mice similar to that of AAV-mediated CLN3 gene therapy, which has already been approved by the Food and Drug Administration for clinical trials. However, two of the major obstacles facing CLN3 gene therapy are proper gene dosing and ubiquitous expression throughout the CNS. Previous studies have shown that low CLN3 expression is required for cellular homeostasis,⁵⁶^,⁵⁷ and that overexpression can be non-therapeutic⁵⁵ or toxic.⁵⁸ Additionally, while the use of the scAAV9 serotype is attractive due to its ability to cross the blood-brain barrier, this serotype is ineffective at transducing microglia.⁵⁹^,⁶⁰ SSOs avoid some of these challenges in that they target and corrects endogenous CLN3 expression and SSOs are taken up broadly across the CNS.⁶¹

The critical age for therapeutic intervention necessary for disease impact is an important consideration for the development of any therapeutic strategy aimed at treating CLN3 Batten disease. In the case of CLN2 Batten disease, treatment with cerliponase alfa (Brineura) has been shown to attenuate but likely does not reverse disease progression, making early intervention essential for optimal treatment outcomes.⁶² Importantly, the Cln3^Δex5/7/8 mouse model allows some evaluation of the potential therapeutic benefit of prenatal expression of the Cln3^Δex5/7/8 isoform as opposed to postnatal treatment. Though the comparisons are indirect, the phenotypic rescue seen in Cln3^{Δex7/8/Δex5/7/8} and homozygote Cln3^Δex5/7/8 mice does not appear to be dramatically improved over that seen in Cln3^Δex7/8 mice treated with an exon 5 skipping SSO shortly after birth.⁹ This result suggests that prenatal SSO treatment may not be necessary to have therapeutic value.

Taken together, the results from this study demonstrate a gene dose effect between the Cln3^Δex5/7/8 allele and disease mitigation, validating our previous report that open reading frame correction is therapeutic in a mouse model of CLN3 Batten disease. In particular, this therapeutic approach may result in a protracted disease course, with beneficial effects of open reading frame correction seen earlier in disease progression. Male patients may also benefit from earlier therapeutic intervention due to their observed earlier disease onset. However, with the less severe disease burden in Cln3^Δex7/8 mice compared with that seen in human patients with the same mutation, it remains possible that any improvements seen in this mouse model would be magnified in patients receiving a similar therapy. Overall, this study supports the use of SSO-induced reading frame correction for the treatment of CLN3 Batten disease.

Material and methods

Plasmids

CLN3 expression plasmids were constructed by amplifying CLN3 WT, CLN3^Δex7/8 and CLN3^Δex5/7/8 cDNA from previously described expression plasmids.⁹ PCR was performed to amplify the inserts and add restriction sites using Q5 DNA polymerase (New England Biolabs, Ipswich, MA) and primers specific for CLN3 exon 1 with a restriction enzyme cleavage site for XhoI and a Kozak sequence (5′-TGCGCTCGAGGCCACCATGGGAGGCTGTGCAGG-3′) and for CLN3 exon 15 with a NotI restriction enzyme site (5′-CAGAATGCGGCCGCTCAGGAGAGCTGGCAGAGGAA-3′). The resulting products were purified, digested with XhoI and NotI, and ligated into similarly digested pCI plasmid using T4 DNA ligase (New England Biolabs, Ipswich, MA). Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) was used to insert the 3xFLAG sequence using forward primer (5′-ACGACGATAAGGATTACAAGGATGACGACGATAAGGGAGGCTGTGCAGGCTCG-3′) and reverse primer (5′-ATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCCATGGTGGCCTCGAGGCTAG-3′) to generate pCI-3xFLAG-CLN3, pCI-3xFLAG-CLN3^Δex7/8, and pCI-3xFLAG-CLN3^Δex5/7/8. All plasmids were sequenced to confirm construction.

Cell culture and transfection

U2OS cells were cultured in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% carbon dioxide. Cells were seeded on coverslips 1 day prior to transfection. One microgram of pCI-3xFLAG-CLN3 WT, pCI-3xFLAG-CLN3^Δex7/8, or pCI-3xFLAG-CLN3^Δex5/7/8 was transiently transfected using Lipofectamine 3000 (Life Technologies, Carlsbad, CA) and incubated with cells for 42 h prior to collection.

Immunoblot analysis

Transfected cells were lysed in 150 mM NaCl, 50 mM Tris-Cl pH 7.6, 1% NP-40, 0.5% Na-DOC, 0.1% SDS, with protease inhibitor cocktail and cleared by centrifugation at 10,000 × g for 20 min at 4°C. Protein concentration was determined by Bradford Assay (1863028, Thermo Fisher Scientific, Waltham, MA) and sonicated. Lysates (20 μg per lane) in NuPAGE lithium dodecyl sulfate 4x sample buffer (NP0007, Invitrogen, Carlsbad, CA) with NuPAGE sample reducing agent (NP0009, Invitrogen, Carlsbad, CA) were heated at 37°C for 20 min and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% Bis-Tris gels and transferred to 0.45 μm polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat dry milk. The blot was probed overnight at 4°C with primary antibodies against FLAG (1:2,000, F3165; Sigma) and β-catenin (1:1,000, 610153, BD Biosciences, Franklin Lakes, NJ) diluted in blocking solution. Horseradish peroxidase-conjugated secondary antibody (32230, Invitrogen, Carlsbad, CA) diluted in blocking solution was incubated with the membrane at room temperature for 1–3 h and protein was detected by chemiluminescence.

Mice

All experimental procedures with mice were performed under protocols approved by the Institutional Animal Care and Use Committee of Rosalind Franklin University (RFUMS). Animals were maintained at 19°C with a 12 h light/dark cycle with food and water available ad libitum. Cln3^Δex7/8 mice were obtained from The Jackson Laboratory [B6.129(Cg)- Cln3tm1.1Mem/J, stock number 017895, Bar Harbor, ME]. Mice were bred at RFUMS and pups were weaned at postnatal day 21, at which time they were genotyped by extraction of DNA from ear tissue using Red Extract-N-Amp (Sigma-Aldrich, Saint Louis, MO) followed by PCR with mouse Cln3 primers specific for WT (forward 5′-GCCTTCTACCCCAGGTAAGC-3′ and reverse 5′-CAGGGATCCCCAACATAGAA-3′), Cln3^Δex7/8 (forward 5′-TTTGTTCTGCTGGGAGCTTT-3′ and reverse 5′-CAGTCTCTGCCTCGTTTTCC-3′), and Cln3^Δex5/7/8 (forward 5′-CAACTCCATCTCCACAGC-3′ and reverse 5′-ACTCTCCAGACTCCCGCTTC-3′).

The Cln3^Δex5/7/8 mouse model was generated using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology (Applied StemCell, Inc., San Francisco, CA). A cocktail of guide RNAs targeting intron 4/exon 5 (5′-CTAGGAGCACCGCCTGAGAC-3′) and exon 5/intron 5 (5′-ACTTGCTGCCTTACAGGTCT-3′) and CRISPR-associated protein (Cas) 9 mRNA were injected into the cytoplasm of Cln3^Δex7/8 embryos³⁶ and implanted into surrogate mice. Progeny were screened for knockout of Cln3 exon 5 using forward primer 5′-ACCCCACGATCCTTGCCTTT-3′ and reverse 5′-GGGAGACTACAGCACATCACTCTG-3′ and confirmed by sequencing.

To assess therapeutic efficacy, the Cln3^Δex5/7/8 mouse model was compared with the previously characterized Cln3^Δex7/8 mouse model, which has been shown to have sensorimotor coordination deficits and histopathological signs including storage material accumulation and gliosis in the brain.³⁶^,⁶³^,⁶⁴ Because CLN3 Batten disease is recessively inherited, mice heterozygous for the deletion of exons 7 and 8 are unaffected by the disease, and thus Cln3^+/Δex7/8 littermates were used as the control group.

RNA isolation and analysis

Mice were anesthetized with urethane and underwent transcardial perfusion with phosphate-buffered saline (PBS). Tissue was extracted and flash frozen in liquid nitrogen. Hippocampal tissue was homogenized in TRIzol reagent (Life Technologies) and RNA was isolated according to the manufacturer’s protocol. RNA was reverse transcribed using GoScript reverse transcriptase (Promega, Madison, WI) and oligo-dT primer. Radiolabeled PCR of the cDNA was performed using GoTaq Green (Promega) with α-³²P-deoxycytidine triphosphate, and primers specific for mouse Cln3: exon 4 forward (5′-CAACTCCATCTCCACAGC-3′) and exon 10 reverse (5′-AGAGGTCCCAGCTGGCAC-3′). PCR products were separated on a 6% nondenaturing polyacrylamide gel.

Open field test

Locomotion, anxiety-like behavior, and spatial processing were assessed in 3-month-old mice using an open field test. Overhead lighting was dimmed in the behavior room, and mice were acclimated to the room for 1 h pre-assessment. Individual mice were placed in the center of the open field chamber (a 54 × 54-cm white acrylic square box with 20-cm-high walls on all sides) and digitally recorded for 10 min using VideoMot2 (TSE Systems GmbH, Bad Homburg, Germany). All mice were assessed at age postnatal day 90 ± 3 days. Assessments were performed during the morning hours of the light cycle (9:00 AM–11:30 AM).

Accelerating rotarod

Motor coordination in 3-month-old mice was determined using an accelerating rotarod (Rotamex-5, Columbus Instruments, Columbus, OH) with a rotation speed that started at 4 revolutions per minute (rpm) and increased to 48 rpm over 240 s with an acceleration of 0.4 rpm/s, for each trial. During the morning of the light cycle, mice were habituated to the testing room for 10 min then tested in three sets of trials consisting of two consecutive runs with a 15-min rest between trials. The latency to passive rotation (rotate once around the rod without falling) and latency to fall from the rod was digitally recorded and was averaged for each mouse from the six trials.

String pulling

Fine motor control in 111-day-old mice was determined using a string-pulling behavioral assessment. Two days prior to the string-pull assessment, a high-value food reward (Honey Nut Cheerios, General Mills, Golden Valley, MN) was presented in the home cage of each mouse to give the mice a chance to familiarize themselves with the intended reward and overcome any possible neophobia they might experience from encountering a novel food item. Twenty-four hours prior to string-pulling assessment, mice were individually placed into standard housing cages that had been pre-prepared with 20 strings varying in length from 30 cm to 100 cm, half of which were baited with the previously acclimated food reward (Honey Nut Cheerio). After 1 h, the number of strings each mouse pulled fully into their cage was recorded, and mice were returned to their home cages. One hour prior to the assessment, mice were habituated to the behavioral room. For the string-pull assessment, mice were placed individually inside a 12.5-cm × 7.5-cm × 9-cm clear plastic chamber with a single, 100-cm, baited string. Mice were assessed during three consecutive trials of 15 min each or until the Cheerio was retrieved, whichever occurred first. Trials were recorded using a high-speed camera (GoPro HERO8 Black, GoPro Inc., San Mateo, CA). Successful reward retrieval, latency to the first bout of string pulling, latency to time of reward retrieval, hand contacts, hand misses, mouth contacts, and mouth misses were scored by hand and reported as the average across two trials.

Grip strength

A grip strength meter (Columbus Instruments, Columbus, OH) was used to measure forelimb grip strength in 115 ± 3-day-old mice according to a published protocol.³⁰ As a mouse grasped the bar, the peak pull force in ounces was recorded on a digital force transducer. Mice were allowed to grasp a bar on the vertically mounted force gauge and the tail was slowly pulled downward by a researcher. Peak grip strength was recorded by the gauge as the maximum force exerted prior to a mouse releasing its forepaws from the bar. Five consecutive measurements were performed at 1-min intervals. Test sessions were performed during the afternoon hours of the light cycle (12:00 PM–1:00 PM).

Histology

Mice were anesthetized with 100 mg/mL urethane and perfused transcardially using PBS. Brains were isolated and one hemisphere was drop fixed in 4% paraformaldehyde for 48 h before being stored in 30% sucrose at 4°C. Brain tissue was sectioned with a Leica SM 2010R microtome or a Leica VT1000S vibratome (Leica Microsystems Inc., Wetzlar, Germany) in the sagittal plane at a thickness of 50 μm.

Immunohistochemistry was performed on free-floating tissue slices using two to three slices per animal for each antibody. Slices were washed in 1% hydrogen peroxide in Tris-buffered saline (TBS) (20 mM Tris, 150 mM sodium chloride) for at least 15 min followed by three 5-min washes in TBS. Non-specific protein binding was blocked using 15% normal serum in TBS-T (TBS with 0.03% Triton X-100) for 30 min. Slices were incubated overnight at 4°C in primary antibodies against ATP synthase subunit C (SCMAS; 1:1,000, ab181243, Abcam, Cambridge, MA), CD68 (1:2,000, MCA1957, BioRad AbD Serotec, Raleigh, NC), or GFAP (1:8,000, Z0334, Dako, Carpinteria, CA) diluted in TBS-T + 10% goat serum. Slices were washed 3 × 5 min in TBS followed by a 2-h incubation in the appropriate biotinylated secondary antibodies, anti-rabbit and anti-rat (1:1,000, BA-1000, BA-9400, Vector Labs, Burlingame, CA) diluted in TBS-T + 10% goat serum. Slices were washed 3 × 5 min in TBS and incubated for 2 h in ABC reagent (Vector Labs). The slices were washed 3 × 5 min in TBS and then incubated in 0.05% DAB solution (D5905, Sigma-Aldrich), washed 3 × 5 min in TBS, mounted on slides, incubated in xylene for 10 min before applying a coverslip using DPX (BDH 36029 4H, Poole, England). Sections were imaged and analyzed using an Aperio AT2 Digital Pathology Slide Scanner (Leica Microsystems Inc.) and associated software. Threshold analysis was performed to quantify SCMAS, GFAP, and CD68 immunoreactivity in ImageJ.

Statistical analyses

To determine the sample size for behavioral tests, a priori power analysis was conducted using G∗Power⁶⁵ based on data from pilot and published studies⁹^,²¹ using alpha of 0.5, power of at least 0.80, and a medium to large effect size according to Cohen’s (1988) criteria. Statistical analyses of animal behavior were performed using Prism 9 (GraphPad Software, Inc, San Diego, CA). Two-way ANOVA was performed to determine whether or not there was a significant interaction between sex and genotype. When a significant sex interaction was found (p < 0.05), data were separated on the basis of sex. When no significant sex-genotype interaction was found, male and female animal data were grouped together for analysis. Once sex effects were determined, data were analyzed using one-way ANOVA along with Dunnett’s multiple comparisons test. Due to data nesting, immunohistochemistry data were analyzed with R 4.2.1 (R Core Team, Vienna, Austria) using linear mixed effects modeling and the lme4 package.⁶⁶ All models were estimated using Restricted Maximum Likelihood estimation, but model comparisons were conducted using traditional maximum likelihood estimation. Data were parametrically bootstrapped to account for non-normality and fit to three separate models: one accounting for sex-genotype interactions, one accounting for a main sex effect, and one examining the effect of genotype alone. The model of best fit was utilized for subsequent Tukey’s post hoc analysis. Analysis details are noted in the figure legends. Data are expressed in histograms as mean ± SEM. In box and whisker plots, boxes represent median ± 25^th-75^th percentile and whiskers represent Tukey whiskers. Differences were designated significant with p < 0.05. Measures of effect size can facilitate assessment of how large or small an observed effect could actually be in a population of interest, and therefore how clinically important it could be.⁶⁷^,⁶⁸^,⁶⁹ Thus, to better assess potential therapeutic benefit, effect sizes were calculated for all comparisons in addition to statistical hypothesis testing (Table S2).

Data availability

The data underlying this article are available in the article and in its online supplemental material.

Acknowledgments

The authors thank Estela Viera for technical support and members of the Hastings lab for stimulating discussions. This work was supported by the National Institutes of Health [NS113233], the Batten Disease Support and Research Association, and the ForeBatten Research Foundation. Quantitation of radioactive PCR products was performed on an instrument in the shared Proteomics facility at RFUMS obtained with support from the National Institutes of Health [S10 OD010662].

Author contributions

J.L.C.: Conceptualization; data curation; formal analysis; investigation; project administration; validation; visualization; writing – original draft; writing – review & editing; M.P.S.: Conceptualization; data curation; formal analysis; investigation; project administration; software; validation; visualization; writing – original draft; writing – review & editing; M.A.P.: data curation; formal analysis; validation; J.R.O.O.: data curation; formal analysis; methodology; software; validation; writing – review & editing; D.G.W.: formal analysis; methodology; resources; software; validation; S.A.M.: formal analysis; resources; software; validation; writing – review & editing; J.M.W.: Conceptualization; funding acquisition; resources; writing – review & editing; M.L.H.: Conceptualization; funding acquisition; project administration; resources; supervision; writing – original draft; writing – review & editing.

Declaration of interests

M.L.H. is an inventor on patents on SSOs filed by RFUMS and Ionis Pharmaceuticals and may be entitled to benefits from licensing of the associated intellectual property. J.M.W is an employee of Amicus Therapeutics, Inc. and holds equity in the company in the form of stock-based compensation; Amicus had no input into this piece of work.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.omtn.2023.05.025.

Supplemental information

Document S1. Figures S1–S7 and Table S2

mmc1.pdf^{(9.3MB, pdf)}

Table S1. The excel document “Table S1” incudes the effect sizes for the immunohistochemistry, separated by target protein (tabs 1–3)

mmc2.xlsx^{(38.1KB, xlsx)}

Document S2. Article plus supplemental information

mmc6.pdf^{(13.1MB, pdf)}

References

1.Munroe P.B., Mitchison H.M., O'Rawe A.M., Anderson J.W., Boustany R.M., Lerner T.J., Taschner P.E., de Vos N., Breuning M.H., Gardiner R.M., Mole S.E. Spectrum of mutations in the Batten disease gene, CLN3. Am. J. Hum. Genet. 1997;61:310–316. doi: 10.1086/514846. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Sleat D.E., Gedvilaite E., Zhang Y., Lobel P., Xing J. Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis. Gene. 2016;593:284–291. doi: 10.1016/j.gene.2016.08.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Masten M.C., Williams J.D., Vermilion J., Adams H.R., Vierhile A., Collins A., Marshall F.J., Augustine E.F., Mink J.W. The CLN3 Disease Staging System: a new tool for clinical research in Batten disease. Neurology. 2020;94:e2436–e2440. doi: 10.1212/WNL.0000000000009454. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Ostergaard J.R., Rasmussen T.B., Mølgaard H. Cardiac involvement in juvenile neuronal ceroid lipofuscinosis (Batten disease) Neurology. 2011;76:1245–1251. doi: 10.1212/WNL.0b013e31821435bd. [DOI] [PubMed] [Google Scholar]
5.Mink J.W., Augustine E.F., Adams H.R., Marshall F.J., Kwon J.M. Classification and natural history of the neuronal ceroid lipofuscinoses. J. Child Neurol. 2013;28:1101–1105. doi: 10.1177/0883073813494268. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Nielsen A.K., Østergaard J.R. Do females with juvenile ceroid lipofuscinosis (Batten disease) have a more severe disease course? The Danish experience. Eur. J. Paediatr. Neurol. 2013;17:265–268. doi: 10.1016/j.ejpn.2012.10.011. [DOI] [PubMed] [Google Scholar]
7.Cialone J., Adams H., Augustine E.F., Marshall F.J., Kwon J.M., Newhouse N., Vierhile A., Levy E., Dure L.S., Rose K.R., et al. Females experience a more severe disease course in Batten disease. J. Inherit. Metab. Dis. 2012;35:549–555. doi: 10.1007/s10545-011-9421-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Miller J.N., Chan C.H., Pearce D.A. The role of nonsense-mediated decay in neuronal ceroid lipofuscinosis. Hum. Mol. Genet. 2013;22:2723–2734. doi: 10.1093/hmg/ddt120. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Centa J.L., Jodelka F.M., Hinrich A.J., Johnson T.B., Ochaba J., Jackson M., Duelli D.M., Weimer J.M., Rigo F., Hastings M.L. Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease. Nat. Med. 2020;26:1444–1451. doi: 10.1038/s41591-020-0986-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Kida E., Kaczmarski W., Golabek A.A., Kaczmarski A., Michalewski M., Wisniewski K.E. Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro. Mol. Genet. Metabol. 1999;66:265–271. doi: 10.1006/mgme.1999.2837. [DOI] [PubMed] [Google Scholar]
11.Kousi M., Lehesjoki A.E., Mole S.E. Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses. Hum. Mutat. 2012;33:42–63. doi: 10.1002/humu.21624. [DOI] [PubMed] [Google Scholar]
12.Wang F., Wang H., Tuan H.F., Nguyen D.H., Sun V., Keser V., Bowne S.J., Sullivan L.S., Luo H., Zhao L., et al. Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements. Hum. Genet. 2014;133:331–345. doi: 10.1007/s00439-013-1381-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Le B.T., Agarwal S., Veedu R.N. Evaluation of DNA segments in 2'-modified RNA sequences in designing efficient splice switching antisense oligonucleotides. RSC Adv. 2021;11:14029–14035. doi: 10.1039/d1ra00878a. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Nilsen T.W., Graveley B.R. Expansion of the eukaryotic proteome by alternative splicing. Nature. 2010;463:457–463. doi: 10.1038/nature08909. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Yang P., Wang D., Kang L. Alternative splicing level related to intron size and organism complexity. BMC Genom. 2021;22:853. doi: 10.1186/s12864-021-08172-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Marasco L.E., Kornblihtt A.R. The physiology of alternative splicing. Nat. Rev. Mol. Cell Biol. 2023;24:242–254. doi: 10.1038/s41580-022-00545-z. [DOI] [PubMed] [Google Scholar]
17.Cunningham F., Allen J.E., Allen J., Alvarez-Jarreta J., Amode M.R., Armean I.M., Austine-Orimoloye O., Azov A.G., Barnes I., Bennett R., et al. Ensembl 2022. Nucleic Acids Res. 2022;50:D988–D995. doi: 10.1093/nar/gkab1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.O'Leary N.A., Wright M.W., Brister J.R., Ciufo S., Haddad D., McVeigh R., Rajput B., Robbertse B., Smith-White B., Ako-Adjei D., et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44:D733–D745. doi: 10.1093/nar/gkv1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kovács A.D., Pearce D.A. Finding the most appropriate mouse model of juvenile CLN3 (Batten) disease for therapeutic studies: the importance of genetic background and gender. Dis. Model. Mech. 2015;8:351–361. doi: 10.1242/dmm.018804. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Finn R., Kovács A.D., Pearce D.A. Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Deltaex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis. Neurochem. Int. 2011;58:648–655. doi: 10.1016/j.neuint.2011.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Blackwell A.A., Schell B.D., Osterlund Oltmanns J.R., Whishaw I.Q., Ton S.T., Adamczyk N.S., Kartje G.L., Britten R.A., Wallace D.G. Skilled movement and posture deficits in rat string-pulling behavior following low dose space radiation ((28)Si) exposure. Behav. Brain Res. 2021;400 doi: 10.1016/j.bbr.2020.113010. [DOI] [PubMed] [Google Scholar]
22.Whishaw I.Q., Pellis S.M., Gorny B.P., Pellis V.C. The impairments in reaching and the movements of compensation in rats with motor cortex lesions: an endpoint, videorecording, and movement notation analysis. Behav. Brain Res. 1991;42:77–91. doi: 10.1016/s0166-4328(05)80042-7. [DOI] [PubMed] [Google Scholar]
23.Blackwell A.A., Banovetz M.T., Qandeel, Whishaw I.Q., Whishaw I.Q., Wallace D.G. The structure of arm and hand movements in a spontaneous and food rewarded on-line string-pulling task by the mouse. Behav. Brain Res. 2018;345:49–58. doi: 10.1016/j.bbr.2018.02.025. [DOI] [PubMed] [Google Scholar]
24.Cui M., Zhang S., Liu Y., Gang X., Wang G. Grip strength and the risk of cognitive decline and dementia: a systematic review and meta-analysis of longitudinal cohort studies. Front. Aging Neurosci. 2021;13 doi: 10.3389/fnagi.2021.625551. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Kunutsor S.K., Isiozor N.M., Voutilainen A., Laukkanen J.A. Handgrip strength and risk of cognitive outcomes: new prospective study and meta-analysis of 16 observational cohort studies. Geroscience. 2022;44:2007–2024. doi: 10.1007/s11357-022-00514-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Duchowny K.A., Ackley S.F., Brenowitz W.D., Wang J., Zimmerman S.C., Caunca M.R., Glymour M.M. Associations between handgrip strength and dementia risk, cognition, and neuroimaging outcomes in the UK biobank cohort study. JAMA Netw. Open. 2022;5 doi: 10.1001/jamanetworkopen.2022.18314. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Herold F., Labott B.K., Grässler B., Halfpaap N., Langhans C., Müller P., Ammar A., Dordevic M., Hökelmann A., Müller N.G. A link between handgrip strength and executive functioning: a cross-sectional study in older adults with mild cognitive impairment and healthy controls. Healthcare (Basel) 2022;10 doi: 10.3390/healthcare10020230. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Bohannon R.W. Muscle strength: clinical and prognostic value of hand-grip dynamometry. Curr. Opin. Clin. Nutr. Metab. Care. 2015;18:465–470. doi: 10.1097/MCO.0000000000000202. [DOI] [PubMed] [Google Scholar]
29.Carson R.G. Get a grip: individual variations in grip strength are a marker of brain health. Neurobiol. Aging. 2018;71:189–222. doi: 10.1016/j.neurobiolaging.2018.07.023. [DOI] [PubMed] [Google Scholar]
30.Takeshita H., Yamamoto K., Nozato S., Inagaki T., Tsuchimochi H., Shirai M., Yamamoto R., Imaizumi Y., Hongyo K., Yokoyama S., et al. Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice. Sci. Rep. 2017;7 doi: 10.1038/srep42323. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Honingh A.K., Kruithof Y.L., Kuper W.F.E., van Hasselt P.M., Sterkenburg P.S. Towards understanding behaviour and emotions of children with CLN3 disease (batten disease): patterns, problems and support for child and family. Int. J. Environ. Res. Publ. Health. 2022;19 doi: 10.3390/ijerph19105895. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Jana M., Dutta D., Poddar J., Pahan K. Activation of PPARalpha exhibits therapeutic efficacy in a mouse model of juvenile neuronal ceroid lipofuscinosis. J. Neurosci. 2023;43:1814–1829. doi: 10.1523/JNEUROSCI.2447-21.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Seibenhener M.L., Wooten M.C. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J. Vis. Exp. 2015:e52434. doi: 10.3791/52434. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Rowan S.A., Lake B.D. Tissue and cellular distribution of subunit c of ATP synthase in Batten disease (neuronal ceroid-lipofuscinosis) Am. J. Med. Genet. 1995;57:172–176. doi: 10.1002/ajmg.1320570213. [DOI] [PubMed] [Google Scholar]
35.Tyynelä J., Cooper J.D., Khan M.N., Shemilts S.J.A., Haltia M. Hippocampal pathology in the human neuronal ceroid-lipofuscinoses: distinct patterns of storage deposition, neurodegeneration and glial activation. Brain Pathol. 2004;14:349–357. doi: 10.1111/j.1750-3639.2004.tb00077.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Cotman S.L., Vrbanac V., Lebel L.A., Lee R.L., Johnson K.A., Donahue L.R., Teed A.M., Antonellis K., Bronson R.T., Lerner T.J., MacDonald M.E. Cln3(Deltaex7/8) knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth. Hum. Mol. Genet. 2002;11:2709–2721. doi: 10.1093/hmg/11.22.2709. [DOI] [PubMed] [Google Scholar]
37.Katz M.L., Shibuya H., Liu P.C., Kaur S., Gao C.L., Johnson G.S. A mouse gene knockout model for juvenile ceroid-lipofuscinosis (Batten disease) J. Neurosci. Res. 1999;57:551–556. [PubMed] [Google Scholar]
38.Mitchison H.M., Bernard D.J., Greene N.D., Cooper J.D., Junaid M.A., Pullarkat R.K., de Vos N., Breuning M.H., Owens J.W., Mobley W.C., et al. Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected] Neurobiol. Dis. 1999;6:321–334. doi: 10.1006/nbdi.1999.0267. [DOI] [PubMed] [Google Scholar]
39.Pontikis C.C., Cella C.V., Parihar N., Lim M.J., Chakrabarti S., Mitchison H.M., Mobley W.C., Rezaie P., Pearce D.A., Cooper J.D. Late onset neurodegeneration in the Cln3-/- mouse model of juvenile neuronal ceroid lipofuscinosis is preceded by low level glial activation. Brain Res. 2004;1023:231–242. doi: 10.1016/j.brainres.2004.07.030. [DOI] [PubMed] [Google Scholar]
40.Ahrens-Nicklas R.C., Tecedor L., Hall A.F., Kane O., Chung R.J., Lysenko E., Marsh E.D., Stein C.S., Davidson B.L. Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation. Mol. Ther. 2022;30:2464–2473. doi: 10.1016/j.ymthe.2022.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Meiman E.J., Kick G.R., Jensen C.A., Coates J.R., Katz M.L. Characterization of neurological disease progression in a canine model of CLN5 neuronal ceroid lipofuscinosis. Dev. Neurobiol. 2022;82:326–344. doi: 10.1002/dneu.22878. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Cabaraux P., Agrawal S.K., Cai H., Calabro R.S., Casali C., Damm L., Doss S., Habas C., Horn A.K.E., Ilg W., et al. Consensus paper: ataxic gait. Cerebellum. 2022 doi: 10.1007/s12311-022-01373-9. [DOI] [PubMed] [Google Scholar]
43.Williams C.T., De Jesus O. StatPearls. 2022. Friedreich ataxia. (Treasure Island (FL)) [Google Scholar]
44.Nuevo-Tapioles C., Santacatterina F., Sánchez-Garrido B., de Arenas C.N., Robledo-Bérgamo A., Martínez-Valero P., Cantarero L., Pardo B., Hoenicka J., Murphy M.P., et al. Effective therapeutic strategies in a preclinical mouse model of Charcot-Marie-Tooth disease. Hum. Mol. Genet. 2021;30:2441–2455. doi: 10.1093/hmg/ddab207. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Bruun I., Reske-Nielsen E., Oster S. Juvenile ceroid-lipofuscinosis and calcifications of the CNS. Acta Neurol. Scand. 1991;83:1–8. doi: 10.1111/j.1600-0404.1991.tb03951.x. [DOI] [PubMed] [Google Scholar]
46.Chen X.J., Levedakou E.N., Millen K.J., Wollmann R.L., Soliven B., Popko B. Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene. J. Neurosci. 2007;27:14515–14524. doi: 10.1523/JNEUROSCI.4338-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Urban R.J., Dillon E.L., Choudhary S., Zhao Y., Horstman A.M., Tilton R.G., Sheffield-Moore M. Translational studies in older men using testosterone to treat sarcopenia. Trans. Am. Clin. Climatol. Assoc. 2014;125:27–42. discussion 42-4; discussion 42-24. [PMC free article] [PubMed] [Google Scholar]
48.Qin W., Pan J., Wu Y., Bauman W.A., Cardozo C. Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1alpha. Biochem. Biophys. Res. Commun. 2010;403:473–478. doi: 10.1016/j.bbrc.2010.11.061. [DOI] [PubMed] [Google Scholar]
49.Ngo S.T., Steyn F.J., McCombe P.A. Gender differences in autoimmune disease. Front. Neuroendocrinol. 2014;35:347–369. doi: 10.1016/j.yfrne.2014.04.004. [DOI] [PubMed] [Google Scholar]
50.Poppens M.J., Cain J.T., Johnson T.B., White K.A., Davis S.S., Laufmann R., Kloth A.D., Weimer J.M. Tracking sex-dependent differences in a mouse model of CLN6-Batten disease. Orphanet J. Rare Dis. 2019;14:19. doi: 10.1186/s13023-019-0994-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Holmes A.D., White K.A., Pratt M.A., Johnson T.B., Likhite S., Meyer K., Weimer J.M. Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8(mnd) mice. Orphanet J. Rare Dis. 2022;17:411. doi: 10.1186/s13023-022-02564-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Makoukji J., El-Sitt S., Makhoul N.J., Soueid J., Kadara H., Boustany R.M. Sex differences in gene expression with galactosylceramide treatment in Cln3Deltaex7/8 mice. PLoS One. 2020;15 doi: 10.1371/journal.pone.0239537. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Toro C.A., Zhang L., Cao J., Cai D. Sex differences in Alzheimer's disease: understanding the molecular impact. Brain Res. 2019;1719:194–207. doi: 10.1016/j.brainres.2019.05.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Liu W., Kleine-Holthaus S.M., Herranz-Martin S., Aristorena M., Mole S.E., Smith A.J., Ali R.R., Rahim A.A. Experimental gene therapies for the NCLs. Biochim. Biophys. Acta, Mol. Basis Dis. 2020;1866 doi: 10.1016/j.bbadis.2020.165772. [DOI] [PubMed] [Google Scholar]
55.Bosch M.E., Aldrich A., Fallet R., Odvody J., Burkovetskaya M., Schuberth K., Fitzgerald J.A., Foust K.D., Kielian T. Self-complementary AAV9 gene delivery partially corrects pathology associated with juvenile neuronal ceroid lipofuscinosis (CLN3) J. Neurosci. 2016;36:9669–9682. doi: 10.1523/JNEUROSCI.1635-16.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Pearce D.A., Sherman F. A yeast model for the study of Batten disease. Proc. Natl. Acad. Sci. USA. 1998;95:6915–6918. doi: 10.1073/pnas.95.12.6915. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Eliason S.L., Stein C.S., Mao Q., Tecedor L., Ding S.L., Gaines D.M., Davidson B.L. A knock-in reporter model of Batten disease. J. Neurosci. 2007;27:9826–9834. doi: 10.1523/JNEUROSCI.1710-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Vitiello S.P., Benedict J.W., Padilla-Lopez S., Pearce D.A. Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for batten disease. Hum. Mol. Genet. 2010;19:931–942. doi: 10.1093/hmg/ddp560. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Foust K.D., Nurre E., Montgomery C.L., Hernandez A., Chan C.M., Kaspar B.K. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 2009;27:59–65. doi: 10.1038/nbt.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Aschauer D.F., Kreuz S., Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013;8 doi: 10.1371/journal.pone.0076310. [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Geary R.S., Norris D., Yu R., Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 2015;87:46–51. doi: 10.1016/j.addr.2015.01.008. [DOI] [PubMed] [Google Scholar]
62.Kohlschütter A., Schulz A., Bartsch U., Storch S. Current and emerging treatment strategies for neuronal ceroid lipofuscinoses. CNS Drugs. 2019;33:315–325. doi: 10.1007/s40263-019-00620-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Osório N.S., Sampaio-Marques B., Chan C.H., Oliveira P., Pearce D.A., Sousa N., Rodrigues F. Neurodevelopmental delay in the Cln3Deltaex7/8 mouse model for Batten disease. Gene Brain Behav. 2009;8:337–345. doi: 10.1111/j.1601-183X.2009.00478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Burkovetskaya M., Karpuk N., Xiong J., Bosch M., Boska M.D., Takeuchi H., Suzumura A., Kielian T. Evidence for aberrant astrocyte hemichannel activity in juvenile neuronal ceroid lipofuscinosis (JNCL) PLoS One. 2014;9 doi: 10.1371/journal.pone.0095023. [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Faul F., Erdfelder E., Buchner A., Lang A.G. Statistical power analyses using G∗Power 3.1: tests for correlation and regression analyses. Behav. Res. Methods. 2009;41:1149–1160. doi: 10.3758/BRM.41.4.1149. [DOI] [PubMed] [Google Scholar]
66.Bates D.W., Mächler M., Bolker B., Walker S. Fitting linear mixed-effects models using lme4. BMJ Qual. Saf. 2015;24:1–3. doi: 10.18637/jss.v067.i01. [DOI] [Google Scholar]
67.Page P. Beyond statistical significance: clinical interpretation of rehabilitation research literature. Int. J. Sports Phys. Ther. 2014;9:726–736. [PMC free article] [PubMed] [Google Scholar]
68.Citrome L. Quantifying clinical relevance. Innov. Clin. Neurosci. 2014;11:26–30. [PMC free article] [PubMed] [Google Scholar]
69.Schober P., Bossers S.M., Schwarte L.A. Statistical significance versus clinical importance of observed effect sizes: what do P values and confidence intervals really represent? Anesth. Analg. 2018;126:1068–1072. doi: 10.1213/ANE.0000000000002798. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Representative video of a Cln3^+/∆ex7/8 control mouse completing the string pull task

Download video file^{(8MB, mp4)}

Video S2. Representative video of a Cln3^∆ex7/8 mouse frequently missing when attempting to grab the string

Download video file^{(11.2MB, mp4)}

Video S3. Representative video of a Cln3^∆ex7/8 mouse using its mouth to pull the string with increased frequency

Download video file^{(7.8MB, mp4)}

Document S1. Figures S1–S7 and Table S2

mmc1.pdf^{(9.3MB, pdf)}

Table S1. The excel document “Table S1” incudes the effect sizes for the immunohistochemistry, separated by target protein (tabs 1–3)

mmc2.xlsx^{(38.1KB, xlsx)}

Document S2. Article plus supplemental information

mmc6.pdf^{(13.1MB, pdf)}

Data Availability Statement

The data underlying this article are available in the article and in its online supplemental material.

[bib1] 1.Munroe P.B., Mitchison H.M., O'Rawe A.M., Anderson J.W., Boustany R.M., Lerner T.J., Taschner P.E., de Vos N., Breuning M.H., Gardiner R.M., Mole S.E. Spectrum of mutations in the Batten disease gene, CLN3. Am. J. Hum. Genet. 1997;61:310–316. doi: 10.1086/514846. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib2] 2.Sleat D.E., Gedvilaite E., Zhang Y., Lobel P., Xing J. Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis. Gene. 2016;593:284–291. doi: 10.1016/j.gene.2016.08.031. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Masten M.C., Williams J.D., Vermilion J., Adams H.R., Vierhile A., Collins A., Marshall F.J., Augustine E.F., Mink J.W. The CLN3 Disease Staging System: a new tool for clinical research in Batten disease. Neurology. 2020;94:e2436–e2440. doi: 10.1212/WNL.0000000000009454. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Ostergaard J.R., Rasmussen T.B., Mølgaard H. Cardiac involvement in juvenile neuronal ceroid lipofuscinosis (Batten disease) Neurology. 2011;76:1245–1251. doi: 10.1212/WNL.0b013e31821435bd. [DOI] [PubMed] [Google Scholar]

[bib5] 5.Mink J.W., Augustine E.F., Adams H.R., Marshall F.J., Kwon J.M. Classification and natural history of the neuronal ceroid lipofuscinoses. J. Child Neurol. 2013;28:1101–1105. doi: 10.1177/0883073813494268. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Nielsen A.K., Østergaard J.R. Do females with juvenile ceroid lipofuscinosis (Batten disease) have a more severe disease course? The Danish experience. Eur. J. Paediatr. Neurol. 2013;17:265–268. doi: 10.1016/j.ejpn.2012.10.011. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Cialone J., Adams H., Augustine E.F., Marshall F.J., Kwon J.M., Newhouse N., Vierhile A., Levy E., Dure L.S., Rose K.R., et al. Females experience a more severe disease course in Batten disease. J. Inherit. Metab. Dis. 2012;35:549–555. doi: 10.1007/s10545-011-9421-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Miller J.N., Chan C.H., Pearce D.A. The role of nonsense-mediated decay in neuronal ceroid lipofuscinosis. Hum. Mol. Genet. 2013;22:2723–2734. doi: 10.1093/hmg/ddt120. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Centa J.L., Jodelka F.M., Hinrich A.J., Johnson T.B., Ochaba J., Jackson M., Duelli D.M., Weimer J.M., Rigo F., Hastings M.L. Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease. Nat. Med. 2020;26:1444–1451. doi: 10.1038/s41591-020-0986-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Kida E., Kaczmarski W., Golabek A.A., Kaczmarski A., Michalewski M., Wisniewski K.E. Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro. Mol. Genet. Metabol. 1999;66:265–271. doi: 10.1006/mgme.1999.2837. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Kousi M., Lehesjoki A.E., Mole S.E. Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses. Hum. Mutat. 2012;33:42–63. doi: 10.1002/humu.21624. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Wang F., Wang H., Tuan H.F., Nguyen D.H., Sun V., Keser V., Bowne S.J., Sullivan L.S., Luo H., Zhao L., et al. Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements. Hum. Genet. 2014;133:331–345. doi: 10.1007/s00439-013-1381-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Le B.T., Agarwal S., Veedu R.N. Evaluation of DNA segments in 2'-modified RNA sequences in designing efficient splice switching antisense oligonucleotides. RSC Adv. 2021;11:14029–14035. doi: 10.1039/d1ra00878a. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Nilsen T.W., Graveley B.R. Expansion of the eukaryotic proteome by alternative splicing. Nature. 2010;463:457–463. doi: 10.1038/nature08909. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Yang P., Wang D., Kang L. Alternative splicing level related to intron size and organism complexity. BMC Genom. 2021;22:853. doi: 10.1186/s12864-021-08172-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.Marasco L.E., Kornblihtt A.R. The physiology of alternative splicing. Nat. Rev. Mol. Cell Biol. 2023;24:242–254. doi: 10.1038/s41580-022-00545-z. [DOI] [PubMed] [Google Scholar]

[bib17] 17.Cunningham F., Allen J.E., Allen J., Alvarez-Jarreta J., Amode M.R., Armean I.M., Austine-Orimoloye O., Azov A.G., Barnes I., Bennett R., et al. Ensembl 2022. Nucleic Acids Res. 2022;50:D988–D995. doi: 10.1093/nar/gkab1049. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.O'Leary N.A., Wright M.W., Brister J.R., Ciufo S., Haddad D., McVeigh R., Rajput B., Robbertse B., Smith-White B., Ako-Adjei D., et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44:D733–D745. doi: 10.1093/nar/gkv1189. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19.Kovács A.D., Pearce D.A. Finding the most appropriate mouse model of juvenile CLN3 (Batten) disease for therapeutic studies: the importance of genetic background and gender. Dis. Model. Mech. 2015;8:351–361. doi: 10.1242/dmm.018804. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20.Finn R., Kovács A.D., Pearce D.A. Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Deltaex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis. Neurochem. Int. 2011;58:648–655. doi: 10.1016/j.neuint.2011.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Blackwell A.A., Schell B.D., Osterlund Oltmanns J.R., Whishaw I.Q., Ton S.T., Adamczyk N.S., Kartje G.L., Britten R.A., Wallace D.G. Skilled movement and posture deficits in rat string-pulling behavior following low dose space radiation ((28)Si) exposure. Behav. Brain Res. 2021;400 doi: 10.1016/j.bbr.2020.113010. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Whishaw I.Q., Pellis S.M., Gorny B.P., Pellis V.C. The impairments in reaching and the movements of compensation in rats with motor cortex lesions: an endpoint, videorecording, and movement notation analysis. Behav. Brain Res. 1991;42:77–91. doi: 10.1016/s0166-4328(05)80042-7. [DOI] [PubMed] [Google Scholar]

[bib23] 23.Blackwell A.A., Banovetz M.T., Qandeel, Whishaw I.Q., Whishaw I.Q., Wallace D.G. The structure of arm and hand movements in a spontaneous and food rewarded on-line string-pulling task by the mouse. Behav. Brain Res. 2018;345:49–58. doi: 10.1016/j.bbr.2018.02.025. [DOI] [PubMed] [Google Scholar]

[bib24] 24.Cui M., Zhang S., Liu Y., Gang X., Wang G. Grip strength and the risk of cognitive decline and dementia: a systematic review and meta-analysis of longitudinal cohort studies. Front. Aging Neurosci. 2021;13 doi: 10.3389/fnagi.2021.625551. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25.Kunutsor S.K., Isiozor N.M., Voutilainen A., Laukkanen J.A. Handgrip strength and risk of cognitive outcomes: new prospective study and meta-analysis of 16 observational cohort studies. Geroscience. 2022;44:2007–2024. doi: 10.1007/s11357-022-00514-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib26] 26.Duchowny K.A., Ackley S.F., Brenowitz W.D., Wang J., Zimmerman S.C., Caunca M.R., Glymour M.M. Associations between handgrip strength and dementia risk, cognition, and neuroimaging outcomes in the UK biobank cohort study. JAMA Netw. Open. 2022;5 doi: 10.1001/jamanetworkopen.2022.18314. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib27] 27.Herold F., Labott B.K., Grässler B., Halfpaap N., Langhans C., Müller P., Ammar A., Dordevic M., Hökelmann A., Müller N.G. A link between handgrip strength and executive functioning: a cross-sectional study in older adults with mild cognitive impairment and healthy controls. Healthcare (Basel) 2022;10 doi: 10.3390/healthcare10020230. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 28.Bohannon R.W. Muscle strength: clinical and prognostic value of hand-grip dynamometry. Curr. Opin. Clin. Nutr. Metab. Care. 2015;18:465–470. doi: 10.1097/MCO.0000000000000202. [DOI] [PubMed] [Google Scholar]

[bib29] 29.Carson R.G. Get a grip: individual variations in grip strength are a marker of brain health. Neurobiol. Aging. 2018;71:189–222. doi: 10.1016/j.neurobiolaging.2018.07.023. [DOI] [PubMed] [Google Scholar]

[bib30] 30.Takeshita H., Yamamoto K., Nozato S., Inagaki T., Tsuchimochi H., Shirai M., Yamamoto R., Imaizumi Y., Hongyo K., Yokoyama S., et al. Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice. Sci. Rep. 2017;7 doi: 10.1038/srep42323. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Honingh A.K., Kruithof Y.L., Kuper W.F.E., van Hasselt P.M., Sterkenburg P.S. Towards understanding behaviour and emotions of children with CLN3 disease (batten disease): patterns, problems and support for child and family. Int. J. Environ. Res. Publ. Health. 2022;19 doi: 10.3390/ijerph19105895. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32.Jana M., Dutta D., Poddar J., Pahan K. Activation of PPARalpha exhibits therapeutic efficacy in a mouse model of juvenile neuronal ceroid lipofuscinosis. J. Neurosci. 2023;43:1814–1829. doi: 10.1523/JNEUROSCI.2447-21.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33.Seibenhener M.L., Wooten M.C. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J. Vis. Exp. 2015:e52434. doi: 10.3791/52434. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34.Rowan S.A., Lake B.D. Tissue and cellular distribution of subunit c of ATP synthase in Batten disease (neuronal ceroid-lipofuscinosis) Am. J. Med. Genet. 1995;57:172–176. doi: 10.1002/ajmg.1320570213. [DOI] [PubMed] [Google Scholar]

[bib35] 35.Tyynelä J., Cooper J.D., Khan M.N., Shemilts S.J.A., Haltia M. Hippocampal pathology in the human neuronal ceroid-lipofuscinoses: distinct patterns of storage deposition, neurodegeneration and glial activation. Brain Pathol. 2004;14:349–357. doi: 10.1111/j.1750-3639.2004.tb00077.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36.Cotman S.L., Vrbanac V., Lebel L.A., Lee R.L., Johnson K.A., Donahue L.R., Teed A.M., Antonellis K., Bronson R.T., Lerner T.J., MacDonald M.E. Cln3(Deltaex7/8) knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth. Hum. Mol. Genet. 2002;11:2709–2721. doi: 10.1093/hmg/11.22.2709. [DOI] [PubMed] [Google Scholar]

[bib39] 37.Katz M.L., Shibuya H., Liu P.C., Kaur S., Gao C.L., Johnson G.S. A mouse gene knockout model for juvenile ceroid-lipofuscinosis (Batten disease) J. Neurosci. Res. 1999;57:551–556. [PubMed] [Google Scholar]

[bib40] 38.Mitchison H.M., Bernard D.J., Greene N.D., Cooper J.D., Junaid M.A., Pullarkat R.K., de Vos N., Breuning M.H., Owens J.W., Mobley W.C., et al. Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected] Neurobiol. Dis. 1999;6:321–334. doi: 10.1006/nbdi.1999.0267. [DOI] [PubMed] [Google Scholar]

[bib41] 39.Pontikis C.C., Cella C.V., Parihar N., Lim M.J., Chakrabarti S., Mitchison H.M., Mobley W.C., Rezaie P., Pearce D.A., Cooper J.D. Late onset neurodegeneration in the Cln3-/- mouse model of juvenile neuronal ceroid lipofuscinosis is preceded by low level glial activation. Brain Res. 2004;1023:231–242. doi: 10.1016/j.brainres.2004.07.030. [DOI] [PubMed] [Google Scholar]

[bib42] 40.Ahrens-Nicklas R.C., Tecedor L., Hall A.F., Kane O., Chung R.J., Lysenko E., Marsh E.D., Stein C.S., Davidson B.L. Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation. Mol. Ther. 2022;30:2464–2473. doi: 10.1016/j.ymthe.2022.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 41.Meiman E.J., Kick G.R., Jensen C.A., Coates J.R., Katz M.L. Characterization of neurological disease progression in a canine model of CLN5 neuronal ceroid lipofuscinosis. Dev. Neurobiol. 2022;82:326–344. doi: 10.1002/dneu.22878. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 42.Cabaraux P., Agrawal S.K., Cai H., Calabro R.S., Casali C., Damm L., Doss S., Habas C., Horn A.K.E., Ilg W., et al. Consensus paper: ataxic gait. Cerebellum. 2022 doi: 10.1007/s12311-022-01373-9. [DOI] [PubMed] [Google Scholar]

[bib45] 43.Williams C.T., De Jesus O. StatPearls. 2022. Friedreich ataxia. (Treasure Island (FL)) [Google Scholar]

[bib46] 44.Nuevo-Tapioles C., Santacatterina F., Sánchez-Garrido B., de Arenas C.N., Robledo-Bérgamo A., Martínez-Valero P., Cantarero L., Pardo B., Hoenicka J., Murphy M.P., et al. Effective therapeutic strategies in a preclinical mouse model of Charcot-Marie-Tooth disease. Hum. Mol. Genet. 2021;30:2441–2455. doi: 10.1093/hmg/ddab207. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib47] 45.Bruun I., Reske-Nielsen E., Oster S. Juvenile ceroid-lipofuscinosis and calcifications of the CNS. Acta Neurol. Scand. 1991;83:1–8. doi: 10.1111/j.1600-0404.1991.tb03951.x. [DOI] [PubMed] [Google Scholar]

[bib48] 46.Chen X.J., Levedakou E.N., Millen K.J., Wollmann R.L., Soliven B., Popko B. Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene. J. Neurosci. 2007;27:14515–14524. doi: 10.1523/JNEUROSCI.4338-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib49] 47.Urban R.J., Dillon E.L., Choudhary S., Zhao Y., Horstman A.M., Tilton R.G., Sheffield-Moore M. Translational studies in older men using testosterone to treat sarcopenia. Trans. Am. Clin. Climatol. Assoc. 2014;125:27–42. discussion 42-4; discussion 42-24. [PMC free article] [PubMed] [Google Scholar]

[bib50] 48.Qin W., Pan J., Wu Y., Bauman W.A., Cardozo C. Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1alpha. Biochem. Biophys. Res. Commun. 2010;403:473–478. doi: 10.1016/j.bbrc.2010.11.061. [DOI] [PubMed] [Google Scholar]

[bib51] 49.Ngo S.T., Steyn F.J., McCombe P.A. Gender differences in autoimmune disease. Front. Neuroendocrinol. 2014;35:347–369. doi: 10.1016/j.yfrne.2014.04.004. [DOI] [PubMed] [Google Scholar]

[bib52] 50.Poppens M.J., Cain J.T., Johnson T.B., White K.A., Davis S.S., Laufmann R., Kloth A.D., Weimer J.M. Tracking sex-dependent differences in a mouse model of CLN6-Batten disease. Orphanet J. Rare Dis. 2019;14:19. doi: 10.1186/s13023-019-0994-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib53] 51.Holmes A.D., White K.A., Pratt M.A., Johnson T.B., Likhite S., Meyer K., Weimer J.M. Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8(mnd) mice. Orphanet J. Rare Dis. 2022;17:411. doi: 10.1186/s13023-022-02564-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib54] 52.Makoukji J., El-Sitt S., Makhoul N.J., Soueid J., Kadara H., Boustany R.M. Sex differences in gene expression with galactosylceramide treatment in Cln3Deltaex7/8 mice. PLoS One. 2020;15 doi: 10.1371/journal.pone.0239537. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib55] 53.Toro C.A., Zhang L., Cao J., Cai D. Sex differences in Alzheimer's disease: understanding the molecular impact. Brain Res. 2019;1719:194–207. doi: 10.1016/j.brainres.2019.05.031. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib56] 54.Liu W., Kleine-Holthaus S.M., Herranz-Martin S., Aristorena M., Mole S.E., Smith A.J., Ali R.R., Rahim A.A. Experimental gene therapies for the NCLs. Biochim. Biophys. Acta, Mol. Basis Dis. 2020;1866 doi: 10.1016/j.bbadis.2020.165772. [DOI] [PubMed] [Google Scholar]

[bib57] 55.Bosch M.E., Aldrich A., Fallet R., Odvody J., Burkovetskaya M., Schuberth K., Fitzgerald J.A., Foust K.D., Kielian T. Self-complementary AAV9 gene delivery partially corrects pathology associated with juvenile neuronal ceroid lipofuscinosis (CLN3) J. Neurosci. 2016;36:9669–9682. doi: 10.1523/JNEUROSCI.1635-16.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib58] 56.Pearce D.A., Sherman F. A yeast model for the study of Batten disease. Proc. Natl. Acad. Sci. USA. 1998;95:6915–6918. doi: 10.1073/pnas.95.12.6915. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib59] 57.Eliason S.L., Stein C.S., Mao Q., Tecedor L., Ding S.L., Gaines D.M., Davidson B.L. A knock-in reporter model of Batten disease. J. Neurosci. 2007;27:9826–9834. doi: 10.1523/JNEUROSCI.1710-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib60] 58.Vitiello S.P., Benedict J.W., Padilla-Lopez S., Pearce D.A. Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for batten disease. Hum. Mol. Genet. 2010;19:931–942. doi: 10.1093/hmg/ddp560. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib61] 59.Foust K.D., Nurre E., Montgomery C.L., Hernandez A., Chan C.M., Kaspar B.K. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 2009;27:59–65. doi: 10.1038/nbt.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib62] 60.Aschauer D.F., Kreuz S., Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013;8 doi: 10.1371/journal.pone.0076310. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib63] 61.Geary R.S., Norris D., Yu R., Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 2015;87:46–51. doi: 10.1016/j.addr.2015.01.008. [DOI] [PubMed] [Google Scholar]

[bib64] 62.Kohlschütter A., Schulz A., Bartsch U., Storch S. Current and emerging treatment strategies for neuronal ceroid lipofuscinoses. CNS Drugs. 2019;33:315–325. doi: 10.1007/s40263-019-00620-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib65] 63.Osório N.S., Sampaio-Marques B., Chan C.H., Oliveira P., Pearce D.A., Sousa N., Rodrigues F. Neurodevelopmental delay in the Cln3Deltaex7/8 mouse model for Batten disease. Gene Brain Behav. 2009;8:337–345. doi: 10.1111/j.1601-183X.2009.00478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib66] 64.Burkovetskaya M., Karpuk N., Xiong J., Bosch M., Boska M.D., Takeuchi H., Suzumura A., Kielian T. Evidence for aberrant astrocyte hemichannel activity in juvenile neuronal ceroid lipofuscinosis (JNCL) PLoS One. 2014;9 doi: 10.1371/journal.pone.0095023. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib67] 65.Faul F., Erdfelder E., Buchner A., Lang A.G. Statistical power analyses using G∗Power 3.1: tests for correlation and regression analyses. Behav. Res. Methods. 2009;41:1149–1160. doi: 10.3758/BRM.41.4.1149. [DOI] [PubMed] [Google Scholar]

[bib68] 66.Bates D.W., Mächler M., Bolker B., Walker S. Fitting linear mixed-effects models using lme4. BMJ Qual. Saf. 2015;24:1–3. doi: 10.18637/jss.v067.i01. [DOI] [Google Scholar]

[bib69] 67.Page P. Beyond statistical significance: clinical interpretation of rehabilitation research literature. Int. J. Sports Phys. Ther. 2014;9:726–736. [PMC free article] [PubMed] [Google Scholar]

[bib70] 68.Citrome L. Quantifying clinical relevance. Innov. Clin. Neurosci. 2014;11:26–30. [PMC free article] [PubMed] [Google Scholar]

[bib71] 69.Schober P., Bossers S.M., Schwarte L.A. Statistical significance versus clinical importance of observed effect sizes: what do P values and confidence intervals really represent? Anesth. Analg. 2018;126:1068–1072. doi: 10.1213/ANE.0000000000002798. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Protracted CLN3 Batten disease in mice that genetically model an exon-skipping therapeutic approach

Jessica L Centa

Matthew P Stratton

Melissa A Pratt

Jenna R Osterlund Oltmanns

Douglas G Wallace

Steven A Miller

Jill M Weimer

Michelle L Hastings

Abstract

Graphical abstract

Introduction

Results

Generation of Cln3Δex5/7/8 mice

Figure 1.

Sensorimotor coordination deficits associated with murine CLN3 disease are absent in a Cln3Δex5/7/8 therapeutic model

Figure 2.

Fine motor control is intact in Cln3Δex5/7/8 therapeutic model compared with CLN3 disease mice

Figure 3.

Grip strength deficits seen in CLN3 disease mice are less severe in Cln3Δex5/7/8 mice

Figure 4.

Cln3Δex5/7/8 mice do not exhibit anxiety-like behavior seen in CLN3 disease mice

Figure 5.

Cln3Δex5/7/8 mice have delayed histopathological signs of CLN3 Batten disease compared with Cln3Δex7/8 disease mice

Figure 6.

Discussion

Material and methods

Plasmids

Cell culture and transfection

Immunoblot analysis

Mice

RNA isolation and analysis

Open field test

Accelerating rotarod

String pulling

Grip strength

Histology

Statistical analyses

Data availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Generation of Cln3^Δex5/7/8 mice

Sensorimotor coordination deficits associated with murine CLN3 disease are absent in a Cln3^Δex5/7/8 therapeutic model

Fine motor control is intact in Cln3^Δex5/7/8 therapeutic model compared with CLN3 disease mice

Grip strength deficits seen in CLN3 disease mice are less severe in Cln3^Δex5/7/8 mice

Cln3^Δex5/7/8 mice do not exhibit anxiety-like behavior seen in CLN3 disease mice

Cln3^Δex5/7/8 mice have delayed histopathological signs of CLN3 Batten disease compared with Cln3^Δex7/8 disease mice