Brain-Directed AAV Gene Therapy Rescues a Mouse Model of the CLN5 Form of Neuronal Ceroid Lipofuscinosis Disease and Normalizes a Blood Plasma Biomarker of Neurodegeneration
Restricted accessResearch articleFirst published online March, 2026
Brain-Directed AAV Gene Therapy Rescues a Mouse Model of the CLN5 Form of Neuronal Ceroid Lipofuscinosis Disease and Normalizes a Blood Plasma Biomarker of Neurodegeneration
CLN5 disease, caused by mutations in the CLN5 gene, is a form of neuronal ceroid lipofuscinoses (Batten disease). Patients suffer progressive motor dysfunction, vision loss, seizures, and dementia, leading to premature death. Here, we report a preclinical study of AAV9-mediated gene therapy in a Cln5−/− mouse model. Single-dose AAV9 carrying human CLN5 driven by the CAG or human synapsin 1 promoter (hSYN) was administered via intracerebroventricular injection into neonatal and juvenile Cln5−/− mice. Treatment efficacy was evaluated by assessment of neurodegeneration, neuroinflammation, locomotor function, and survival. AAV9 expressing CLN5 driven by the hSYN promoter significantly alleviated neurodegeneration, improved biochemical and glycosphingolipid profiles, neuropathological and locomotor function, and extended lifespan of the Cln5−/− mice. However, gene transfer employing the CAG promoter demonstrated limited therapeutic efficacy. Furthermore, delayed intervention in juveniles provided superior therapeutic response compared with early neonatal intervention and normalized lifespan. Finally, blood plasma neurofilament light that is significantly elevated in the Cln5−/− mice is restored to normal wildtype levels following treatment. These results indicate that brain-directed adeno-associated virus (AAV) gene therapy could be a promising treatment strategy for CLN5 disease and efficacy might be monitored using a noninvasive blood plasma biomarker.
SimonatiA, WilliamsRE. Neuronal Ceroid Lipofuscinosis: The multifaceted approach to the clinical issues, an overview. Front Neurol, 2022; 13:811686.
2.
WilliamsRE, MoleSE. New nomenclature and classification scheme for the neuronal ceroid lipofuscinoses. Neurology, 2012; 79(2):183–191.
3.
GardnerE, MoleSE. The genetic basis of phenotypic heterogeneity in the neuronal ceroid lipofuscinoses. Front Neurol, 2021; 12:754045.
4.
MoleSE, AndersonG, BandHA, et al.Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis. Lancet Neurol, 2019; 18(1):107–116.
5.
MedohUN, HimsA, ChenJY, et al.The Batten disease gene product CLN5 is the lysosomal bis(monoacylglycero)phosphate synthase. Science, 2023; 381(6663):1182–1189.
6.
SimonatiA, WilliamsRE, NardocciN, et al.Phenotype and natural history of variant late infantile ceroid-lipofuscinosis 5. Dev Med Child Neurol, 2017; 59(8):815–821.
7.
XinW, MullenTE, KielyR, et al.CLN5 mutations are frequent in juvenile and late-onset non-Finnish patients with NCL. Neurology, 2010; 74(7):565–571.
8.
ManciniC, NassaniS, GuoY, et al.Adult-onset autosomal recessive ataxia associated with neuronal ceroid lipofuscinosis type 5 gene (CLN5) mutations. J Neurol, 2015; 262(1):173–178.
FrugierT, MitchellNL, TammenI, et al.A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571 + 1G>A) leading to excision of exon 3. Neurobiol Dis, 2008; 29(2):306–315.
11.
GilliamD, KolicheskiA, JohnsonGS, et al.Golden Retriever dogs with neuronal ceroid lipofuscinosis have a two-base-pair deletion and frameshift in CLN5. Mol Genet Metab, 2015; 115(2–3):101–109.
12.
JollyRD, ArthurDG, KayGW, et al.Neuronal ceroid-lipofuscinosis in Borderdale sheep. N Z Vet J, 2002; 50(5):199–202.
13.
KolicheskiA, JohnsonGS, O’BrienDP, et al.Australian cattle dogs with neuronal ceroid Lipofuscinosis are homozygous for a CLN5 nonsense mutation previously identified in border collies. J Vet Intern Med, 2016; 30(4):1149–1158.
14.
KopraO, VesaJ, von SchantzC, et al.A mouse model for Finnish variant late infantile neuronal ceroid lipofuscinosis, CLN5, reveals neuropathology associated with early aging. Hum Mol Genet, 2004; 13(23):2893–2906.
15.
MizukamiK, KawamichiT, KoieH, et al.Neuronal ceroid lipofuscinosis in Border collie dogs in Japan: Clinical and molecular epidemiological study (2000–2011). ScientificWorldJournal, 2012; 2012:383174.
von SchantzC, KielarC, HansenSN, et al.Progressive thalamocortical neuron loss in Cln5 deficient mice: Distinct effects in Finnish variant late infantile NCL. Neurobiol Dis, 2009; 34(2):308–319.
18.
SchmiedtML, BessaC, HeineC, et al.The neuronal ceroid lipofuscinosis protein CLN5: New insights into cellular maturation, transport, and consequences of mutations. Hum Mutat, 2010; 31(3):356–365.
19.
MoharirA, PeckSH, BuddenT, et al.The role of N-glycosylation in folding, trafficking, and functionality of lysosomal protein CLN5. PLoS One, 2013; 8(9):e74299.
20.
JulesF, SauvageauE, Dumaresq-DoironK, et al.CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein. Exp Cell Res, 2017; 357(1):40–50.
21.
IsosomppiJ, VesaJ, JalankoA, et al.Lysosomal localization of the neuronal ceroid lipofuscinosis CLN5 protein. Hum Mol Genet, 2002; 11(8):885–891.
22.
De SilvaB, AdamsJ, LeeSY. Proteolytic processing of the neuronal ceroid lipofuscinosis related lysosomal protein CLN5. Exp Cell Res, 2015; 338(1):45–53.
23.
MurraySJ, WellbyMP, BarrellGK, et al.Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease. Front Pharmacol, 2023; 14:1212235.
24.
MurraySJ, RussellKN, MelzerTR, et al.Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease. Exp Eye Res, 2021; 207:108600.
25.
MitchellNL, RussellKN, WellbyMP, et al.Longitudinal in vivo monitoring of the CNS demonstrates the efficacy of gene therapy in a sheep model of CLN5 Batten disease. Mol Ther, 2018; 26(10):2366–2378.
26.
MitchellNL, MurraySJ, WellbyMP, et al.Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease. Front Genet, 2023; 14:1212228.
27.
KimJY, GrunkeSD, LevitesY, et al.Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J Vis Exp, 2014(91):51863.
28.
GundersenHJ, JensenEB, KieuK, et al.The efficiency of systematic sampling in stereology–reconsidered. J Microsc, 1999; 193(Pt 3):199–211.
29.
RahimAA, WongAM, AhmadiS, et al.In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression. Gene Ther, 2012; 19(9):936–946.
30.
NevilleDCA, CoquardV, PriestmanDA, et al.Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling. Anal Biochem, 2004; 331(2):275–282.
31.
FearnleyIM, WalkerJE, MartinusRD, et al.The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase. Biochem J, 1990; 268(3):751–758.
32.
HaltiaM. The neuronal ceroid-lipofuscinoses: From past to present. Biochim Biophys Acta, 2006; 1762(10):850–856.
33.
PalmerDN, BarnsG, HusbandsDR, et al.Ceroid lipofuscinosis in sheep. II. The major component of the lipopigment in liver, kidney, pancreas, and brain is low molecular weight protein. J Biol Chem, 1986; 261(4):1773–1777.
34.
PalmerDN, FearnleyIM, WalkerJE, et al.Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease). Am J Med Genet, 1992; 42(4):561–567.
35.
PalmerDN, MartinusRD, CooperSM, et al.Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence. J Biol Chem, 1989; 264(10):5736–5740.
36.
VarkiA, CummingsRD, EskoJD, StanleyP, HartGW, AebiM, et al., editors. Essentials of Glycobiology. 4th ed. Cold Spring Harbor (NY); 2022.
37.
RapolaJ, LahdetieJ, IsosomppiJ, et al.Prenatal diagnosis of variant late infantile neuronal ceroid lipofuscinosis (vLINCL[Finnish]; CLN5). Prenat Diagn, 1999; 19(7):685–688.
38.
WangY, CaoX, LiuP, et al.KCTD7 mutations impair the trafficking of lysosomal enzymes through CLN5 accumulation to cause neuronal ceroid lipofuscinoses. Sci Adv, 2022; 8(31):eabm5578.
39.
WangY, WangH, WangC. Lysosomal dysfunction, autophagic defects, and CLN5 accumulation underlie the pathogenesis of KCTD7-mutated neuronal ceroid lipofuscinoses. Autophagy, 2023; 19(6):1876–1878.
40.
HughesSM, HopeKM, XuJB, et al.Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy. Neurobiol Dis, 2014; 62:543–550.
41.
HuberRJ, KimWD, Wilson-SmillieM. Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD. Traffic, 2024; 25(1):e12925.
42.
HuberRJ, MathavarajahS. Secretion and function of Cln5 during the early stages of Dictyostelium development. Biochim Biophys Acta Mol Cell Res, 2018; 1865(10):1437–1450.
43.
McLeanJR, SmithGA, RochaEM, et al.Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection. Neurosci Lett, 2014; 576:73–78.
44.
HammondSL, LeekAN, RichmanEH, et al.Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection. PLoS One, 2017; 12(12):e0188830.
45.
MitchellNL, RussellKN, BarrellGK, et al.Characterization of neuropathology in ovine CLN5 and CLN6 neuronal ceroid lipofuscinoses (Batten disease). Dev Neurobiol, 2023; 83(5–6):127–142.
46.
KhalilM, TeunissenCE, OttoM, et al.Neurofilaments as biomarkers in neurological disorders. Nat Rev Neurol, 2018; 14(10):577–589.
47.
GiacomucciG, MazzeoS, BagnoliS, et al.Plasma Neurofilament light chain as a biomarker of Alzheimer’s disease in subjective cognitive decline and mild cognitive impairment. J Neurol, 2022; 269(8):4270–4280.
48.
Stevenson-HoareJ, HeslegraveA, LeonenkoG, et al.Plasma biomarkers and genetics in the diagnosis and prediction of Alzheimer’s disease. Brain, 2023; 146(2):690–699.
49.
JungY, DamoiseauxJS. The potential of blood neurofilament light as a marker of neurodegeneration for Alzheimer’s disease. Brain, 2024; 147(1):12–25.
50.
PiccaA, GuerraF, CalvaniR, et al.Mitochondrial dysfunction, protein misfolding and neuroinflammation in Parkinson’s disease: Roads to biomarker discovery. Biomolecules, 2021; 11(10):1508.
51.
VerdeF, OttoM, SilaniV. Neurofilament light chain as biomarker for amyotrophic lateral sclerosis and frontotemporal dementia. Front Neurosci, 2021; 15:679199.
52.
ZanardiniR, SaracenoC, BenussiL, et al.Exploring Neurofilament light chain and exosomes in the genetic forms of frontotemporal dementia. Front Neurosci, 2022; 16:758182.
53.
ZetterbergH, TeunissenC, van SwietenJ, et al.The role of neurofilament light in genetic frontotemporal lobar degeneration. Brain Commun, 2023; 5(1):fcac310.
54.
SenMK, HossainMJ, MahnsDA, et al.Validity of serum neurofilament light chain as a prognostic biomarker of disease activity in multiple sclerosis. J Neurol, 2023; 270(4):1908–1930.
55.
DesuHL, SawickaKM, WuerchE, et al.A rapid review of differences in cerebrospinal neurofilament light levels in clinical subtypes of progressive multiple sclerosis. Front Neurol, 2024; 15:1382468.
56.
BayoumyS, VerberkIMW, VermuntL, et al.Neurofilament light protein as a biomarker for spinal muscular atrophy: A review and reference ranges. Clin Chem Lab Med, 2024; 62(7):1252–1265.
57.
IwanK, PatelN, HeslegraveA, et al.Cerebrospinal fluid neurofilament light chain levels in CLN2 disease patients treated with enzyme replacement therapy normalise after two years on treatment. F1000Res, 2021; 10:614.
58.
FanM, SidhuR, FujiwaraH, et al.Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling. J Lipid Res, 2013; 54(10):2800–2814.
59.
TobiasF, OlsonMT, ColognaSM. Mass spectrometry imaging of lipids: Untargeted consensus spectra reveal spatial distributions in Niemann-Pick disease type C1. J Lipid Res, 2018; 59(12):2446–2455.
60.
TrilckM, PeterF, ZhengC, et al.Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons. Brain Res, 2017; 1657:52–61.
61.
SomogyiA, PetcherskiA, BeckertB, et al.Altered expression of ganglioside metabolizing enzymes results in GM3 ganglioside accumulation in cerebellar cells of a mouse model of juvenile neuronal ceroid lipofuscinosis. Int J Mol Sci, 2018; 19(2):625.
62.
JabsS, QuitschA, KäkeläR, et al.Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis. J Neurochem, 2008; 106(3):1415–1425.
63.
ShowalterMR, BergAL, NagourneyA, et al.The emerging and diverse roles of Bis(monoacylglycero) phosphate lipids in cellular physiology and disease. Int J Mol Sci, 2020; 21(21):8067.
64.
GallalaHD, SandhoffK. Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion. Neurochem Res, 2011; 36(9):1594–1600.
65.
Hullin-MatsudaF, TaguchiT, GreimelP, et al.Lipid compartmentalization in the endosome system. Semin Cell Dev Biol, 2014; 31:48–56.
66.
AnheuserS, BreidenB, SchwarzmannG, et al.Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity. J Lipid Res, 2015; 56(9):1747–1761.
67.
SandhoffK, HarzerK. Gangliosides and gangliosidoses: Principles of molecular and metabolic pathogenesis. J Neurosci, 2013; 33(25):10195–10208.
68.
CaughlinS, HepburnJD, ParkDH, et al.Increased expression of simple ganglioside species GM2 and GM3 detected by MALDI imaging mass spectrometry in a combined rat model of Abeta toxicity and stroke. PLoS One, 2015; 10(6):e0130364.
69.
CaughlinS, MaheshwariS, AgcaY, et al.Membrane-lipid homeostasis in a prodromal rat model of Alzheimer’s disease: Characteristic profiles in ganglioside distributions during aging detected using MALDI imaging mass spectrometry. Biochim Biophys Acta Gen Subj, 2018; 1862(6):1327–1338.
70.
PernberZ, BlennowK, BogdanovicN, et al.Altered distribution of the gangliosides GM1 and GM2 in Alzheimer’s disease. Dement Geriatr Cogn Disord, 2012; 33(2–3):174–188.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
