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Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60-90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs -/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs +/- control mice. Mice given the highest dose (5.6 × 10 11 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials., (© 2021 SSIEM.)

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2 , in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort ( P < 0.04) and European natural history cohort ( P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A method is presented for quantitative analysis of the biodistribution of adeno-associated virus (AAV) gene transfer vectors following in vivo administration. We used iodine-124 (I-124) radiolabeling of the AAV capsid and positron emission tomography combined with compartmental modeling to quantify whole-body and organ-specific biodistribution of AAV capsids from 1 to 72 h following administration. Using intravenous (IV) and intracisternal (IC) routes of administration of AAVrh.10 and AAV9 vectors to nonhuman primates in the absence or presence of anticapsid immunity, we have identified novel insights into initial capsid biodistribution and organ-specific capsid half-life. Neither I-124-labeled AAVrh.10 nor AAV9 administered intravenously was detected at significant levels in the brain relative to the administered vector dose. Approximately 50% of the intravenously administered labeled capsids were dispersed throughout the body, independent of the liver, heart, and spleen. When administered by the IC route, the labeled capsid had a half-life of ∼10 h in the cerebral spinal fluid (CSF), suggesting that by this route, the CSF serves as a source with slow diffusion into the brain. For both IV and IC administration, there was significant influence of pre-existing anticapsid immunity on I-124-capsid biodistribution. The methodology facilitates quantitative in vivo viral vector dosimetry, which can serve as a technique for evaluation of both on- and off-target organ biodistribution, and potentially accelerate gene therapy development through rapid prototyping of novel vector designs.

Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell-mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease-inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.

Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction ( p  < 2 × 10 -4 ) and 71% reduction in stress-related fractional shortening ( p  < 10 -5 ). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls ( p  < 0.02). A one-time intravenous administration of 10 11 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN , corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls ( p  > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.

The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function. This is particularly challenging for products based on the adeno-associated virus (AAV) platform, which has poor in vitro infectivity, limiting the sensitivity and thus the usefulness of cell-based assays. A rigorous in vivo assay has been established that separately evaluates infection, transcription, and resulting protein levels with specifications for each based on real time polymerase chain reaction (DNA and RNA) and standard protein assays. For an acceptance criterion, an administered vector must have vector DNA, transgene mRNA, and transgene expressed protein each concurrently meet individual specifications or the production lot fails. Using the AAVrh.10 serotype as a model vector and three different transgenes as examples, the assay is based on intravenous administration of the vector to male mice. At 2 weeks, the harvested liver is homogenized and assessed for vector genome levels (to assess for vector delivery), mRNA (to assess vector infectivity and transcription), and protein in the liver or serum (to assess protein expression). For all AAV vectors, the assay is robust and reproducible: vector DNA (linearity 10 2 -10 9 copies, coefficient of variation) intra-assay <0.8%, inter-assay <0.5%; mRNA intra-assay <3.3%, inter-assay <3.4%. The reproducibility of the assay for transgene expressed protein is product specific. This in vivo potency assay is a strategy for characterization and a quantitative lot release test, providing a path forward to meet regulatory drug requirements for any AAV gene therapy vectors.

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-β (Aβ) peptide deposition and amyloid burden (ε4 >ε3 >ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)-mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aβ levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aβ burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aβ and amyloid deposition. We further demonstrate that a widespread reduction of brain Aβ burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rationale: Anti-angiogenesis therapies such as bevacizumab, the monoclonal antibody to vascular endothelial growth factor (VEGF), have been used against ovarian cancer, but transient and low peritoneal drug levels are likely a factor in treatment failure. We hypothesized that a single administration of adeno-associated virus (AAV)-mediated intraperitoneal expression of bevacizumab would direct persistent expression and suppress growth and metastasis of ovarian cancer., Methods: AAVrh.10BevMab, a rhesus serotype 10 adeno-associated viral vector coding for bevacizumab, was evaluated for the capacity of a single intraperitoneal administration to persistently suppress peritoneal tumor growth in an intraperitoneal model of ovarian carcinomatosis with human ovarian cancer cells in nude immunodeficient mice., Results: The data demonstrates that AAVrh10.BevMab mediates persistent and high levels of bevacizumab in the peritoneal cavity following a single intraperitoneal administration in mice. In AAVrh10.BevMab treated A2780 human ovarian cancer-bearing mice, tumor growth was significantly suppressed (p<0.05) and the area of blood vessels in the tumor was decreased (p<0.04). Survival of mice with A2780 xenografts or SK-OV3 xenografts was greatly prolonged in the presence of AAVrh10.BevMab (p<0.001). Administration of AAVrh10.BevMab 4days after A2780-luciferase cell implantation reduced tumor growth (p<0.01) and increased mouse survival (p<0.0001). Combination of AAVrh10.BevMab with cytotoxic reagents paclitaxel or topotecan proved to be more effective in increasing survival than treatment with cytotoxic reagent alone., Conclusion: A single administration of AAVrh10.BevMab provides sustained and high local expression of bevacizumab in the peritoneal cavity, and significantly suppresses peritoneal carcinomatosis and increases survival in an ovarian cancer murine model., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS administration into the white matter is likely to be safe and yields the widest distribution of ARSA, making it the most suitable route of vector delivery.

Alpha-1 antitrypsin (α1AT) deficiency is a common autosomal recessive disorder characterized by a marked reduction in serum α1AT levels, lung and liver disease. α1AT is mainly produced and secreted by hepatocytes, with its primary function to protect the lung against the proteolytic activity of neutrophil elastase. Serum α1AT levels <11 μM are associated with progressive destruction of lung parenchyma and early-onset of panacinar emphysema in the age range 35-45. The current approved treatment for α1AT deficiency is a costly protein augmentation therapy requiring weekly intravenous infusion of purified α1AT from pooled human plasma. Gene therapy offers the advantage of a single vector administration, eliminating the burden of the repeated purified protein infusions, with the consequent reduced overall drug cost and improved compliance. We have developed a novel, highly efficient gene therapy approach for α1AT deficiency based on the administration of AAVrh.10hα1AT, an adeno-associated viral vector serotype rh.10 coding for normal M-type human α1AT via the intrapleural route. On the basis of prior murine studies, this approach provides sustained α1AT proximal to the lung with a highly efficient vector. In support of a clinical trial for this approach, we carried out a study to assess the safety of intrapleural administration of AAVrh.10hα1AT to 280 mice and 36 nonhuman primates. The data demonstrate that this approach is safe, with no toxicity issues. Importantly, there was persistent expression of the human α1AT mRNA in chest cavity cells for the duration of the study (6 months in mice and 1 year in nonhuman primates). Together, these data support the initiation of a clinical trial of intrapleural human AAVrh.10hα1AT for the treatment of α1AT deficiency.

Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.

Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of neovascular age-related macular degeneration and diabetic retinopathy. Bevacizumab, an anti-VEGF monoclonal antibody, is efficacious for these disorders, but requires monthly intravitreal administration, with associated discomfort, cost, and adverse event risk. We hypothesized that a single intravitreal administration of adeno-associated virus (AAV) vector expressing bevacizumab would result in persistent eye expression of bevacizumab and suppress VEGF-induced retinal neovascularization. We constructed an AAV rhesus serotype rh.10 vector to deliver bevacizumab (AAVrh.10BevMab) and assessed its ability to suppress neovascularization in transgenic mice overexpressing human VEGF165 in photoreceptors. Intravitreal AAVrh.10BevMab directed long-term bevacizumab expression in the retinal pigmented epithelium. Treated homozygous mice had reduced levels of neovascularization, with 90±4% reduction 168 days following treatment. Thus, a single administration of AAVrh.10BevMab provides long-term suppression of neovascularization without the costs and risks associated with the multiple administrations required for the current conventional bevacizumab monoclonal drug delivery.

Object: The authors conducted a phase I study of late infantile neuronal ceroid lipofuscinosis using an adenoassociated virus serotype 2 (AAV2) vector containing the deficient CLN2 gene (AAV2(CU)hCLN2). The operative technique, radiographic changes, and surgical complications are presented., Methods: Ten patients with late infantile neuronal ceroid lipofuscinosis disease each underwent infusion of AAV2(CU)hCLN2 (3 x 10(12) particle units) into 12 distinct cerebral locations (2 depths/bur hole, 75 minutes/infusion, and 2 microl/minute). Innovative surgical techniques were developed to overcome several obstacles for which little or no established techniques were available. Successful infusion relied on preoperative stereotactic planning to optimize a parenchymal target and diffuse administration. Six entry sites, each having 2 depths of injections, were used to reduce operative time and enhance distribution. A low-profile rigid fixation system with 6 integrated holding arms was utilized to perform simultaneous infusions within a practical time frame. Dural sealant with generous irrigation was used to avoid CSF egress with possible subdural hemorrhage or altered stereotactic registration., Results: Radiographically demonstrated changes were seen in 39 (65%) of 60 injection sites, confirming localization and infusion. There were no radiographically or clinically defined complications., Conclusions: The neurosurgical considerations and results of this study are presented to offer guidance and a basis for the design of future gene therapy or other clinical trials in children that utilize direct therapeutic delivery.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal childhood neurodegenerative lysosomal storage disease with no known therapy. There are estimated to be 200 to 300 children in the United States at any one time with the disease. LINCL is a genetic disease resulting from a deficiency of tripeptidyl peptidase I (TPP-I), a proteolytic enzyme encoded by CLN2, the gene that is mutated in individuals with LINCL. The subjects are chronically ill, with a progressive CNS disorder that invariably results in death, typically by age 8 to 12 years. The strategy of this clinical study is based on the concept that persistent expression in the CNS of the normal CLN2 cDNA with production of sufficient amounts of TPP-I should prevent further loss of neurons, and hence limit disease progression. To assess this concept, an adeno-associated virus vector (AAV2CUh-CLN2) will be used to transfer to and express the human CLN2 cDNA in the brain of children with LINCL. The vector consists of the AAV2 capsid enclosing the 4278-base single-stranded genome consisting of the two inverted terminal repeats of AAV serotype 2 and an expression cassette composed of the human cytomegalovirus (CMV) enhancer, the chicken beta-actin promoter/splice donor and 5' end of the intron, the 3' end of the rabbit P-globin intron and splice acceptor, the human CLN2 cDNA with an optimized Kozak translation initiation signal, and the polyadenylation/transcription stop codon from rabbit 3-globin. The proposed study will include 10 individuals and will be divided into two parts. Group A, to be studied first, will include four individuals with the severe form of the disease. Group B of the trial will include six individuals with a moderate form of the disease. After direct intracranial administration of the vector, there will be neurological assessment based on the LINCL clinical rating scale and magnetic resonance imaging/magnetic resonance spectroscopy assessment of the brain in regions of vector administration. The data generated will help evaluate two hypotheses: (1) that it is safe to carry out direct intracranial administration of the AAV2cuhCLN2 vector to the CNS of individuals with LINCL, and (2) that administration of the AAV2cuhCLN2 vector will slow down or halt the progression of the disease in the central nervous system.