Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

Positron Emission Tomography Quantitative Assessment of Off-Target Whole-Body Biodistribution of I-124-Labeled Adeno-Associated Virus Capsids Administered to Cerebral Spinal Fluid.

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.

Assessment of Safety and Biodistribution of AAVrh.10hCLN2 Following Intracisternal Administration in Nonhuman Primates for the Treatment of CLN2 Batten Disease.

CLN2 disease is a fatal, childhood autosomal recessive disorder caused by mutations in ceroid lipofuscinosis type 2 (CLN2) gene, encoding tripeptidyl peptidase 1 (TPP-1). Loss of TPP-1 activity leads to accumulation of storage material in lysosomes and resultant neuronal cell death with neurodegeneration. Genotype/phenotype comparisons suggest that the phenotype should be ameliorated with increase of TPP-1 levels to 5-10% of normal with wide central nervous system (CNS) distribution. Our previous clinical study showed that intraparenchymal (IPC) administration of AAVrh.10hCLN2, an adeno-associated vector serotype rh.10 encoding human CLN2, slowed, but did not stop disease progression, suggesting that this may be insufficient to distribute the therapy throughout the CNS (Sondhi 2020). In this study, we assessed whether the less invasive intracisternal delivery route would be safe and provide a wider distribution of TPP-1. A study was conducted in nonhuman primates (NHPs) with intracisternal delivery to cerebrospinal fluid (CSF) of AAVrh.10hCLN2 (5 × 1013 genome copies) or phosphate buffered saline (PBS). No abnormal behavior was noted. CNS magnetic resonance imaging and clinical chemistry data were all unremarkable. Histopathology of major organs had no abnormal finding attributable to the intervention or the vector, except that in one out of two animals treated with AAVrh.10hCLN2, dorsal root ganglia showed mild-to-moderate mononuclear cell infiltrates and neuronal degeneration. In contrast to our previous NHP study (Sondhi 2012) with IPC administration where TPP-1 activity was >2 × above controls in 30% of treated brains, in the two intracisternal treated NHPs, the TPP-1 activity was >2 × above controls in 50% and 41% of treated brains, and 52% and 84% of brain had >1,000 vector genomes/µg DNA, compared to 0% in the two PBS NHP. CSF TPP1 levels in treated animals were 43-62% of normal human levels. Collectively, these data indicate that AAVrh.10hCLN2 delivered by intracisternal route is safe and widely distributes TPP-1 in brain and CSF at levels that are potentially therapeutic. Clinical Trial Registration: NCT02893826, NCT04669535, NCT04273269, NCT03580083, NCT04408625, NCT04127578, and NCT04792944.

Assessment of Residual Full-Length SV40 Large T Antigen in Clinical-Grade Adeno-Associated Virus Vectors Produced in 293T Cells.

Efficient production of adeno-associated virus (AAV) vectors is a significant challenge. Human embryonic kidney HEK293T cells are widely used in good manufacturing practice facilities, producing higher yield of AAV vectors for clinical applications than HEK293 through the addition of a constitutive expression of SV40 large T antigen (SV40T), which stimulates Rep expression. However, the theoretical potential for tumorigenic consequences of a clinical AAV product containing residual DNA encoding SV40T, which may inhibit p53 growth suppressive functions is a safety concern. Although the risk is theoretical, to assure a low risk/high confidence of safety for clinical drug development, we have established a sensitive assay for assessment of functional full-length transcription competent SV40T DNA in HEK293T cell-produced AAV vectors. Using HEK293T generated 8, 9, and rh.10 serotype AAV vectors, the presence of SV40T in purified vector was assessed in vitro using quantitative polymerase chain reaction (qPCR) targeting a 129 bp amplicon combined with nested PCR targeting full-length SV40T DNA. Although low levels of the smaller amplicon were present in each AAV serotype, the full-length SV40T was undetectable. No transcription competent full-length SV40T DNA was observed by reverse transcription-quantitative polymerase chain reaction using an in vivo amplification of signal in mouse liver administered (2-10 × 1010 gc) 129 bp amplicon-positive AAV vectors. As a control for gene transfer, high levels of expressed transgene mRNAs were observed from each serotype AAV vector, yet, SV40T mRNA was undetectable. In vivo assessment of these three liver-tropic AAV serotypes, each with amplicon-positive qPCR SV40T DNA, demonstrated high transgene mRNA expression but no SV40T mRNA, that is, detection of small segments of SV40T DNA in 293T cell produced AAV inappropriately leads to the conclusion of residuals with the potential to express SV40T. This sensitive assay can be used to assess the level, if any, of SV40T antigen contaminating AAV vectors generated by HEK293T cells. ClinicalTrials.gov identifier: NCT03634007; NCT05302271; NCT01414985; NCT01161576.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

Preclinical safety and biodistribution assessment of Ad-KCNH2-G628S administered via atrial painting in New Zealand white rabbits.

Post-operative atrial fibrillation (POAF) is the most common complication after cardiac surgery. Despite implementation of several pharmacological strategies, incidence of POAF remains at approximately 30%. An adenovirus vector encoding KCNH2-G628S has proven efficacious in a porcine model of AF. In this preclinical study, 1.5 × 1010 or 1.5 × 1012 Ad-KCNH2-G628S vector particles (vp) were applied to the atrial epicardium or 1.5 × 1012 vp were applied to the whole epicardial surface of New Zealand White rabbits. Saline and vector vehicle served as procedure controls. Animals were followed for up to 42 days. Vector genomes persisted in the atria up to 42 days, with no distribution to extra-thoracic organs. There were no adverse effects attributable to test article on standard toxicological endpoints or on blood pressure, left atrial or ventricular ejection fractions, electrocardiographic parameters, or serum IL-6 or troponin concentrations. Mononuclear infiltration of the myocardium of the atrial free walls of low-dose, but not high-dose animals was observed at 7 and 21 days, but these changes did not persist or affect cardiac function. After scaling for heart size, results indicate the test article is safe at doses up to 25 times the maximum proposed for the human clinical trial.

Safety of Intravenous Administration of an AAV8 Vector Coding for an Oxidation-Resistant Human α1-Antitrypsin for the Treatment of α1-Antitrypsin Deficiency.

α1-antitrypsin (AAT) deficiency is a common autosomal recessive hereditary disorder, with a high risk for the development of early-onset panacinar emphysema. AAT, produced primarily in the liver, functions to protect the lung from neutrophil protease; with AAT deficiency, unimpeded neutrophil proteases destroy the lung parenchyma. AAT is susceptible to oxidative damage resulting in an inability to inhibit its target proteases, neutrophil elastase, and cathepsin G. The major sites of oxidative modification on the AAT molecule are methionine residues 351 and 358. We have previously demonstrated that an engineered variant of AAT that resists oxidation by modifying both protein surface methionines (M351V and M358L) provides antiprotease protection, despite oxidative stress. In mice, intravenous delivery of the modified AAT(AVL) variant by AAV serotype 8, AAV8hAAT(AVL), primarily to the liver resulted in long-term expression of an AAT that resists oxidative inactivation. In this study, we evaluated the safety of intravenous administration of AAV8hAAT(AVL) in a dose-escalating, blinded, placebo-controlled toxicology study in wild-type mice. The study assessed organ histology and clinical pathology findings of mice, intravenously administered AAV8hAAT(AVL) at three doses (5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 genome copies [gc]/kg), compared to control mice injected intravenously with phosphate-buffered saline. As previously demonstrated, administration of AAV8hAAT(AVL) resulted in dose-dependent expression of high, potentially therapeutic, levels of serum human AAT protein that persist for at least 6 months. Antibodies against the AAV8 capsid were elicited as expected, but there was no antibody detected against the AAT(AVL) protein generated by the AAV8hAAT(AVL) vector. There was no morbidity or mortality observed in the study. The data demonstrate that intravenous administration of AAV8hAAT(AVL) is safe with no significant adverse effect attributed to AAV8hAAT(AVL) vector at any dose. This study demonstrates that AAV8hAAT(AVL) has a safety profile consistent with the requirements for proceeding to a clinical study.

Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results demonstrate that AAVrh.10hFXN may induce expression of therapeutic levels of mature hFXN in mice.

Genetic Modification of the AAV5 Capsid with Lysine Residues Results in a Lung-Tropic Liver-Detargeted Gene Transfer Vector.

Intravenous (IV) administration of naturally occurring adeno-associated virus (AAV) vectors are liver tropic, with a significant proportion of the total vector dose mediating gene expression in liver hepatocytes. AAV capsids that are directed toward other organs such as lung may be useful for therapy of nonliver-based diseases. Based on the knowledge that the lung capillary endothelium is the first capillary bed encountered by an intravenously administered AAV vector, and that the lung endothelium glycocalyx is enriched in negatively charged sialic acid, we hypothesized that adding positively changed lysine residues to the AAV capsid would enhance AAV biodistribution to the lung after IV administration. Using site-directed mutagenesis, two lysine residues were inserted into variable loop VIII of the AAV serotype 5 capsid (AAV5-PK2). Organ distribution of AAV5-PK2 was compared with that of AAV5, AAV2, and AAV2-7m8 4 weeks after IV administration (1011 gc) to C57Bl/6 male mice. As predicted, after IV administration, AAV5-PK2 had the highest biodistribution in the lung (p < 0.02 compared with AAV5, AAV2, and AAV2-7m8). Furthermore, biodistribution to liver of AAV5-PK2 was 2 logs decreased compared with AAV5 (p < 10-4) with a ratio of AAV5-PK2 lung to liver of 62-fold compared with AAV5 of 0.2-fold (p < 0.0003). The AAV5-PK2 capsid represents a lung-tropic AAV vector that is also significantly detargeted from the liver, a property that may be useful in lung-directed gene therapies.

Long-term functional correction of cystathionine ß-synthase deficiency in mice by adeno-associated viral gene therapy.

Cystathionine ß-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 × 1011 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.

Automated Retinal Layer Segmentation in CLN2-Associated Disease: Commercially Available Software Characterizing a Progressive Maculopathy.

Purpose: CLN2-associated disease is a hereditary, fatal lysosomal storage disorder characterized by progressive brain and retinal deterioration. Here, we characterize the inner and outer retinal degeneration using automated segmentation software in optical coherence tomography scans, providing an objective, quantifiable metric for monitoring subtle changes previously identified with a validated disease classification scale (the Weill Cornell Batten Scale). Methods: This study is a retrospective, single-center cohort review of images from examinations under anesthesia in treatment-naïve patients with CLN2-associated disease. Automated segmentation software was used to delineate retinal nerve fiber, ganglion cell layer (GCL), and outer nuclear layer (ONL) thickness measurements in the fovea, parafovea, and perifovea based on age groups (months): 30 to 38, 39 to 45, 46 to 52, 53 to 59, 60 to 66, and 67 or older. Results: Twenty-seven eyes from 14 patients were included, with 8 serial images yielding 36 interpretable optical coherence tomography scans. There was a significant difference in parafoveal ONL thickness between 39 to 45 and 46 to 52 months of age (P = 0.032) not seen in other regions or retinal layers. Perifoveal ONL demonstrated a difference in thickness between the 60 to 66 and greater than 67 months age cohorts (P = 0.047). There was strong symmetry between eyes, and high segmentation repeatability. Conclusions: Parafoveal ONL thickness represents a sensitive, early age indicator of CLN2-associated degeneration. Outer retinal degeneration is apparent at younger ages than inner retinal changes though in treatment-naïve patients all retinal layers showed significant differences between 60 to 66 and more than 67 months of age. Translational Relevance: This study establishes sensitive, quantitative biomarkers for assessing retinal degeneration in a large cohort natural history study in anticipation of future clinical trials.

Safety of Direct Intraparenchymal AAVrh.10-Mediated Central Nervous System Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy, a fatal pediatric neurodegenerative lysosomal storage disease caused by mutations in the arylsulfatase A (ARSA) gene, is characterized by intracellular accumulation of sulfatides in the lysosomes of cells of the central nervous system (CNS). In previous studies, we have demonstrated efficacy of AAVrh.10hARSA, an adeno-associated virus (AAV) serotype rh.10 vector coding for the human ARSA gene to the CNS of a mouse model of the disease, and that catheter-based intraparenchymal administration of AAVrh.10hARSA to the CNS of nonhuman primates (NHPs) white matter results in widespread expression of ARSA. As a formal dose-escalating safety/toxicology study, we assessed the safety of intraparenchymal delivery of AAVrh.10hARSA vector to 12 sites in the white matter of the CNS of NHPs at 2.85 × 1010 (total low dose, 2.4 × 109 genome copies [gc]/site) and 1.5 × 1012 (total high dose, 1.3 × 1011 gc/site) gc, compared to AAVrh.10Null (1.5 × 1012 gc total, 1.3 × 1011 gc/site) as a vector control, and phosphate buffered saline for a sham surgical control. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA. However, animals treated with the high dose AAVrh.10ARSA and the high dose Null vector had highly localized CNS abnormalities on magnetic resonance imaging scans at the sites of catheter infusions, and histopathology demonstrated that these sites were associated with infiltrates of T cells, B cells, microglial cells, and/or macrophages. Although these findings had no clinical consequences, these safety data contribute to understanding the dose limits for CNS white matter direct intraparenchymal administration of AAVrh.10 vectors for treatment of CNS disorders.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

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.

Cocaine vaccine dAd5GNE protects against moderate daily and high-dose "binge" cocaine use.

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose.

Quantitative Whole-Body Imaging of I-124-Labeled Adeno-Associated Viral Vector Biodistribution in Nonhuman Primates.

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.

Gene therapy for alpha 1-antitrypsin deficiency with an oxidant-resistant human alpha 1-antitrypsin.

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.

Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy.

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 1011 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.

Symmetric Age Association of Retinal Degeneration in Patients with CLN2-Associated Batten Disease.

PURPOSE: Mutations in the CLN2 gene lead to a neurodegenerative and blinding lysosomal storage disorder: late infantile neuronal ceroid lipofucinosis, also known as "CLN2 disease." The purpose of the current study was to characterize the evolution of CLN2-associated retinal manifestations using the Weill Cornell Batten Scale (WCBS) and the age association of the retinal degeneration using central subfield thickness (CST) measurements and then correlate these findings with fundus photography and OCT to determine a critical period for retinal intervention. DESIGN: Retrospective, single-center cohort. PARTICIPANTS: Eighty-four eyes of 42 treatment-naïve patients with CLN2 disease. METHODS: Clinical records, fundus photographs, and OCT imaging for patients with CLN2 disease collected during examinations under anesthesia were reviewed. Imaging was categorized per WCBS criteria by 3 masked graders. MAIN OUTCOME MEASURES: CLN2-associated retinopathy assessed using WCBS scores, fundus photographs, and OCT imaging, correlated with patient age. RESULTS: Eighty-four eyes of 42 patients had baseline fundus photographs, with baseline OCT in 31 eyes of 16 patients. Fundus photographs were obtained serially for 26 eyes of 13 patients, and serial OCT scans were obtained in 10 eyes of 5 patients. At baseline, bilateral WCBS scores were highly correlated for OCT and fundus photographs (r = 0.96 and 0.82, respectively). Central subfield thickness was negatively correlated with left and right eye WCBS OCT scores (r = -0.92 and -0.83, respectively; P < 0.001) and fundus photograph scores (r = -0.80 and -0.83, respectively; P < 0.001). OCT thickness was symmetrical between each eye. Baseline OCT data with age fit using a sigmoid function demonstrated a period of accelerated loss between 48 and 72 months of age. CONCLUSIONS: Retinal degeneration associated with CLN2 disease manifests as a progressive, symmetrical decline, which appears to accelerate during a critical period at 48 to 72 months of age, suggesting intervention with retina-specific CLN2 gene therapy should occur ideally before or as early as possible within this critical period. The WCBS is a valuable tool and is highly correlated with the extent of retinal degeneration observed in OCT or fundus photographs; by using the fellow eye as a control, this grading scale can be used to monitor the effect of CLN2 gene therapy in future trials.

Anti-Phospho-Tau Gene Therapy for Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors' characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.