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Adeno-associated virus (AAV)-mediated gene therapies have provided promising treatments for numerous neurological disorders. Redosing of AAV to the central nervous system (CNS) is an attractive research area due to both the somewhat immunologically privileged status of the CNS as well as the possibility of reduced glial transgene expression over time following a single injection. Continued study of the immune responses to both intraparenchymal and intra-CSF delivery of AAV mediated gene therapies, as well as the continued study of immunosuppressive regimens, could allow for eventual redosing in patients.

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Thalamic infusion of adeno-associated viral (AAV) vectors has been shown to have therapeutic effects in neuronopathic lysosomal storage diseases. Preclinical studies in sheep model of Tay-Sachs disease demonstrated that bilateral thalamic injections of AAV gene therapy are required for maximal benefit. Translation of thalamic injection to patients carries risks in that (1) it has never been done in humans, and (2) dosing scale-up based on brain weight from animals to humans requires injection of larger volumes. To increase the safety margin of this infusion, a flexible cannula was selected to enable simultaneous bilateral thalamic infusion in infants while monitoring by imaging and/or to enable awake infusions for injection of large volumes at low infusion rates. In this study, we tested various infusion volumes (200-800 μL) and rates (0.5-5 μL/min) to determine the maximum tolerated combination of injection parameters. Animals were followed for ∼1 month postinjection with magnetic resonance imaging (MRI) performed at 14 and 28 days. T1-weighted MRI was used to quantify thalamic damage followed by histopathological assessment of the brain. Trends in data show that infusion volumes of 800 μL (2 × the volume required in sheep based on thalamic size) resulted in larger lesions than lower volumes, where the long infusion times (between 13 and 26 h) could have contributed to the generation of larger lesions. The target volume (400 μL, projected to be sufficient to cover most of the sheep thalamus) created the smallest lesion size. Cannula placement alone did result in damage, but this is likely associated with an inherent limitation of its use in a small brain due to the length of the distal rigid portion and lack of stable fixation. An injection rate of 5 μL/min at a volume ∼1/3 of the thalamus (400-600 μL) appears to be well tolerated in sheep both clinically and histopathologically.

Adeno-associated virus (AAV) gene therapy for neurological diseases was revolutionized by the discovery that AAV9 crosses the blood-brain barrier (BBB) after systemic administration. Transformative results have been documented in various inherited diseases, but overall neuronal transduction efficiency is relatively low. The recent development of AAV-PHP.B with ∼60-fold higher efficiency than AAV9 in transducing the adult mouse brain was the major first step toward acquiring the ability to deliver genes to the majority of cells in the central nervous system (CNS). However, little is known about the mechanism utilized by AAV to cross the BBB, and how it may diverge across species. In this study, we show that AAV-PHP.B is ineffective for systemic CNS gene transfer in the inbred strains BALB/cJ, BALB/cByJ, A/J, NOD/ShiLtJ, NZO/HILtJ, C3H/HeJ, and CBA/J mice, but it is highly potent in C57BL/6J, FVB/NJ, DBA/2J, 129S1/SvImJ, and AKR/J mice and also the outbred strain CD-1. We used the power of classical genetics to uncover the molecular mechanisms AAV-PHP.B engages to transduce CNS at high efficiency, and by quantitative trait locus mapping we identify a 6 Mb region in chromosome 15 with an logarithm of the odds (LOD) score ∼20, including single nucleotide polymorphisms in the coding region of 9 different genes. Comparison of the publicly available data on the genome sequence of 16 different mouse strains, combined with RNA-seq data analysis of brain microcapillary endothelia, led us to conclude that the expression level of Ly6a is likely the determining factor for differential efficacy of AAV-PHP.B in transducing the CNS across different mouse strains.

Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and the central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease, since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse (B6;129-Gaa Tm1Rabn /J), this study sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. Three-month-old Gaa -/- animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6 months later. Both AAV vectors prolonged survival. AAVB1-treated animals had a robust weight gain compared to the AAV9-treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated that both vectors transduced the heart efficiently, leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1-treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1-treated animals showed improved respiratory function comparable to wild-type animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and β subunits separately (TSD α + β) injected at high (1.3 × 10 13 vector genomes) or low (4.2 × 10 12 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + β sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + β), and ganglioside clearance was most widespread in the TSD α + β high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in β-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or β-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and β-subunits. Three doses (3.2 × 10 12 vg [n = 3]; 3.2 × 10 11 vg [n = 2]; or 1.1 × 10 11 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/β developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/β, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/β intracranial injection among different species, despite encoding for self-proteins.

Recently, the gene therapy field has begun to experience clinical successes in a number of different diseases using various approaches and vectors. The workshop Gene Therapy: Charting a Future Course, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities, brought together early and mid-career researchers to discuss the key scientific challenges and opportunities, ethical and communication issues, and NIH and foundation resources available to facilitate further clinical advances.

Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function.

Convection-enhanced delivery (CED) of recombinant adeno-associated virus (rAAV) vectors is a promising approach for delivery of therapeutic transgenes to the brain. In this study we have systematically examined vector dosing in vivo. Infusions of rAAV serotypes 2/1, 2/2, 2/rh8, 2/9, and 2/rh10 expressing an enhanced green fluorescent protein reporter gene were undertaken into the striatum of rats and pigs using CED. Vector distribution, as defined by the volume of distribution and number of transduced cells following each infusion, was determined using stereological methods. Immunohistochemistry was used to determine the transductional tropism of serotypes and to evaluate for the presence of immune cell infiltration into the brain. Vector distribution was highly variable between serotypes. Infusion rate had no significant effect on vector distribution or the occurrence of tissue damage. For serotypes 2/1, 2/2 and 2/rh10, as the vector concentration was increased beyond 10(12) vg/ml, no increase in vector distribution was observed. In contrast, for serotypes 2/rh8 and 2/9, retrograde axonal transport was observed above this threshold concentration. Cell transduction was principally neuronal for all serotypes and was associated with a low-level immune response. In planning clinical trials it is critical that these observations are considered in order to achieve optimal vector dosing.

The development and use of gene transfer techniques creates an opportunity to achieve better treatment modalities for numerous disease entities. Promising results for treatment in tumor cells in culture and in small animal models have been reported. Nevertheless, the lack of widespread vector distribution throughout tumor tissue is one of the current limitations for successful clinical application of gene therapy paradigms. The use of migratory tumor cells themselves as vector delivery vehicles may allow wider vector distribution in tumors. In addition, continuous release of retrovirus vectors on-site could generate a high local virion concentration over an extended time period with consequent increases in transduction efficiency. In this paper, we present in culture and in vivo data of a herpes simplex virus-Epstein-Barr virus hybrid amplicon vector containing retrovirus vector components (tribrid vector) that allows conversion of tumor cells into retroviral producer cells. With this method, we were able to achieve a local fourfold amplification of stable transgene expression in tumors. The application of this system, which can integrate a transgene cassette into tumors with therapeutic bystander effects, could increase the local amplification effect to a level of clinical relevance.

Gene therapy for the nervous system is a newly emerging field with special issues related to modes of delivery, potential toxicity, and realistic expectations for treatment of this vital and highly complex tissue. This review focuses on the potential for gene delivery to the brain, as well as possible risks and benefits of these procedures. This includes discussion of appropriate vectors, such as adeno-associated virus, lentivirus, gutless adenovirus, and herpes simplex virus hybrid amplicons, and cell vehicles, such as neuroprogenitor cells. Routes of delivery for focal and global diseases are enumerated, including use of migratory cells, facilitation of vascular delivery across the blood-brain barrier, cerebrospinal fluid delivery, and convection injection. Attention is given to examples of diseases falling into different etiologic types: metabolic deficiency states, including Canavan disease and lysosomal storage disorders; and degenerative conditions, including Parkinson's disease and other neurodegenerative conditions.

Mutations in the alpha-chain of lysosomal hexosaminidase (EC 3.2.1.52) underlie two distinct biochemical phenotypes known as variant B and variant B1 of G(M2) gangliosidosis. This paper shows that the transduction of human B1-type fibroblasts (producing catalytically inactive alpha-chains) with a retroviral vector encoding the human hexosaminidase alpha-chain leads to a complete correction of HexA (alpha beta dimer) activity with both synthetic and natural substrates. The alpha-subunit overexpression leads to a partial HexB (beta beta dimer) depletion corresponding to about 10% of control HexB activity. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. The high levels of recombinant enzyme correctly produced the metabolic defect, enabling the cells efficiently to degrade the accumulated storage product present in lysosomes. The transduced fibroblasts are also able to secrete HexA efficiently into the culture medium. Moreover, transfer of the human transgene product to B1-type deficient fibroblasts lead to an increase of activity against 4MUGS, the alpha-chain specific synthetic substrate, up to 30% of the control mean activity level. This level of activity might be sufficient to restore the normal ganglioside G(M2) metabolism in recipient cells. The data obtained demonstrate that B1-type phenotype can be efficiently corrected by retrovirus-mediated gene transfer.

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.

Brain tumors that have disseminated into cerebrospinal fluid (CSF) pathways are an unresolved therapeutic problem, especially in pediatric neurooncology. Here a gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) paradigm was tested using an HSV vector in a rodent model of disseminated central nervous system tumors. 9L-gliosarcoma cells were implanted simultaneously into the brain and the CSF of syngeneic rats. Five days later, resulting intracerebral and leptomeningeal tumors were treated by intrathecal injection of a replication-conditional HSV vector. This vector was defective for the ribonucleotide reductase gene, but contained an intact HSV-tk gene. Systemic GCV treatment was started 2 days after vector application and continued for 14 days. Tumor-free, long-term survival (LTS) was achieved in 90% of the animals treated with this combined therapeutic approach, whereas only 30% LTS was found in animals that had received the vector alone and 10% LTS in untreated animals. This therapeutic response probably involves oncolytic, on-site replication of the vector, activation of GCV by a HSV-TK, and a strong immune response both to the vector and to 9L cells. Apparent vector-related mortality was observed in 20% of animals without subsequent GCV therapy, but no vector-related mortality was found when the animals were treated with GCV after vector application. Given the successful outcome of this experimental treatment and the apparent potential of GCV to control HSV-related toxicity, intrathecal application of HSV vectors combined with GCV treatment may be a promising approach for treatment of disseminated brain tumors.

The present study investigated the ability of a recombinant herpes simplex virus type 1 (HSV) vector to deliver genes into disseminated brain tumor foci through intrathecal injection of the vector. The animal model was designed to simulate brain tumors with cerebrospinal fluid (CSF) metastases, which are found especially in the pediatric population. 9L gliosarcoma cells were injected both into the right frontal lobe and in through the cisterna magna of adult rats. The HSV vector, hrR3, was inoculated intrathecally 5 days later. This vector is defective in the gene for ribonucleotide reductase, and, therefore, replicates preferentially in dividing cells; it retains an intact HSV-thymidine kinase gene (HSV-tk). Two days after injection of the vector, immunohistochemical staining for HSV thymidine kinase (HSV-TK) revealed expression in frontal tumors, as well as in leptomeningeal tumor foci along the entire neuroaxis. HSV-TK-immunopositive cells were most frequent in small tumors contacting the CSF pathways. Frontal lobe tumors showed the highest density of HSV-TK-immunopositive cells around their periphery with little expression in central parts. Some paraventricular neurons temporarily showed HSV-TK-immunolabeling at this early time point. The number of HSV-TK-immunopositive tumor cells markedly decreased 5 days after injection of the HSV vector. In all animals, some toxicity was observed in the first 2-4 days after virus injection with extensive leptomeningeal inflammation. In conclusion, intrathecal application of HSV vectors can mediate widespread transfer of the therapeutic HSV-tk gene into disseminated tumors throughout the brain and CSF pathways. Although there was marked toxicity associated with intrathecal injection of this vector, this mode of gene delivery offers a promising approach for treatment of CSF-metastases in conjunction with development of less toxic vectors.