Richard O. Snyder

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.

Haemophilia B, or factor IX deficiency, is an X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5–30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery1. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral2 and adenoviral3 vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression3,4. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells5. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues6‐12. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Epitope Mapping of Human Anti-Adeno-Associated Virus Type 2 Neutralizing Antibodies: Implications for Gene Therapy and Virus Structure

ABSTRACT Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

ABSTRACT Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Adeno-Associated Virus Vector Genomes Persist as Episomal Chromatin in Primate Muscle

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 1012 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint.

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease.

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinsons disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.

Gene therapy for aromatic L-amino acid decarboxylase deficiency.

Gene therapy can restore some motor function in patients with aromatic l-amino acid decarboxylase deficiency. Gene Therapy for AADC Deficiency Patients with aromatic l-amino acid decarboxylase (AADC) deficiency cannot produce the neurotransmitter dopamine from its precursor l-DOPA in the brain. Dopamine is a crucial molecule required for normal motor function. There are few treatment options for AADC deficiency, and most patients afflicted with this rare disease die in childhood. In a phase 1 clinical trial, Hwu and colleagues use an adeno-associated virus (AAV) type 2 vector to deliver the AADC gene into a brain area called the putamen in four children with AADC deficiency. Although at first the patients exhibited dyskinesias (abnormal muscle movements), these resolved after a few months and the patients showed improved motor function. One patient after 16 months was able to stand, and the other three patients were able to sit upright with support. Several other symptoms improved as well including mood and oculogyric crises. There were a number of translational challenges for this gene therapy clinical trial. For example, the authors had to work out how to deliver the viral vector carrying the therapeutic gene directly into the putamen, and even then only a small part of the putamen became transduced with the AADC gene. Also, because the patients’ brains had not been able to make dopamine, it was not clear how the neurons would respond once dopamine started to be produced. Despite these challenges, this first-in-human gene therapy clinical trial suggests that targeting localized areas in the brain with a therapeutic gene delivered by an AAV vector could help ameliorate the symptoms of AADC deficiency and may also be useful for treating other diseases caused by lack of a crucial enzyme in brain tissue. Aromatic l-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector–mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[18F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.

Nerve Growth Factor Expressed in the Medial Septum Followingin VivoGene Delivery Using a Recombinant Adeno-Associated Viral Vector Protects Cholinergic Neurons from Fimbria-Fornix Lesion-Induced Degeneration

Nerve growth factor (NGF) has been shown to support the survival of axotomized medial septal cholinergic neurons after aspirative lesions of the fimbria-fornix (FF). This survival effect has been achieved utilizing intraventricular and intraparenchymal delivery of the NGF protein. While the use of NGF for the treatment of the cholinergic deficits present in Alzheimers disease shows promise based on its efficacy in animal models, concerns about side-effects of intraventricular NGF delivery in humans have been raised. In the present study, NGF was delivered directly to the medial septum via a recombinant adeno-associated viral vector (rAAV) encoding the cDNA for human NGF prior to a FF lesion in rats. This rAAV-mediated NGF delivery was shown to significantly attenuate the medial septal cholinergic cell loss observed in animals receiving an equivalent injection of a control rAAV vector.

Safety and Efficacy of Regional Intravenous (RI) Versus Intramuscular (IM) Delivery of rAAV1 and rAAV8 to Nonhuman Primate Skeletal Muscle

We developed a drug-free regional intravenous (RI) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (IM) delivery of the same dose of vector. We show that RI delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After IM, muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although RI delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that RI is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.We developed a drug-free regional intravenous (r.i.) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (i.m.) delivery of the same dose of vector. We show that r.i. delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After i.m., muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although r.i. delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that r.i. is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.