Search

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8 + T cell activation. Alternatively, pDCs may also promote CD8 + T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.

Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans. For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefit with a single treatment. This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity. There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials. The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic l-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Barth syndrome (BTHS) is a rare mitochondrial disease that affects heart and skeletal muscle and has no curative treatment. It is caused by recessive mutations in the X-linked gene TAZ, which encodes tafazzin. To develop a clinically relevant gene therapy to restore tafazzin function and treat BTHS, three different adeno-associated virus serotype 9 vectors were tested and compared to identify the optimal promoter-cytomegalovirus (CMV), desmin (Des), or a native tafazzin promoter (Taz)-for TAZ expression following intravenous administration of 1 × 10 13 vector genomes/kilogram to a mouse model of BTHS as either neonates (1-2 days of age) or adults (3 months of age). At 5 months of age, evaluations of biodistribution and TAZ expression levels, mouse activity assessments, fatigue in response to exercise, muscle strength, cardiac function, mitochondrial structure, oxygen consumption, and electron transport chain complex activity assays were performed to measure the extent of improvement in treated mice. Each promoter was scored for significant improvement over untreated control mice and significant improvement compared with the other two promoters for every measurement and within each age of administration. All three of the promoters resulted in significant improvements in a majority of the assessments compared with untreated BTHS controls. When scored for overall effectiveness as a gene therapy, the Des promoter was found to provide improvement in the most assessments, followed by the CMV promoter, and finally Taz regardless of injection age. This study provides substantial support for translation of an adeno-associated virus serotype 9-mediated TAZ gene replacement strategy using a Des promoter for human BTHS patients in the clinic.

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.

Background: Pompe disease is a fatal neuromuscular disorder caused by a deficiency in acid α-glucosidase, an enzyme responsible for glycogen degradation in the lysosome. Currently, the only approved treatment for Pompe disease is enzyme replacement therapy (ERT), which increases patient survival, but does not fully correct the skeletal muscle pathology. Skeletal muscle pathology is not corrected with ERT because low cation-independent mannose-6-phosphate receptor abundance and autophagic accumulation inhibits the enzyme from reaching the lysosome. Thus, a therapy that more efficiently targets skeletal muscle pathology, such as adeno-associated virus (AAV), is needed for Pompe disease., Objective: The goal of this project was to deliver a rAAV9-coGAA vector driven by a tissue restrictive promoter will efficiently transduce skeletal muscle and correct autophagic accumulation., Methods: Thus, rAAV9-coGAA was intravenously delivered at three doses to 12-week old Gaa-/- mice. 1 month after injection, skeletal muscles were biochemically and histologically analyzed for autophagy-related markers., Results: At the highest dose, GAA enzyme activity and vacuolization scores achieved therapeutic levels. In addition, resolution of autophagosome (AP) accumulation was seen by immunofluorescence and western blot analysis of autophagy-related proteins. Finally, mice treated at birth demonstrated persistence of GAA expression and resolution of lysosomes and APs compared to those treated at 3 months., Conclusion: In conclusion, a single systemic injection of rAAV9-coGAA ameliorates vacuolar accumulation and prevents autophagic dysregulation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10 14 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10 15 VG in crude cell harvests and an average of 3.5 × 10 14 total VG of purified rAAV9 material, resulting in greater than 1 × 10 5 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa -/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 10 12 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step. Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.

Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice.

A biodistribution and toxicology study was performed to test the acute toxicities of intradiaphragmatic injection of a recombinant adeno-associated virus (rAAV) 2/1-human acid alpha-Glucosidase (hGAA) driven by a cytomegalovirus (CMV) promoter (rAAV1-CMV-hGAA) in New Zealand white rabbits and in the rodent Pompe disease model by injecting at the right quadriceps. Studies performed using fluoroscopy and AAV2-GFP demonstrated spread upon intradiaphragmatic injection, and the ability of AAV to infect and express acid α-glucosidase (GAA) throughout the diaphragm. For the preclinical study, 10 rabbits (5 male, 5 female) were divided into two groups, vehicle control (Lactated Ringer's) and test article (1.5×10(12) vector genomes [vg] rAAV1-CMV-hGAA), and euthanized on day 21. After direct visualization, the left hemidiaphragm was injected at three locations. There was up to a 2,500-fold increase in circulating anti-AAV1 antibodies directed to the vector capsids. In addition, up to an 18-fold increase in antibodies against the GAA protein was generated. Injection sites maintained up to 1.0×10(5) vg/μg genomic DNA (gDNA), while uninjected sites had up to 1.0×10(4) vg/μg gDNA. Vector DNA was present in blood at 24 hr postinjection at up to 1.0×10(6) vg/μg gDNA, followed by a decrease to 1.0×10(3) vg/μg gDNA at euthanization on day 21. Nominal amounts of vector DNA were present in peripheral organs, including the brain, spinal cord, gonads, and skeletal muscle. Upon histopathological examination, fibroplasias of the serosal surface were noted at diaphragm injections sites of both groups. In addition, an increase in mononuclear cell infiltration in the diaphragm and esophagus in vector-dosed animals was found. Elevated creatine phosphokinase levels, an indicator of muscle repair, was observed in all animals postprocedure but persisted in vector-injected rabbits until euthanization. A follow-up study suggested that this was directed against the human transgene expression in a foreign species. Overall, this study demonstrates diffusion of vector throughout the diaphragm after localized injections.

Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools. Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 after direct tongue injection in 129SVE mice as well as a mouse model that displays neuromuscular pathology (Gaa(-/-)). Hypoglossal (XII) motoneurons were histologically evaluated 8 weeks after tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken β-actin promoter (1 × 10(11) vector genomes). On average, GFP expression was detected in 234 ± 43 XII motoneurons 8 weeks after AAV9-GFP tongue injection. In contrast, tongue injection with a highly efficient retrograde anatomical tracer (cholera toxin β subunit, CT-β) resulted in infection of 818 ± 88 XII motoneurons per mouse. The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with neuromuscular disease (Gaa(-/-)). Routine hematoxylin and eosin staining and cluster of differentiation (CD) immunostaining for T cells (CD3) indicated no persistent inflammation within the tongue or XII nucleus after AAV9 injection. Additional experiments indicated no adverse effects of AAV9 on the pattern of breathing. We conclude that AAV9 can retrogradely infect a significant portion of a given motoneuron pool in normal and dystrophic mice, and that its transduction efficiency is approximately 30% of what can be achieved with CT-β.

Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.

Since the first demonstration of in vivo gene transfer into myocardium there have been a series of advancements that have driven the evolution of cardiac gene delivery from an experimental tool into a therapy currently at the threshold of becoming a viable clinical option. Innovative methods have been established to address practical challenges related to tissue-type specificity, choice of delivery vehicle, potency of the delivered material, and delivery route. Most importantly for therapeutic purposes, these strategies are being thoroughly tested to ensure safety of the delivery system and the delivered genetic material. This review focuses on the development of recombinant adeno-associated virus (rAAV) as one of the most valuable cardiac gene transfer agents available today. Various forms of rAAV have been used to deliver "pre-event" cardiac protection and to temper the severity of hypertrophy, cardiac ischemia, or infarct size. Adeno-associated virus (AAV) vectors have also been functional delivery tools for cardiac gene expression knockdown studies and successfully improving the cardiac aspects of several metabolic and neuromuscular diseases. Viral capsid manipulations along with the development of tissue-specific and regulated promoters have greatly increased the utility of rAAV-mediated gene transfer. Important clinical studies are currently underway to evaluate AAV-based cardiac gene delivery in humans.

We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-alpha. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver.

Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.

Background: Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein., Major Findings: AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal) was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+) T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer., Conclusions: These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method.

Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30-90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 0.99). Comparisons are drawn between the present work and two other studies of ocular gene therapy for RPE65-LCA that were carried out contemporaneously and reported.

Hepatic gene transfer is envisioned as a substitute for protein replacement therapies, many of which are derived from blood products. Thus, the target populations may have a high prevalence of blood-borne pathogens, such as hepatitis C virus (HCV). We sought to determine whether the safety of recombinant adeno-associated virus serotype 2 (rAAV2) would be altered by preexisting HCV infection. Doses of approximately 1 x 10(13) vector genomes of an rAAV2-chimpanzee alpha(1)-antitrypsin (rAAV2-cAAT) vector were injected into the portal vein of each of three HCV genome-positive (HCV+) chimpanzees and three HCV-negative (HCV-) controls. Acute safety studies were performed up to 90 days after vector administration, along with analyses of the peripheral blood and liver tissue for rAAV2-cAAT genomes. Vector genome copy numbers in blood and liver tissue were similar in both groups. All animals demonstrated increases in liver and muscle enzyme levels after the pretreatment liver biopsy (5 days before vector injection) and after the vector injection. However, HCV+ animals demonstrated a substantially greater rise in aspartate aminotransferase, alanine aminotransferase, and creatinine phosphokinase values than HCV- animals. Histopathology demonstrated abnormal lipid accumulation (steatosis) in the hepatocytes of HCV+ animals, both before and after vector injection. These data indicate an increased susceptibility to subclinical liver toxicity from portal vein injection of rAAV2 in the presence of HCV infection.

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.

To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

Pompe disease is caused by a lack of functional lysosomal acid alpha-glucosidase (GAA) and can ultimately lead to fatal hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore the therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity in vivo in a mouse model of Pompe disease. We have further characterized cardiac and respiratory function in rAAV2/1-treated animals 1 year post-treatment. Similar to the patient population, electrocardiogram measurements (P-R interval) are significantly shortened in the Pompe mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.34+/-1.6 ms, as compared to untreated controls (35.58+/-0.57 ms) (P

Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935-1943) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a "riboswitch," a self-cleaving ribozyme controlled by toyocamycin in the "ON" mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.

Objective: To characterize the biological effects of IM administration of a recombinant adeno-associated virus serotype 2 (rAAV2) vector containing feline erythropoietin (fEPO) cDNA and determine whether readministration of the vector or removal of muscle tissue at the injection sites alters those effects., Animals: 10 healthy 7-week-old specific pathogen-free cats., Procedure: Cats received 1 X 10(7) infective units (iU; n = 3), 1 X 10(8) iU (3), or 1 X 10(9) iU (2) of rAAV2-fEPO vector IM (day 0). Two control cats received an rAAV2 vector containing the LacZ gene (1 X 10(9) iU, IM). In all cats, hematologic variables and serum fEPO concentration were measured at intervals; anti-rAAV2 antibody titer was measured on day 227. In cats that did not respond to treatment, the rAAV2-fEPO vector was readministered. Injection sites were subsequently surgically removed., Results: Compared with control cats, cats treated with 1 X 10(9) iU of rAAV2-fEPO vector had increased Hct and serum fEPO concentrations. One of these cats developed pure RBC aplasia; its Hct normalized following injection site excision. Cats receiving lower doses of vector had no response; on retreatment, 1 of those cats developed sustained erythrocytosis that persisted despite injection site removal and the others did not respond or responded transiently. Antibodies against rAAV2 were detected in all vector-treated cats., Conclusions and Clinical Relevance: Gene therapy may be an effective treatment for cats with hypoproliferative anemia. However, rAAV2-fEPO vector administration may result in pure RBC aplasia or pathologic erythrocytosis, and injection site removal does not consistently abolish the biological response.

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases. We hypothesized that delivery of vectors in a viscous solution would increase transduction by providing a longer exposure time to target cells. We demonstrate that gel-mediated delivery of rAAV serotypes 1, 2, and 5 results in higher transduction efficiencies than free vectors alone when administered in vivo to mouse diaphragms. We further establish greater tropism of rAAV1 vectors for the diaphragm compared to serotypes 2 and 5. This report describes a novel method for efficient delivery of rAAV vectors to the mouse diaphragm and is the first demonstration of gene transfer to the diaphragm using recombinant adeno-associated virus vectors.

A recombinant virus vector constructed from adeno-associated virus (AAV) that has been altered to carry the human alpha1-antitrypsin (hAAT) gene expressed from a hybrid chicken beta-actin promoter with a cytomegalovirus enhancer has been developed. The construct has been shown to initiate the production of hAAT in animal models closely matching the proposed human trial. The proposed clinical trial is an open-label, phase I study administering recombinant adeno-associated virus alpha1-antitrypsin (rAAV2-CB-hAAT) gene vector intramuscularly to AAT-deficient human subjects where gene expression can be measured directly in blood samples to assess safety. Safety parameters will be measurement of changes in serum chemistries and hematology, urinalysis, pulmonary function testing, semen assay for vector genomes, immunologic response to AAT, and AAV, as well as reported subject history of any symptoms.

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A. From in vitro work, it was determined that coinfection with both vectors is required as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were achieved throughout the duration of the study (22 weeks). Intravenous and intramuscular delivery resulted in a maximal average expression of 31.4 +/- 6.4 and 29 +/- 6.5% of normal murine factor VIII levels, respectively. Western blots of cryoprecipitate as well as immunostaining of injection sites with an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII. Because the murine form of the gene was used in the mouse model, less than 1 Bethesda unit of inhibitors was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A.

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., (Copyright 2002 Elsevier Science (USA))

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy.

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Within the past decade, gene therapy strategies have come to the forefront of novel therapeutics. Tremendous advances in vector technology along with deeper understandings of vector biology and the molecular mechanisms of disease have significantly advanced the field of human gene therapy. This manuscript will discuss the viral-based subset of current gene transfer vectors. In particular, the most established viral vectors to date, including parvovirus, adenovirus, retrovirus, lentivirus, and herpesvirus-based vectors, are described, as well as the current innovative improvements being made to each. From past experience, it has become evident that in addition to optimising the vectors in terms of transgene expression, minimising vector-related immunology, and vector production, methods of vector delivery resulting in optimum vector transduction of target cells need to be established. This review will also illustrate several current improved physical delivery systems for optimal vector administration.

Efficient and targeted delivery of a DNA payload is vital for developing safe gene therapy. Owing to the recent success of commercial oncolytic vector and multiple COVID-19 vaccines, adenovirus vectors are back in the spotlight. Adenovirus vectors can be used in gene therapy by altering the wild-type virus and making it replication-defective; specific viral genes can be removed and replaced with a segment that holds a therapeutic gene, and this vector can be used as delivery vehicle for tissue specific gene delivery. Modified conditionally replicative–oncolytic adenoviruses target tumors exclusively and have been studied in clinical trials extensively. This comprehensive review seeks to offer a summary of adenovirus vectors, exploring their characteristics, genetic enhancements, and diverse applications in clinical and preclinical settings. A significant emphasis is placed on their crucial role in advancing cancer therapy and the latest breakthroughs in vaccine clinical trials for various diseases. Additionally, we tackle current challenges and future avenues for optimizing adenovirus vectors, promising to open new frontiers in the fields of cell and gene therapies. [ABSTRACT FROM AUTHOR]

In 2017, Nick cofounded a second AAV gene therapy company, Lacerta Therapeutics, which is developing gene therapies for central nervous system and lysosomal storage diseases. Nick was also the founding director of the Powell Gene Therapy Center at the University of Florida. In 1999, Nick cofounded an AAV gene therapy company, Applied Genetic Technologies Corporation, focused on gene therapy of retinal diseases. [Extracted from the article]

AbstractLysosomal storage diseases are a group of rare inborn errors of metabolism resulting from deficiency in normal lysosomal function. These diseases are characterized by progressive accumulation of storage material within the lysosomes of affected cells, ultimately leading to cellular dysfunction. Multiple tissues ranging from musculoskeletal and visceral to tissues of the central nervous system are typically involved in disease pathology. Since the advent of enzyme replacement therapy (ERT) to manage some LSDs, general clinical outcomes have significantly improved; however, treatment with infused protein is lifelong and continued disease progression is still evident in patients. Viral gene therapy may provide a viable alternative or adjunctive therapy to current management strategies for LSDs. In this review, we discuss the various viral vector systems that have been developed and some of the strategy designs for the treatment of LSDs. [ABSTRACT FROM AUTHOR]

Recently, adeno-associated virus (AAV)-mediated gene therapies have attracted clinical interest for treating neurodegenerative diseases including spinal muscular atrophy (SMA), Canavan disease (CD), Parkinson's disease (PD), and Friedreich's ataxia (FA). The influx of clinical findings led to the first approved gene therapy for neurodegenerative disorders in 2019 and highlighted new safety concerns for patients. Large doses of systemically administered AAV stimulate host immune responses, resulting in anti-capsid and anti-transgene immunity with implications for transgene expression, treatment longevity, and patient safety. Delivering lower doses directly to the central nervous system (CNS) is a promising alternative, resulting in higher transgene expression with decreased immune responses. However, neuroinflammatory responses after CNS-targeted delivery of AAV are a critical concern. Reported signs of AAV-associated neuroinflammation in preclinical studies include dorsal root ganglion (DRG) and spinal cord pathology with mononuclear cell infiltration. In this review, we discuss ways to manage neuroinflammation, including choice of AAV capsid serotypes, CNS-targeting routes of delivery, genetic modifications to the vector and/or transgene, and adding immunosuppressive strategies to clinical protocols. As additional gene therapies for neurodegenerative diseases enter clinics, tracking biomarkers of neuroinflammation will be important for understanding the impact immune reactions can have on treatment safety and efficacy. [ABSTRACT FROM AUTHOR]