ReGenX

AAV9 encoding microdystrophin under the control of a cardiac promoter was injected intravenously into mdx mice, resulting in protection of cardiac morphology and function. This could be developed into a therapeutic for DMD-associated cardiomyopathy.

In a rat model of lysosomal storage disease, the extent of disease did not impact transduction of the liver with AAV8, suggesting that efficiency of gene transfer in humans will not be impacted by age. Inclusion of several additional regulatory elements were deemed to be unnecessary, as they did not improve efficiency nor reduce the immune response to the transgene product

In order to understand how to treat genetic diseases that affect newborns, it is critical to understand hepatic gene transfer in neonates. This study demonstrates that heptic gene transfer with AAV8 in newborn rhesus monkeys is extremely efficient, but transgene expression is less stable than in adolescent monkeys.

A mouse model of familial hypercholesterolemia (LDL receptor-deficiency) was created that has a lipoprotein phenotype that much more closely resembles that of humans than previous mouse models. In this model, gene therapy with AAV8 encoding the human LDL receptor significantly corrected the high plasma cholesterol levels at doses of vector that are able to be developed for human use. In addition, the vector had beneficial effects in treating the more common heterozygous FH phenotype.

A gene therapy for OTC deficiency is being developed using AAV8 for gene replacement. This paper delineates vector optimization that further increased gene expression to improve efficacy and lower the dose of vector required for therapeutic effect.

The authors compare intravenous and intracoronary delivery of AAV5, AAV6, and AAV9. Optimal vector/delivery was determined by quantitating both the percentage of cells positive for GFP expression and GFP intensity. AAV9 was found to be superior in both methods of delivery, with IV delivery resulting in more homogeneous but low-level cardiac expression, and IC delivery resulting in more intense but less homogenous cardiac expression.

GSD-Ia mice were injected intravenously with either AAV2, AAV7, AAV8, or AAV9 encoding a normal copy of the deficient enzyme, glucose-6-phosphatase (G6Pase). While AAV7, AAV8, and AAV9 all correced the hepatic phenotype of G6Pase deficiency, treatment with AAV9 also reduced the renal complications of the disease.

AAV8 was used to deliver a cDNA encoding the cholesterol metabolic pathway enzyme LCAT to mouse liver; expression was stable for at least 32 weeks.  The gene was delivered to a mouse strain that was genetically modified to have a more human-like metabolic pathway, to determine that LCAT improved lipid profiles.

Dr. David Baltimore at California Institute of Technology and his colleagues reported that NAV rAAV8 vectors encoding genes for anti-HIV neutralizing antibodies, when injected into the muscle of mice, can direct the synthesis and secretion of therapeutic levels of those antibodies into the circulation. They further demonstrated that the antibodies are capable of protecting the experimental animals from HIV challenge.  This approach is designated “passive immunization”, in which an individual is given high levels of an antibody that has already been demonstrated to be capable of neutralizing HIV to prevent infection.  To avoid repetitive delivery of purified antibodies over a lifetime, researchers adopted gene delivery with NAV rAAV8 vectors involving a one-time injection of the vector, which directs cells to synthesize and secrete the antibody long-term.

AAV9 was shown to efficiently deliver a therapeutic gene to a large animal heart.  Heart failure in the pig was rescued with AAV9-mediated delivery of S100A1, which regulates calcium-controlled myocardial contractility.

The mouse model of OTC deficiency was made to more closely resemble the human disease by rAAV8-mediated delivery of an shRNA to knockdown residual endogenous OTC mRNA, causing the mice to develop hyperammonemia.  Furthermore, co-administration of rAAV8 encoding a “rescue” OTC cDNA determined the doses required to control the manifestations of OTC deficiency.

The mouse model of multiple sulfatase deficiency (MSD) was treated with rAAV9 delivered both systemically and into the CNS.  This demonstrates that delivery of a single therapeutic by two routes could be used for treating severe whole-body disorders.

The mouse model of primary hyperoxaluria type I (PH1) was treated with rAAV8 or rAAV5 delivered systemically.  Both were able to normalize metabolism, with AAV8 showing higher levels of transgene expression in the liver.

This form of severe combined immunodeficiency (SCID) has previously been treated using enzyme replacement therapy or using virally-transduced T cells.  In this paper, AAV1 delivered IM is compared to rAAV9 delivered IV, and shows enzyme replacement and immune reconstitution of ADA-deficient mice using AAV9.

rAAV8 was used to deliver shRNA to ApoB to mouse liver, showing significant reduction in circulating LDL levels.

NAV Technology-based AAV vectors were shown to be efficiently released into the medium during manufacturing, increasing vector yields and simplifying production.

See also accompanying article, Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale.  Hum. Gene Ther. 2010 21:1259-1271

rAAV9 delivered IV to MPS IIIB mice corrects lysosomal storage defects and provides neurological benefit and survival benefit.

Using engineered AAV capsids with an ovalbumin immunodominant peptide, this study demonstrates that AAV2 capsid stimulates a T-cell response in mice, but that AAV8 does not. This is despite equivalent levels and kinetics of capsid antigen presentation. These data do not support a previously proposed hypothesis from a hemophilia B clinical trial that CTLs to AAV capsid clear transduced cells.

Treatment of hemophilia A (Factor VIII deficiency) is complicated by the development of antibodies to Factor VIII. AAV8 was used to deliver a copy of the Factor VIII gene to hemophilia A mice. The optimal approach of using a codon-optimized Factor VIII transgene coupled to a liver-specific promoter resulted in sustained therapeutic expression, and tolerance to Factor VIII without additional manipulations.

This editorial accompanied the article on the Phase I clinical trial of AAV8 for Hemophilia B. It describes the history of Hemophilia B (also known as Christmas disease), and why the current clinical trial is a landmark study.

This commentary in Human Gene Therapy describes the NEJM article on the Phase 1 clinical trial of AAV8 in Hemophilia B patients and highlights the potential of AAV8 and the other novel AAV serotypes in clinical gene therapy.

This study examined long-term safety of intravenous administration of rAAV8 encoding the human Factor IX cDNA in non-human primates, demonstrating >5 years of transgene expression.

Researchers report on a combined Phase 1/2 clinical trial involving NAV rAAV8-mediated gene transfer in patients with hemophilia B. In this trial, six hemophilia B patients received a single intravenous injection of NAV rAAV8 vectors encoding a normal copy of the defective gene, Factor IX.  All patients are expressing enough Factor IX to convert their phenotypes from severe to mild-moderate. Four out of six patients have been able to discontinue use of prophylactic, recombinant Factor IX and have not experienced spontaneous bleeding since receiving the therapy. The other two patients have been able to extend the time between prophylactic treatments. Most importantly, the therapeutic was well-tolerated.

AAV8 transduced rods and cones in a mouse model of LCA1, improving visual funct6ion.  AAV8 was shown to be more effective than lentivirus or AAV5, which have been used in previous publications.

This publication demonstrates that AAV8 is more efficient than AAV5 in transducing photoreceptors in a large animal, including both rods and cones.  Along with the recent published data in non-human primates (below), this demonstrates the utility of AAV8 in treating a wide variety of retinal degenerative diseases such as retinitis pigmentosa.

This study in non-human primates demonstrates the abilities of AAV8 and AAV9 to transduce cones as well as rods in the retina. Because the NHP retina is the most similar to humans, it suggests that different AAV serotypes could be used for different retinal degenerations in humans, depending on which cells of the retina need to be most efficiently transduced.

This publication is the first study to analyze the retinal cell types transduced by rAAV8 in non-human primates, which have a retinal structure closest to that of humans.  AAV8 was shown to be approximately 10-fold more efficient in transducing both the RPE and photoreceptors, which are targets for gene therapy of most forms of retinitis pigmentosa and other inherited retinal degenerative diseases.

More detailed commentary: http://regenxbio.com/index.php/science/publications/

Treatment of neurologic diseases with novel AAV serotypes has been gaining momentum, as these vectors have been shown to transduce much of the CNS with less invasive routes of administration. This study demonstrates that Intrathecal delivery of AAV9 as well as AAV7  in non-human primates gives widespread gene expression in the CNS and the peripheral nervous system.

AAVrh10 was used to deliver a therapeutic transgene to treat mice with Krabbe disease (globoid cell leukodystrophy). A combination of intracerebroventricular, intracerebellar, and intravenous injection in newborn mice resulted in nearly normal phenotype for up to 8 months.

The authors show that AAV9 delivery to the CNS by injection into the CSF (including intrathecal injections) is not inhibited by the presence of serum neutralizing antibodies (NAbs) to AAV9, up to titers of at least 1:128. This suggests that for diseases that might be treated through this route of administration, the vast majority of patients would be eligible for treatment, given the low frequency of high titer NAbs to AAV9.

A variety of inherited diseases require treatment of young children; rAAV9, the serotype preferred for IV-based gene transfer to the CNS, was studied in young non-human primates. At all ages studied, from birth through 3 years, AAV9 was efficient at crossing the blood-brain barrier. Cerebrospinal fluid (CSF) injections were performed, which efficiently targeted motor neurons, and restricted gene expression to the CNS.

A total of 10 different AAV serotypes were tested, and rAAVrh.10, as well as rAAV9, were shown to efficiently cross the BBB of neonatal mice.  Transgene expression was carefully documented within regions of the brain and spinal cord.

rAAV8-mediatedgene transfer to the liver of MPS IIIA mice resulted in high and sustained levels of circulating active sulfamidase, and completely corrected lysosomal GAG accumulation in most somatic tissues. Surprisingly, high sustained circulating sulfamidase levels also resulted a 50% reduction of GAG accumulation in the brain of male mice, which were expressing 4x normal levels of enzyme, and these mice had an increased lifespan.

The authors demonstrated the superiority of AAVrh.10 as compared to AAV5 for the treatment of metachromatic leukodystrophy in the mouse model. The efficacy shown supports a proposed clinical trial, which is expected to begin dosing patients shortly. In this month's issue of Human Gene Therapy researchers discuss the signficance of this study.

Commentary:

AAV9: Over the Fence and Into the Woods . . .

Research Articles:

• Correction of Neurological Disease of Mucopolysaccharidosis IIIB in Adult Mice by rAAV9 Trans-Blood–Brain Barrier Gene Delivery
• Preclinical Differences of Intravascular AAV9 Delivery to Neurons and Glia: A Comparative Study of Adult Mice and Nonhuman Primates
• Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer
• A Potential Role of Distinctively Delayed Blood Clearance of Recombinant Adeno-associated Virus Serotype 9 in Robust Cardiac Transduction