AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disease affecting >20 million people worldwide. Loss of daylight vision typically occurs due to the dysfunction/loss of cone photoreceptors, the cell type that initiates our color and high-acuity vision. Currently, there is no effective treatment for RP, other than gene therapy for a limited number of specific disease genes. To develop a disease gene-agnostic therapy, we screened 20 genes for their ability to prolong cone photoreceptor survival in vivo. Here, we report an adeno-associated virus vector expressing Txnip, which prolongs the survival of cone photoreceptors and improves visual acuity in RP mouse models. A Txnip allele, C247S, which blocks the association of Txnip with thioredoxin, provides an even greater benefit. Additionally, the rescue effect of Txnip depends on lactate dehydrogenase b (Ldhb) and correlates with the presence of healthier mitochondria, suggesting that Txnip saves RP cones by enhancing their lactate catabolism.

Retinitis pigmentosa is an inherited eye disease affecting around one in every 4,000 people. It results from genetic defects in light sensitive cells of the retina, called photoreceptor cells, which line the back of the eye. Though vision loss can occur from birth, retinitis pigmentosa usually involves a gradual loss of vision, sometimes leading to blindness. Rod photoreceptors, which are responsible for vision in low light, are impacted first. The disease then affects cone photoreceptors, the cells that detect light during the day, providing both color and sharp vision. Around 100 mutated genes associated with retinitis pigmentosa have been identified, but only a handful of families with one of these mutant genes have been treated with a gene therapy specific for their mutated gene. There are currently no therapies available to treat the vast number of people with this disease. The mutations that cause retinitis pigmentosa directly affect the rod cells that detect dim light, leading to loss of night vision. There is also an indirect effect that causes cone photoreceptors to stop working and die. One theory to explain this two-step disease process relates to the fact that cone photoreceptors are very active cells, requiring a high level of energy, nutrients and oxygen. If surrounding rod cells die, cone photoreceptors may be deprived of some essential supplies, leading to cone cell death and daylight vision loss. To examine this theory, Xue et al. tested a new gene therapy designed to alleviate the potential shortfall in nutrients. The experiments used three different strains of mice that had the same genetic mutations as humans with retinitis pigmentosa. The gene therapy used a virus, called adeno-associated virus (AAV), to deliver 20 different genes to cone cells. Each of the 20 genes tested plays a different role in cells' processing of nutrients to provide energy. After administering the treatment, Xue et al. monitored the mice to see whether or not their vision was affected, and how cone cells responded. Only one of the 20 genes, Txnip, delivered using gene therapy, had a beneficial effect, prolonging cone cell survival in all three mouse strains. The mice that received Txnip also retained their ability to discern moving stripes on vision tests. Further investigations demonstrated that activating Txnip forced the cones to start using a molecule called lactate as an energy source, which could be more available to them than glucose, their usual fuel. These cells also had healthier mitochondria ­ the compartments inside cells that produce and manage energy supplies. This dual effect on fuel use and mitochondrial health is thought to be the basis for the extended cone survival and function. These experiments by Xue et al. have identified a good gene therapy candidate for treating retinitis pigmentosa independently of which genes are causing the disease. Further research will be required to test the safety of the gene therapy, and whether its beneficial effects translate to humans with retinitis pigmentosa, and potentially other diseases with unhealthy photoreceptors.

In Situ Detection of Adeno-associated Viral Vector Genomes with SABER-FISH.

Gene therapy with recombinant adeno-associated viral (AAV) vectors is a promising modality for the treatment of a variety of human diseases. Nonetheless, there remain significant gaps in our understanding of AAV vector biology, due in part to the lack of robust methods to track AAV capsids and genomes. In this study, we describe a novel application of signal amplification by exchange reaction fluorescence in situ hybridization (SABER-FISH) that enabled the visualization and quantification of individual AAV genomes after vector administration in mice. These genomes could be seen in retinal cells within 3 h of subretinal AAV delivery, were roughly full length, and correlated with vector expression in both photoreceptors and the retinal pigment epithelium. SABER-FISH readily detected AAV genomes in the liver and muscle following retro-orbital and intramuscular AAV injections, respectively, demonstrating its utility in different tissues. Using SABER-FISH, we also found that retinal microglia, a cell type deemed refractory to AAV transduction, are in fact efficiently infected by multiple AAV serotypes, but appear to degrade AAV genomes prior to nuclear localization. Our findings show that SABER-FISH can be used to visualize AAV genomes in situ, allowing for studies of AAV vector biology and the tracking of transduced cells following vector administration.

Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a disease that initially presents as night blindness due to genetic deficits in the rod photoreceptors of the retina. Rods then die, causing dysfunction and death of cone photoreceptors, the cell type that mediates high acuity and color vision, ultimately leading to blindness. We investigated immune responses in mouse models of RP and found evidence of microglia activation throughout the period of cone degeneration. Using adeno-associated vectors (AAVs), delivery of genes encoding microglial regulatory signals led to the identification of AAV serotype 8 (AAV8) soluble CX3CL1 (sCX3CL1) as a promising therapy for degenerating cones. Subretinal injection of AAV8-sCX3CL1 significantly prolonged cone survival in three strains of RP mice. Rescue of cones was accompanied by improvements in visual function. AAV8-sCX3CL1 did not affect rod survival, microglia localization, or inflammatory cytokine levels in the retina. Furthermore, although RNA sequencing of microglia demonstrated marked transcriptional changes with AAV8-sCX3CL1, pharmacological depletion of up to ∼99% of microglia failed to abrogate the effect of AAV8-sCX3CL1 on cone survival. These findings indicate that AAV8-sCX3CL1 can rescue cones in multiple mouse models of RP via a pathway that does not require normal numbers of microglia. Gene therapy with sCX3CL1 is a promising mutation-independent approach to preserve vision in RP and potentially other forms of retinal degeneration.

Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission.

The vast majority of new HIV infections result from relatively inefficient transmission of the virus across mucosal surfaces during sexual intercourse. A consequence of this inefficiency is that small numbers of transmitted founder viruses initiate most heterosexual infections. This natural bottleneck to transmission has stimulated efforts to develop interventions that are aimed at blocking this step of the infection process. Despite the promise of this strategy, clinical trials of preexposure prophylaxis have had limited degrees of success in humans, in part because of lack of adherence to the recommended preexposure treatment regimens. In contrast, a number of existing vaccines elicit systemic immunity that protects against mucosal infections, such as the vaccines for influenza and human papilloma virus. We recently demonstrated the ability of vectored immunoprophylaxis (VIP) to prevent intravenous transmission of HIV in humanized mice using broadly neutralizing antibodies. Here we demonstrate that VIP is capable of protecting humanized mice from intravenous as well as vaginal challenge with diverse HIV strains despite repeated exposures. Moreover, animals receiving VIP that expresses a modified VRC07 antibody were completely resistant to repetitive intravaginal challenge by a heterosexually transmitted founder HIV strain, suggesting that VIP may be effective in preventing vaginal transmission of HIV between humans.

Broad protection against influenza infection by vectored immunoprophylaxis in mice.

Neutralizing antibodies that target epitopes conserved among many strains of influenza virus have been recently isolated from humans. Here we demonstrate that adeno-associated viruses (AAV) encoding two such broadly neutralizing antibodies are protective against diverse influenza strains. Serum from mice that received a single intramuscular AAV injection efficiently neutralized all H1, H2 and H5 influenza strains tested. After infection with diverse strains of H1N1 influenza, treated mice showed minimal weight loss and lung inflammation. Protection lasted for at least 11 months after AAV injection. Notably, even immunodeficient and older mice were protected by this method, suggesting that expression of a monoclonal antibody alone is sufficient to protect mice from illness. If translated to humans, this prophylactic approach may be uniquely capable of protecting immunocompromised or elderly patient populations not reliably protected by existing vaccines.

Antibody-based protection against HIV infection by vectored immunoprophylaxis.

Despite tremendous efforts, development of an effective vaccine against human immunodeficiency virus (HIV) has proved an elusive goal. Recently, however, numerous antibodies have been identified that are capable of neutralizing most circulating HIV strains. These antibodies all exhibit an unusually high level of somatic mutation, presumably owing to extensive affinity maturation over the course of continuous exposure to an evolving antigen. Although substantial effort has focused on the design of immunogens capable of eliciting antibodies de novo that would target similar epitopes, it remains uncertain whether a conventional vaccine will be able to elicit analogues of the existing broadly neutralizing antibodies. As an alternative to immunization, vector-mediated gene transfer could be used to engineer secretion of the existing broadly neutralizing antibodies into the circulation. Here we describe a practical implementation of this approach, which we call vectored immunoprophylaxis (VIP), which in mice induces lifelong expression of these monoclonal antibodies at high concentrations from a single intramuscular injection. This is achieved using a specialized adeno-associated virus vector optimized for the production of full-length antibody from muscle tissue. We show that humanized mice receiving VIP appear to be fully protected from HIV infection, even when challenged intravenously with very high doses of replication-competent virus. Our results suggest that successful translation of this approach to humans may produce effective prophylaxis against HIV.