AAV Vectors Research Articles

GDF5 as a rejuvenating treatment for age-related neuromuscular failure.

Sarcopenia involves a progressive loss of skeletal muscle force, quality and mass during ageing, which results in increased inability and death; however, no cure has been established thus far. Growth differentiation factor 5 (GDF5) has been described to modulate muscle mass maintenance in various contexts. For our proof of concept, we overexpressed GDF5 by AAV vector injection in Tibialis Anterior (TA) muscle of adult aged (20 months) mice and performed molecular and functional analysis of skeletal muscle. We analysed human Vastus Lateralis muscle biopsies from adult young (21-42 years) and aged (77-80 years) donors, quantifying the molecular markers modified by GDF5 overexpression (OE) in mouse muscle. We validated the major effects of GDF5 overexpression using human immortalized myotubes and Schwann Cells (SCs). We established a pre-clinical study by treating chronically (for 4 months) aged mice using recombinant GDF5 protein (rGDF5) in systemic administration and evaluated the long-term effect of this treatment on muscle mass and function. Here, we demonstrated that GDF5 OE in the old TAs promoted an increase of 16.5% of muscle weight (P = 0.0471) associated with a higher percentage of 5000-6000 µm2 large fibres (P = 0.0211), without the induction of muscle regeneration. Muscle mass gain was associated with an amelioration of 26.8% of rate of force generation (P = 0.0330) and a better neuromuscular connectivity (P = 0.0098). Moreover, GDF5 OE preserved neuromuscular junction (NMJ) morphology (38.5% of nerve terminal area increase, P < 0.0001) and stimulated the expression of re-innervation-related genes, in particular markers of SCs (fold change 3.19 for S100b gene expression, P = 0.0101). To further characterize the molecular events induced by GDF5 OE during ageing, we performed a genome-wide transcriptomic analysis of treated muscles and showed that this factor leads to a "rejuvenating" transcriptomic signature in aged mice, as 42% of the transcripts dysregulated by ageing reverted to youthful expression levels upon GDF5 OE (P < 0.05). Towards a pre-clinical approach, we performed a long-term systemic treatment using rGDF5 and showed its effectiveness in counteracting age-related muscle wasting, improving muscle function (17,8% of absolute maximal force increase, P = 0.0079), ensuring neuromuscular connectivity and preventing NMJ degeneration (7,96% of AchR area increase, P = 0.0125). In addition, in human muscle biopsies, we found the same age-related alterations than those observed in mice and improved by GDF5 and reproduced its major effects on human cells, suggesting this treatment as efficient in humans. Overall, these data provide a foundation to examine the curative potential of GDF5 drug in clinical trials for sarcopenia and, eventually, other neuromuscular diseases.

Niosomes: A Smart Drug Delivery System for Brain Targeting.

Niosomes are lipid-based nanovesicles that have the potential to act as drug-delivery vehicles for a variety of agents. They are effective drug delivery systems for both ASOs and AAV vectors, with advantages such as improved stability, bioavailability, and targeted administration. In the context of brain-targeted drug delivery, niosomes have been investigated as a drug delivery system for brain targeting, but more research is needed to optimize their formulation to improve their stability and release profile and address the challenges of scale-up and commercialization. Despite these challenges, several applications of niosomes have demonstrated the potential of novel nanocarriers for targeted drug delivery to the brain. This review briefly overviews the current use of niosomes in treating brain disorders and diseases.

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2.

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 × 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viralvector administration.

Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

Abstract 2950: CRISPR/saCas9 and CRISPR/spCas9 systems for combinatorial genetic screens (CRISPR-KO, CRISPRa, CRISPRi)

Abstract This study explores the utilization of orthogonal CRISPR-based gene editing/modulation systems for combinatorial genetic screens using CRISPR knockout (CRISPR-KO), CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) functionalities. S. aureus (sa)Cas9 is an alternative nuclease to S. pyogenes (sp)Cas9 in scenarios where the latter cannot be used, or when multiple independent CRISPR systems need to be simultaneously expressed in the same cell. In this study we set out to explore the feasibility of utilizing different combinations of saCas9 and spCas9 CRISPR systems to achieve the simultaneous inactivation (via CRISPR-KO or CRISPRi) and transactivation (via CRISPRa) of different target genes in the same host cell. For this purpose, a complete set of tools for CRISPR/saCas9 gene editing and gene modulation was developed, compatible with CRISPR/spCas9 co-expression. Specifically, we developed and validated optimized saCas9 and sg(sa)RNA lentiviral and AAV vectors, dual expression (sp)/(sa)sgRNA lentiviral library vectors, as well as (sa)CRISPR-KO, (sa)CRISPRi, (sa)CRISPRa fluorescence-based activity kits for the functional validation of saCas9 expressing cell lines. Results demonstrating the feasibility of the orthogonal screens will be presented, with different combinations of CRISPR-KO, CRISPRa, and CRISPRi systems in multiple cell lines. Citation Format: Nadya Isachenko, Gayane Aleksanyan, Paul Diehl, Donato Tedesco. CRISPR/saCas9 and CRISPR/spCas9 systems for combinatorial genetic screens (CRISPR-KO, CRISPRa, CRISPRi) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2950.

Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

Short-hairpin RNAs (shRNA), targeting knockdown of specific genes, hold enormous promise for precision-based therapeutics to treat numerous neurodegenerative disorders. However, whether shRNA constructed molecules can modify neuronal circuits underlying certain behaviors has not been explored. We designed shRNA to knockdown the human HTR2A gene in vitro using iPSC-differentiated neurons. Multi-electrode array (MEA) results showed that the knockdown of the 5HT-2A mRNA and receptor protein led to a decrease in spontaneous electrical activity. In vivo, intranasal delivery of AAV9 vectors containing shRNA resulted in a decrease in anxiety-like behavior in mice and a significant improvement in memory in both mice (104%) and rats (92%) compared to vehicle-treated animals. Our demonstration of a non-invasive shRNA delivery platform that can bypass the blood–brain barrier has broad implications for treating numerous neurological mental disorders. Specifically, targeting the HTR2A gene presents a novel therapeutic approach for treating chronic anxiety and age-related cognitive decline.

Fine-tuning FAM161A gene augmentation therapy to restore retinal function

For 15 years, gene therapy has been viewed as a beacon of hope for inherited retinal diseases. Many preclinical investigations have centered around vectors with maximal gene expression capabilities, yet despite efficient gene transfer, minimal physiological improvements have been observed in various ciliopathies. Retinitis pigmentosa-type 28 (RP28) is the consequence of bi-allelic null mutations in the FAM161A, an essential protein for the structure of the photoreceptor connecting cilium (CC). In its absence, cilia become disorganized, leading to outer segment collapses and vision impairment. Within the human retina, FAM161A has two isoforms: the long one with exon 4, and the short one without it. To restore CC in Fam161a-deficient mice shortly after the onset of cilium disorganization, we compared AAV vectors with varying promoter activities, doses, and human isoforms. While all vectors improved cell survival, only the combination of both isoforms using the weak FCBR1-F0.4 promoter enabled precise FAM161A expression in the CC and enhanced retinal function. Our investigation into FAM161A gene replacement for RP28 emphasizes the importance of precise therapeutic gene regulation, appropriate vector dosing, and delivery of both isoforms. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

High-yield recombinant adeno-associated viral vector production by multivariate optimization of bioprocess and transfection conditions.

Recombinant adeno-associated viral vectors (rAAVs) are one of the most used vehicles for gene therapy, with five rAAV therapeutics commercially approved by the FDA. To improve product yield, we optimized the suspension production process of rAAV8 vectors carrying a proprietary transgene using a commercially available transfection reagent, FectoVIR-AAV. Using a miniaturized automated 250 mL scale bioreactor system, we generated models of vector genome (vg) titer, capsid (cp) titer, and Vg:Cp percentage from two multivariate design of experiment studies, one centered around bioreactor operating parameters, and another based on the transfection conditions. Using the optimized process returned from these models, the vector genome titer from the bioreactor was improved to beyond 1 × 1012 vg/mL. Five critical parameters were identified that had large effects on the pre-purification vector quantity-the transfection pH, production pH, complexation time, viable cell density at transfection, and transfection reagent to DNA ratio. The optimized process was further assessed for its performance extending to six AAV serotypes, namely AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 carrying a transgene encoding for green fluorescent protein (GFP). Five of the six serotypes returned higher vector genome titers than the control condition. These data suggest that the choice of transfection reagent is a major factor in improving vector yield. The multivariate design of experiment approach is a powerful way to optimize production processes, and the optimized process from one AAV vector can to some extent be generalized to other serotypes and transgenes to accelerate development timelines of new programs.

A humanized mouse model for adeno-associated viral gene therapy

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg−/−/Rag2−/−/Fah−/−/Aavr−/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

CD38 deficient mice are not protected from atherosclerosis

CD38 is a multifunctional enzyme implicated in chemotaxis of myeloid cells and lymphocyte activation, but also expressed by resident cells such as endothelial and smooth muscle cells. CD38 is important for host defense against microbes. However, CD38's role in the pathogenesis of atherosclerosis is controversial with seemingly conflicting results reported so far. To clarify the discrepancy of current literature on the effect of CD38 ablation on atherosclerosis development, we implanted a shear stress modifier around the right carotid artery in CD38−/− and WT mice. Hypercholesterolemia was induced by human gain-of-function PCSK9 (D374Y), introduced using AAV vector (serotype 9), combined with an atherogenic diet for a total of 9 weeks. Atherosclerosis was assessed at the aortic root, aortic arch and the right carotid artery. The findings can be summarized as follows: i) CD38−/− and WT mice had a similar atherosclerotic burden in all three locations, ii) No significant differences in monocyte infiltration or macrophage content could be seen in the plaques, and iii) The amount of collagen deposition in the plaques were also similar between CD38−/− and WT mice. In conclusion, our data suggest that CD38−/− mice are neither protected against nor prone to atherosclerosis compared to WT mice.

Therapeutic Strategy for Fabry Disease by Intravenous Administration of Adeno-Associated Virus 9 in a Symptomatic Mouse Model.

Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A), an enzyme that hydrolyzes glycosphingolipids in lysosome. Accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in tissues, induces cellular dysfunction leading to multi-organ disorder. Gene therapy is a promising strategy that can overcome these problems, and virus vectors such as adeno-associated virus (AAV) have been used for study on gene therapy. We used human Gb3 synthetase-transgenic (TgG3S)/α-Gal A knockout (GLAko) mice. TgG3S/GLAko mice have elevated Gb3 accumulation in the major organs compared with GLAko mice, which have been widely used as a model for FD. At the age of 6 weeks, male TgG3S/GLAko were injected with 2 × 1012 vector genome AAV9 vectors containing human α-Gal A cDNA. Eight weeks after intravenous injection of AAV, α-Gal A enzymatic activity was elevated in the plasma, heart, and liver of TgG3S/GLAko mice to levels corresponding to 224%, 293%, and 105% of wild-type, respectively. Gb3 amount 8 weeks after AAV injection in the heart and liver of this group was successfully reduced to levels corresponding to 16% and 3% of untreated TgG3S/GLAko mice. Although the brain and kidney of AAV9-treated TgG3S/GLAko mice showed no significant increases in α-Gal A activity, Gb3 amount was smaller than untreated littermates (48% and 44%, respectively). In this study, systemic AAV administration did not show significant extension of the lifespan of TgG3S/GLAko mice compared with the untreated littermates. The timing of AAV injection, capsid choice, administration route, and injection volume may be important to achieve sufficient expression of α-Gal A in the whole body for the amelioration of lifespan.

Development of Stable Packaging and Producer Cell Lines for the Production of AAV Vectors.

Today, recombinant adeno-associated virus (rAAV) vectors represent the vector systems which are mostly used for in vivo gene therapy for the treatment of rare and less-rare diseases. Although most of the past developments have been performed by using a transfection-based method and more than half of the authorized rAAV-based treatments are based on transfection process, the tendency is towards the use of stable inducible packaging and producer cell lines because their use is much more straightforward and leads in parallel to reduction in the overall manufacturing costs. This article presents the development of HeLa cell-based packaging/producer cell lines up to their use for large-scale rAAV vector production, the more recent development of HEK293-based packaging and producer cell lines, as well as of packaging cell lines based on the use of Sf9 cells. The production features are presented in brief (where available), including vector titer, specific productivity, and full-to-empty particle ratio.

Preferential transduction of parvalbumin-expressing cortical neurons by AAV-mDLX5/6 vectors.

A major goal of modern neuroscience is to understand the functions of the varied neuronal types that comprise the mammalian brain. Toward this end, some types of neurons can be targeted and manipulated with enhancer-bearing AAV vectors. These vectors hold great promise to advance basic and translational neuroscience, but to realize this potential, their selectivity must be characterized. In this study, we investigated the selectivity of AAV vectors carrying an enhancer of the murine Dlx5 and Dlx6 genes. Vectors were injected into the visual cortex of two macaque monkeys, the frontal cortex of two others, and the somatosensory/motor cortex of three rats. Post-mortem immunostaining revealed that parvalbumin-expressing neurons were transduced efficiently in all cases but calretinin-expressing neurons were not. We speculate that this specificity is a consequence of differential activity of this DLX5/6 enhancer in adult neurons of different developmental lineages.

Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes.

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR

Self-complementary AAV vectors (scAAV) employ a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. Yet, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These non-canonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors termed covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of employing a mutant ITR, we utilized a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vector. Treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to development of improved human gene therapy drugs.

Abstract WP314: Post-Stroke Regulation of α-Synuclein by microRNA miR-7 and Circular RNA CDR1as

α-Synuclein (α-Syn) induced after stroke is a known mediator of ischemic brain damage. We currently evaluated the molecular mechanisms that regulate α-Syn expression in the post-ischemic brain. The miR-7 is an abundantly expressed miRNA in brain that targets α-Syn mRNA with high energy. In adult mice subjected to transient middle cerebral artery occlusion (MCAO), miR-7 levels were down-regulated in the cerebral cortex as early as 3h and late as 3 days of reperfusion which leads to derepression of α-Syn mRNA that facilitates α-Syn protein translation. Treating mice after transient MCAO with a miR-7 mimic significantly curtailed ischemic brain damage and promoted better motor function recovery. However, the pri-miR-7a and -7b levels were unaltered indicating that the post-ischemic reduction in miR-7 levels are not due to reduced expression of miR-7 gene. With bioinformatics, we observed that the circRNA CDR1as contains &gt;70 binding sites with good affinity for miR-7. CDR1as is thought to sequester miR-7 to protect it from degradation and/or to transport it to the sites of its necessity. Transient MCAO also led to down-regulation of CDR1as expression between 3h and 3 days of reperfusion. When CDR1as levels were replenished in the post-ischemic mice with a CDR1as AAV9 vector, the miR-7 levels as well as binding of CDR1as to Ago2/miR-7 increased and α-Syn protein levels decreased significantly in the peri-infarct cortex. Furthermore, the CDR1as AAV9 treated mice showed decreased markers of inflammation and apoptosis, better motor function recovery and smaller infarcts. Our studies thus indicate that α-Syn in the post-ischemic brain is controlled by the mutual interaction of a miRNA (miR-7) and a circRNA (CDR1as). Thus, understanding noncoding RNA interactions and mechanisms might help to design better therapies to alleviate post-stroke neuronal damage.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy

Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.