Molecular Therapy Methods & Clinical Development

Adeno-associated viruses (AAVs) are popular gene therapy vectors, but AAVs can cause toxicity. This is particularly evident following expression of some transgenes, e.g. GFP, in the retinal pigment epithelium (RPE), which leads to loss of RPE cells and photoreceptors. Here, we sought to unravel the toxicity mechanism(s). Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. The CHOP KO partially alleviated toxicity for RPE but not photoreceptors, whereas the type I interferon receptor KO partially alleviated toxicity for photoreceptors but not RPE. In situ hybridization of interferon pathway transcripts (IFNB1, IFNAR1) revealed that the RPE and retina can produce and potentially respond to interferon. These data suggest that transgene-induced cell stress responses in the RPE lead to RPE cell death, while interferon signaling contributes to the death of photoreceptors.

Efficient and cell-specific gene delivery to cochlear inner hair cells (IHCs) remains a major challenge for inner ear gene therapy. Here, we identify and characterize a novel AAV2-derived capsid, AAV-WM04, that enables highly efficient and selective IHC transduction at low doses. Using an in vivo-directed evolution strategy, we generated a randomized AAV2 capsid library with 9-amino acid insertions and performed iterative selection in the adult mouse cochlea. Next-generation sequencing revealed enrichment of several variants, among which AAV-WM04 exhibited superior packaging efficiency and pronounced IHC tropism. AAV-WM04 achieved near-complete IHC transduction throughout the cochlear axis in adult mice, outperforming clinically relevant vectors with minimal off-target expression and no detectable ototoxicity. Robust and exclusive IHC transduction was further validated in non-human primates following round window membrane delivery, underscoring translational potential. Therapeutically, AAV-WM04 enabled efficient dual-AAV trans-splicing delivery of the large OTOF gene, resulting in uniform full-length otoferlin expression in IHCs. In a humanized Otof Q829X/Q829X mouse model, AAV-WM04 restored auditory function across a broad frequency range at relatively low doses and achieved durable hearing recovery. Collectively, these findings establish AAV-WM04 as a next-generation IHC-targeted vector with high efficiency, safety, and cross-species applicability for precision gene therapy of hereditary hearing loss.

Most recessive inherited retinal dystrophies are, in principle, amenable to gene augmentation, yet only one such therapy has received regulatory approval. We sought to identify tractable autosomal recessive retinitis pigmentosa (RP) genes that could be treated using existing non-proprietary adeno-associated virus (AAV) components. More than 100 RP-associated genes were prioritized according to cellular expression, coding-sequence size, and the availability of cell type-specific promoters, yielding 19 candidate genes expressed predominantly in rods and/or retinal pigment epithelium (RPE). In rhesus monkey eyes, the human RHO and BEST1 promoters drove rod- and RPE-specific expression, respectively, whereas the GFAP and RLBP1 promoters were limited by absent Muller glial expression or dose-associated RPE toxicity. We selected PDE6B as a proof-of-concept gene and evaluated AAV8-RHO-PDE6B after neonatal subretinal delivery. The vector effectively preserved outer nuclear layer structure, electroretinography responses, and visually guided behaviors in rd1 and rd10 mice for at least 6 months. Dose-ranging studies identified retinal abnormalities at the higher doses, whereas the lower doses were comparatively well tolerated in eyes of mice and rhesus monkeys. Together, these findings define a preclinical framework for developing gene-augmentation vectors for a subset of early-stage autosomal recessive RP patients.

Cystic fibrosis (CF) is the most common monogenic lung disease and results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). There have been over 2000 variants identified in patients that result in loss of function of the CFTR protein leading to systemic disease and respiratory failure in adolescence. While some variants encode proteins with residual activity that can be corrected or potentiated by CFTR modulators, at least 10% of CF individuals cannot tolerate the modulators or have nonsense mutations which fail to make any protein. For all people with CF, a mutation agnostic gene replacement strategy could provide a cure for CF lung disease. Here, we propose using a systemic route of administration to deliver a functional CFTR minigene cargo with a lung tropic AAV capsid. This would serve to reach multiple organs, most importantly the lung epithelium, and would provide a functional CFTR transgene that could be expressed in any cell type with a ubiquitous promoter. To achieve this, we generated the smallest CFTR minigene tested in an AAV delivery to date. We demonstrate its expression and function following transfection in cell-based assays and restoration of function in primary CF airway cells after viral delivery. Furthermore, we identify an AAV capsid that can transduce alveolar and airway epithelium with systemic delivery in non-human primates. These data provide tools for delivering a functional CFTR minigene that fits within the packaging capacity of an AAV and demonstrate lung transduction with an AAV following systemic delivery in a large animal model. This strategy first and foremost can reach target airway cells by circumventing the strong mucosal barrier in CF airways but may also provide a method by which to restore CFTR function in additional CF affected organs.

To facilitate the translation of NF1 exon 17 skipping as a mutation-specific therapy for Neurofibromatosis type 1 into in vivo testing, we have continued to develop more efficient antisense oligonucleotides (ASOs), humanized mouse models, and explored multiple delivery platforms including an adeno-associated virus (AAV)-U7-SnRNA vector approach. We evaluated both biodistribution and exon skipping efficacy of a U7-SnRNA targeting NF1 exon 17 with an SFFV-driven cassette containing T2A-linked Luciferase (Luc) and eGFP packaged in AAV-9, AAV-F and AAV-B1 capsids. We show that AAV-F is superior to AAV-9 and AAV-B1 for mouse brain delivery based on DNA transduction, GFP expression, and luciferase activity, but AAV-B1 delivers 2-4 fold more to sciatic nerve (SCN). In terms of exon skipping, AAV-F appears to induce the most skipping in liver and optic nerve (ON), while AAV-B1 mediates highest skipping in the liver, SCN, and ON. The identification of AAV serotypes that allow efficient transduction and delivery of transgenes to the mouse CNS and PNS is impactful for preclinical research in murine models of other diseases. Furthermore, this is both the first report of NF1 exon skipping efficacy in vivo and the first successful application of an U7-SnRNA for the restoration of functional neurofibromin for NF1.

Mutations in the Eyes Shut Homolog (EYS) gene are a leading cause of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the EYS coding sequence ([~]9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length EYS gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-STAR (Uni-directional and Site-specific Transgene Assembly by Recombination). The system consists of three AAV constructs carrying discrete EYS segments flanked by engineered, non-compatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system in vitro by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. In vivo, subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete EYS gene and provide a foundation for future therapeutic development targeting EYS-associated retinal degenerations.

Sandhoff Disease (SD), a fatal neurodegenerative disorder, is caused by the absence of {beta}-hexosaminidase (Hex) and subsequent accumulation of GM2 ganglioside in lysosomes. Previous studies have led to adeno-associated virus (AAV) gene therapy for children with GM2 gangliosidosis in both expanded access and Phase I/II clinical trials via intracranial and/or cerebrospinal fluid-based delivery. The current study investigated intravenous (IV) gene therapy of SD cats, treated at one month of age with a bicistronic AAV vector. While untreated SD cats lived to 4.3{+/-}0.2 months, cats treated with low and high doses lived to 8.3{+/-}1.2 and 12.4{+/-}2.7 months, respectively. In-life assessments revealed clear clinical benefit of AAV treatment, with the most dramatic improvement seen in the reduction of overt full-body tremors. Cerebrospinal fluid levels of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were decreased, indicating a reduction of cell damage within the central nervous system. Magnetic resonance imaging (MRI) and spectroscopy (MRS) acquired on a 7 Tesla scanner indicated that structural pathology and metabolite abnormalities are partially normalized by AAV treatment. Dose-dependent reduction of GM2 ganglioside storage and increases in Hex activity were most substantial in the caudal regions of the brain and in the spinal cord. Immunohistochemistry revealed reduction in neuroinflammatory cell populations and partial correction of myelin deficits. These results support the dose-dependent efficacy of AAV delivered IV for significant restoration of clinical metrics and Hex function in a feline model of SD. One Sentence SummaryIntravenous administration of AAV gene therapy is safe and efficacious in a feline model of Sandhoff disease.

Microglia are critical innate immune cells of the brain. In vivo targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors. Recently, there has been some progress demonstrating the feasibility and potential of using AAV to transduce microglia after direct intraparenchymal vector injection. Data suggests that combining specific AAV capsids with microglia-specific gene expression cassettes to reduce neuron off-targeting will be key. However, no groups have developed AAV capsids for microglia transduction after intracerebroventricular (ICV) injection. The ICV route of administration has advantages such as increased brain biodistribution while avoiding issues related to systemic injection. Here, we performed an in vivo selection using an AAV peptide display library that enables recovery of capsids that mediate transgene expression in microglia. Using this approach, we identified a capsid, MC5, which mediated enhanced transduction of microglia after ICV injection compared to AAV9. Furthermore, MC5 enhanced both the efficiency (85%) and specificity (93%) of transduction compared to a recently described evolved AAV9 capsid for microglia targeting after direct injection into the brain parenchyma. Exploration of the use of MC5 in a mouse models of Alzheimers disease revealed transduced microglia surrounding and within plaques. Overall, our results demonstrate that the MC5 capsid is a useful gene transfer tool to target microglia in vivo by direct and ICV routes of administration.

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

Adeno-associated virus (AAV) vectors are widely used for gene delivery, but inefficient transduction can require high vector doses. We tested whether unmodified linear polyvinylpyrrolidone (PVP), a pharmaceutical excipient, can improve AAV formulation without chemical modification of the vector or polymer. PVP10, PVP40, and PVP360 were evaluated in vitro across HEK293, HeLa, MEF, and CHO cells; selected formulations were tested after intravenous delivery, and 3% PVP40 was tested by subretinal delivery. In vitro, 1-3% PVP increased AAV8- and AAV9-mediated GFP expression across multiple cell lines. PVP360 showed broad activity in the initial cross-cell assay, whereas the HEK293 molecular-weight screen identified PVP40 and PVP360 as the most active formulations. The substantial fold increase observed in CHO cells largely reflected the low baseline transduction of the control group. MTT absorbance declined with concentrated PVP360, whereas PVP40 retained transduction-enhancing activity and was selected for local testing. In the HeLa AAV-DJ assay, the response pattern differed between MOI 1,000 and MOI 100; the largest observed increases in GFP-positive area occurred with 1.5% PVP10 and 1.5% PVP360 at MOI 100. Intravenous AAV9 delivery with PVP did not consistently increase ex vivo organ reporter signal. By contrast, subretinal delivery of AAV-PHP.eB with 3% PVP40 produced a 1.79- fold larger mean DsRed-positive area per retinal section, averaged within each eye (Welchs t-test p = 0.063; Bayesian Pr[{Delta} > 0] = 0.952). These findings support further evaluation of PVP40 for local subretinal AAV delivery.

Adeno-associated viruses (AAVs) have emerged as powerful tools for delivery of genes to a variety of cell types including pancreatic endocrine cells. Currently, AAV serotype 8 (AAV8) is the main AAV vector employed for infecting pancreatic cells for transgene transfer. We aimed to address whether alternative serotypes (AAV2, AAV6, and AAV9) commonly used for gene transfer can be effective in transducing pancreatic cells efficiently. We also screened the additives heparin and neuraminidase to further understand the interaction between the individual AAV types included in this work and the cells for optimal infection. Murine pancreatic {beta}-cells and -cells as well as fibroblasts were infected with AAV serotypes 2, 6, and 9 carrying the transgene for enhanced green fluorescent protein (eGFP). AAV2 outperformed AAV9 in transducing pancreatic cells, while AAV6 induced cytotoxicity. Both AAV2 and AAV9 displayed slightly higher tropism for -cells than for {beta}-cells. Compared to the pancreatic cells, the fraction of GFP-expressing cells at various multiplicities of infection was consistently lower for fibroblasts. Incubation of AAV2 with heparin prior to transduction failed to induce any GFP expression in {beta}-cells, indicating that the primary site used for initial interaction with pancreatic cells are heparan sulfate proteoglycans. Treatment of {beta}-cells with neuraminidase prior to AAV9 infection appeared to improve the number of GFP-positive cells, but the increase was not statistically significant. These findings expand the repertoire of available serotypes for AAV-mediated delivery of transgenes to pancreatic endocrine cells and may contribute to gene therapy strategies for pancreas pathologies.

Reprogramming of Muller glial (MG) cells into retinal neurons has the potential to treat vision loss by regenerating the retina. Development of efficient gene delivery systems to target the MG cells is critical. Adeno-associated virus (AAV) serotypes and promoter specificity are important factors that influence AAV transduction profile in the retina. However, studies that optimize these parameters to specifically target MG cells are limited, in particular in rats which are commonly used for eye research. Here we tested 4 AAV serotypes and 14 promoters to optimize gene delivery to human MG cells in vitro and/or rat MG cells in vivo. We showed that the combinatorial use of MG-specific serotypes and promoters achieved high specificity for MG cell targeting, with ShH10Y serotype and the GFAP (gfaABC1D) promoter as the best performing tool to target rat MG cells in vivo. We developed new AAV vectors using known and novel MG-specific promoters and engineered short promoter variants to improve the cargo capacity of AAV delivery. Our results highlighted a number of promoters that can target MG cells in vitro or in vivo. This study further expands the AAV toolbox to target MG cells, which has important implications for retinal gene therapy development.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

The efficacy of adeno-associated virus (AAV)-mediated systemic gene therapy for central nervous system (CNS) diseases is often limited by the blood-brain barrier (BBB). This study systematically evaluated the tissue distribution of three BBB-crossing AAV capsid variants (PHP.eB, CNSRCV300, and BI-hTFR1) following intravenous injection in mice, using either a constitutive promoter (CAG) or a neuron-specific promoter (hSyn) to drive EGFP reporter expression. Compared with AAV9, both PHP.eB and CNSRCV300 demonstrated significantly enhanced BBB penetration and brain transduction efficiency. While the use of the hSyn promoter led to reduced transgene expression in the brain compared with the CAG promoter, and substantially decreased visible reporter expression in peripheral organs, viral deposition in the liver could still be detected via immunohistochemistry. Overall, CNSRCV300 exhibited the most favorable balance between brain-targeting efficiency and biosafety, highlighting its potential as a promising delivery vector. In summary, both the capsid and promoter jointly influence AAV-mediated expression in vivo, and although cell type-specific promoters can reduce off-target expression, residual viral deposition in non-target tissues remains a potential safety concern.

Viral vector technologies based on adeno-associated virus (AAV) have demonstrated promising ability to deliver genetic cargo to a range of organs in vivo, with several novel candidates showing clinical efficacy in human trials over the past decade. However, naturally occurring AAV serotypes are limited in their ability to target skeletal muscle, an important gene therapy target for many neuromuscular disorders. This means that high doses of AAV are often required to achieve therapeutically effective doses in muscle. To overcome this, novel AAV vector capsids have been engineered by inserting targeting peptides into the AAV9 capsid variable region VIII (VRIII) to achieve greater muscle transduction efficiency. Here we describe investigation of a newly reported capsid, called MyoAAV1A combined with clinically validated muscle-specific promoters. We profiled the efficiency of in vivo delivery to murine skeletal muscle and found that the optimal combination of MyoAAV1A capsid with MHCK7 promoter maintains transgene expression in skeletal muscle, and reduces expression in off-target tissues, particularly the liver. This highlights a promising capsid-promoter combination to progress in further preclinical research for skeletal muscle gene therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/605568v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@10bbe78org.highwire.dtl.DTLVardef@5e2e0org.highwire.dtl.DTLVardef@71f4a7org.highwire.dtl.DTLVardef@1750d81_HPS_FORMAT_FIGEXP M_FIG C_FIG

Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it continues to suffer from the high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. In this regard, AAV therapies generally also must be administered as a single dose since neutralizing antibodies develop in patients who receive the virus. Strategies for circumventing these issues remain limited. As a novel solution, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV (VAAV) delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem and perhaps even allow administration of multiple doses, expanding the scope of AAV treatments.

Autosomal recessive thymidine kinase 2 (TK2) mutations causes TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with deoxycytidine and thymidine ameliorates mitochondrial defects and extends lifespan of Tk2 knock-in mouse (TK2-/-); however, efficacy is limited by age- and tissue-dependent expression of the cytosolic enzymes Tk1 and Dck. Thus, therapies aimed at systemic restoration of TK2 activity are needed. Here, we demonstrate that delivery of human TK2 cDNA to Tk2-/- mice using AAV9 efficiently rescued Tk2 activity in all the tissues tested except kidney, delayed disease onset, and increased lifespan. Sequential treatment of Tk2-/- mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2-/- mice dramatically improved mtDNA copy numbers in liver and kidney, animal growth, and lifespan. These data indicate that combined pharmacological and gene therapies may be highly efficacious for human TK2 deficiency.

We report the discovery of novel adeno-associated virus (AAV) capsid variants engineered for superior intravitreal (IVT) gene delivery to the primate retina. Utilizing the REACH platform, we constructed a diverse AAV variant library and employed a multi-stage screening strategy involving in vitro selection on human retinal pigment cells followed by direct in vivo screening in non-human primates (NHPs). Following IVT administration in NHPS of a barcoded variant pool, next-generation sequencing analysis of retinal tissues identified lead candidates (e.g., E52, E54, and E57) that achieved transduction levels in the neural retina and RPE 5-10 fold higher than the benchmark R100. Concurrently, these high-potency variants exhibited an exceptional ocular confinement profile, with minimal to undetectable vector genome distribution in systemic organs. This combination of markedly enhanced retinal transduction and stringent local tropism establishes these engineered capsids as promising next-generation vectors for the treatment of inherited and acquired retinal diseases via a minimally invasive IVT route.

Adeno-associated virus purification by density-gradient ultracentrifugation is labor-intensive and often results in substantial titer loss due to particle aggregation. Here, we present a scalable co-isolation strategy in which AAV is precipitated together with extracellular vesicles secreted by the producer cell line, completely bypassing density-gradient separation. The resulting AAV-EV preparations comprise free AAV, free EVs, and EV-associated AAV. Functionally, AAV-EV vectors (AAV2/1 serotype) support efficient ex vivo genome editing across multiple independent loci in mouse and rat zygotes, achieving a mean targeting efficiency of approximately 26%. Compared with gradient-purified AAV administered at matched doses, AAV-EV formulations yielded 2.34-fold higher embryo viability while maintaining equivalent transgene copy numbers. By leveraging EVs as a biological matrix, this approach enables ultracentrifugation-free AAV isolation without compromising vector functionality. Overall, AAV-EV represents an accessible and embryo-tolerant platform for rodent genome engineering that aligns with the principles of Replacement, Reduction, and Refinement (3R) principles.

Recombinant adeno-associated viral (rAAV) vectors are a potent tool, but their clinical application is restricted by insufficient target tissue transduction and liver toxicity. We employed a novel two-step engineering strategy to create novel rAAV capsids with global tissue de-targeting, then produced strong tissue-specific expression by adding a peptide sequence. We created a novel capsid, AAV.Zero1, with globally de-targeted transduction by loop swapping domains from AAV9 into AAV2. Making an R585A substitution (AAV.Zero2) re-targeted tissues but deleting residues 585-587 (AAV.Zero3) abrogated transduction. Inserting a myogenic peptide into AAV.Zero3 produced a novel capsid (AAV.eM) with strong muscle-specific transgene expression while maintaining minimal off-target expression, including in liver, which was conserved in two mouse strains and non-human primates. AAV.eM showed similar expression as the leading myotropic vector MyoAAV.4A but had a more favorable safety profile. Importantly, AAV.eM was able to functionally rescue a mouse model of Duchenne Muscular Dystrophy following systemic delivery of a micro-dystrophin gene. Thus, AAV.eM is an improved myotropic rAAV capsid that de-targets other tissues, especially the liver, and proof-of-concept for a platform to create capsids with specific properties that translate across species by addition of peptides onto low transduction backbones.