Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B.

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-human primates at doses 1.6× and 4.3× the highest dose proposed for the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution and shedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

miR-181a/b downregulation: a mutation-independent therapeutic approach for inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene-specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR-181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR-181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno-associated viral vector (AAV) that harbors a miR-181a/b inhibitor (sponge) sequence (AAV2/8-GFP-Sponge-miR-181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO-P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR-181a/b downregulation modulates the level of the mitochondrial fission-related protein Drp1 and rescues the mitochondrial fragmentation in RHO-P347S photoreceptors. Overall, these data support the potential use of miR-181a/b downregulation as an innovative mutation-independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene-specific therapeutic approaches.

Therapeutic homology-independent targeted integration in retina and liver.

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

Challenging Safety and Efficacy of Retinal Gene Therapies by Retinogenesis.

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Gene replacement therapy provides benefit in an adult mouse model of Leigh syndrome.

Mutations in nuclear-encoded mitochondrial genes are responsible for a broad spectrum of disorders among which Leigh syndrome is the most common in infancy. No effective therapies are available for this severe disease mainly because of the limited capabilities of the standard adeno-associated viral (AAV) vectors to transduce both peripheral organs and the CNS when injected systemically in adults. Here, we used the brain-penetrating AAV-PHP.B vector to reinstate gene expression in the Ndufs4 knockout mouse model of Leigh syndrome. Intravenous delivery of an AAV.PHP.B-Ndufs4 vector in 1-month-old knockout mice restored mitochondrial complex I activity in several organs including the CNS. This gene replacement strategy extended lifespan, rescued metabolic parameters, provided behavioural improvement, and corrected the pathological phenotype in the brain, retina, and heart of Ndufs4 knockout mice. These results provide a robust proof that gene therapy strategies targeting multiple organs can rescue fatal neurometabolic disorders with CNS involvement.

Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated down-regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

AAV-miR-204 Protects from Retinal Degeneration by Attenuation of Microglia Activation and Photoreceptor Cell Death.

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival.

Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15.

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 (KLF15) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin (RHO) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO-expressing cells). Adeno-associated virus (AAV) vector-mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene-targeted manipulation.

The centrosomal OFD1 protein interacts with the translation machinery and regulates the synthesis of specific targets.

Protein synthesis is traditionally associated with specific cytoplasmic compartments. We now show that OFD1, a centrosomal/basal body protein, interacts with components of the Preinitiation complex of translation (PIC) and of the eukaryotic Initiation Factor (eIF)4F complex and modulates the translation of specific mRNA targets in the kidney. We demonstrate that OFD1 cooperates with the mRNA binding protein Bicc1 to functionally control the protein synthesis machinery at the centrosome where also the PIC and eIF4F components were shown to localize in mammalian cells. Interestingly, Ofd1 and Bicc1 are both involved in renal cystogenesis and selected targets were shown to accumulate in two models of inherited renal cystic disease. Our results suggest a possible role for the centrosome as a specialized station to modulate translation for specific functions of the nearby ciliary structures and may provide functional clues for the understanding of renal cystic disease.

Rhodopsin targeted transcriptional silencing by DNA-binding.

Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

Transdermal spinal catheter placement in piglets: Description and validation of the technique.

BACKGROUND: The swine species represents a perfect model for translational medicine due to its physiological and anatomical resemblance to humans. The development of techniques for spinal catheter insertion in swine is significantly useful but, at the moment, the only technique described requires laminectomy as a surgical approach. NEW METHOD: The proposed techniques represent a transdermal approach for catheter placement in piglets. The study was divided into Phase I (anatomical study on 8 cadavers) and Phase II (in vivo application of the technique in 20 anaesthetised 30-day old piglets). A spinal needle was introduced between the L2 and L3 spinous processes with a ventro-cranial orientation until cerebro-spinal fluid leakage. It was then replaced with a Tuohy needle, used to introduce the catheter into the intrathecal space. Before inserting the catheter, the approximate length from the insertion point to the external projection of the Cisterna Magna was measured using the gradation markings on the device. RESULTS: The technique described allowed spinal catheter placement in all piglets. In Phase I, the correct placement was confirmed using fluoroscopy while, in Phase II, cerebrospinal fluid leakage from the needle was relied on. No clinical alterations were detected either during the procedure or during the following days. COMPARISON WITH EXISTING METHOD: This technique is easy and requires less skilled operators when compared to the other existing method which involves a surgical approach. Moreover, being less invasive, it potentially leads to fewer complications. CONCLUSIONS: In conclusion, the technique can be performed safely in piglets, and provides an easier and less invasive approach for spinal catheter insertion.

Recombinant vectors based on porcine adeno-associated viral serotypes transduce the murine and pig retina.

Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.

Correlation between photoreceptor layer integrity and visual function in patients with Stargardt disease: implications for gene therapy.

PURPOSE: To perform a clinical characterization of Stargardt patients with ABCA4 gene mutation, and to investigate the correlation between the inner and outer segment (IS/OS) junction morphology and visual acuity, fundus lesions, electroretinogram abnormalities, and macular sensitivity. METHODS: Sixty-one patients with Stargardt disease (STGD) were given a comprehensive ophthalmic examination. Inner-outer photoreceptor junction morphology evaluated by spectral-domain optical coherence tomography was correlated with visual acuity, fundus lesions, fundus autofluorescence, full-field and multifocal electroretinography responses, and microperimetric macular sensitivities. We classified STGD patients into three groups: (1) IS/OS junction disorganization in the fovea, (2) IS/OS junction loss in the fovea, and (3) extensive loss of IS/OS junction. Mutation analysis of the ABCA4 gene was carried out by sequencing the complete coding region. RESULTS: A significant difference in visual acuity was observed between IS/OS groups 1 and 2 and between IS/OS groups 2 and 3 (P < 0.0001). A significant difference in microperimetry sensitivity was observed between IS/OS groups 2 and 3, and between IS/OS groups 1 and 3 (P < 0.0001). There was also a statistically significant correlation between IS/OS abnormalities and the extent of fundus lesions (Spearman P ≤ 0.01), as well as with the type of ERG and multifocal ERG results (Spearman P ≤ 0.01). Finally, the degree of IS/OS junction preservation showed a statistically significant correlation with the extension of foveal abnormalities assessed by fundus autofluorescence imaging (Spearman P ≤ 0.01). The G1961E mutation was more frequent in the patients without extensive loss of IS/OS junction (P = 0.01) confirming its association with a milder STGD phenotype. CONCLUSIONS: The results of this study suggest that a comprehensive approach in the examination of Stargardt patients has the potential to improve the understanding of vision loss and may provide a sensitive measure to evaluate the efficacy of future experimental therapies in patients with STGD.

The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina.

Long noncoding RNAs (lncRNAs) are emerging as regulators of many basic cellular pathways. Several lncRNAs are selectively expressed in the developing retina, although little is known about their functional role in this tissue. Vax2os1 is a retina-specific lncRNA whose expression is restricted to the mouse ventral retina. Here we demonstrate that spatiotemporal misexpression of Vax2os1 determines cell cycle alterations in photoreceptor progenitor cells. In particular, the overexpression of Vax2os1 in the developing early postnatal mouse retina causes an impaired cell cycle progression of photoreceptor progenitors toward their final committed fate and a consequent delay of their differentiation processes. At later developmental stages, this perturbation is accompanied by an increase of apoptotic events in the photoreceptor cell layer, in comparison with control retinas, without affecting the proper cell layering in the adult retina. Similar results are observed in mouse photoreceptor-derived 661W cells in which Vax2os1 overexpression results in an impairment of the cell cycle progression rate and cell differentiation. Based on these results, we conclude that Vax2os1 is involved in the control of cell cycle progression of photoreceptor progenitor cells in the ventral retina. Therefore, we propose Vax2os1 as the first example of lncRNA that acts as a cell cycle regulator in the mammalian retina during development.

MicroRNA-restricted transgene expression in the retina.

BACKGROUND: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. METHODOLOGY/PRINCIPAL FINDINGS: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3'UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. CONCLUSIONS: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.

Evaluation of Italian patients with leber congenital amaurosis due to AIPL1 mutations highlights the potential applicability of gene therapy.

PURPOSE: To evaluate the suitability of gene delivery-based approaches as potential treatment of Leber congenital amaurosis 4 (LCA4) due to AIPL1 mutations. METHODS: Genomic DNA from patients was analyzed using a microarray chip and direct sequencing. A detailed clinical evaluation including fundus autofluorescence (FAF) and optical coherence tomography (OCT) was performed in patients with AIPL1 mutations. Aipl1 null mice and porcine eyes were subretinally injected with adeno-associated viral (AAV) vectors harboring the human AIPL1 coding sequence. RESULTS: We identified 10 LCA4 patients with mutations in AIPL1. The p.W278X sequence variation was the one most frequently found. Clinical assessment revealed common features including diffuse retinal dystrophies and maculopathy. However, optical coherence tomography showed partially retained photoreceptors in extramacular regions at all ages. The fundus autofluorescence was elicitable at the posterior pole and absent in the fovea. AAV-mediated gene transfer in Aipl1 -/- mice was associated with restoration of AIPL1 and ßPDE expression in photoreceptors and protection from degeneration. Administration of a clinically relevant dose of AAV2/8-AIPL1 to the preclinical large porcine retina resulted in high level of AIPL1 photoreceptor expression in the absence of toxicity. CONCLUSIONS: Using advanced imaging diagnostics we showed that maculopathy is a main feature of LCA4. We identified retinal areas at the posterior pole with surviving photoreceptors present even in adult LCA4 patients, which could be the target of gene therapy. The possible use of gene therapy for LCA4 is additionally supported by the protection from photoreceptor degeneration observed in Aipl 1-/- mice and by the high levels of photoreceptor transduction in the absence of toxicity observed after AAV2/8 delivery to the large porcine retina.

Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa.

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a 'repression-replacement' strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.