Gene Therapy For Retinal Diseases Research Articles

Gene therapy for retinal diseases: From genetics to treatment.

The gene therapy approach for retinal disorders has been considered largely over the last decade owing to the favorable outcomes of the US Food and Drug Administration-approved commercial gene therapy, Luxturna. Technological advances in recent years, such as next-generation sequencing, research in molecular pathogenesis of retinal disorders, and precise correlations with their clinical phenotypes, have contributed to the progress of gene therapies for various diseases worldwide, and more recently in India as well. Thus, considerable research is being conducted for the right choice of vectors, transgene engineering, and accessible and cost-effective large-scale vector production. Many retinal disease-specific clinical trials are presently being conducted, thereby necessitating the collation of such information as a ready reference for the scientific and clinical community. In this article, we present an overview of existing gene therapy research, which is derived from an extensive search across PubMed, Google Scholar, and clinicaltrials.gov sources. This contributes to prime the understanding of basic aspects of this cutting-edge technology and information regarding current clinical trials across many different conditions. This information will provide a comprehensive evaluation of therapies in existing use/research for personalized treatment approaches in retinal disorders.

Optogenetics and Targeted Gene Therapy for Retinal Diseases: Unravelling the Fundamentals, Applications, and Future Perspectives.

With a common aim of restoring physiological function of defective cells, optogenetics and targeted gene therapies have shown great clinical potential and novelty in the branch of personalized medicine and inherited retinal diseases (IRDs). The basis of optogenetics aims to bypass defective photoreceptors by introducing opsins with light-sensing capabilities. In contrast, targeted gene therapies, such as methods based on CRISPR-Cas9 and RNA interference with noncoding RNAs (i.e., microRNA, small interfering RNA, short hairpin RNA), consists of inducing normal gene or protein expression into affected cells. Having partially leveraged the challenges limiting their prompt introduction into the clinical practice (i.e., engineering, cell or tissue delivery capabilities), it is crucial to deepen the fields of knowledge applied to optogenetics and targeted gene therapy. The aim of this in-depth and novel literature review is to explain the fundamentals and applications of optogenetics and targeted gene therapies, while providing decision-making arguments for ophthalmologists. First, we review the biomolecular principles and engineering steps involved in optogenetics and the targeted gene therapies mentioned above by bringing a focus on the specific vectors and molecules for cell signalization. The importance of vector choice and engineering methods are discussed. Second, we summarize the ongoing clinical trials and most recent discoveries for optogenetics and targeted gene therapies for IRDs. Finally, we then discuss the limits and current challenges of each novel therapy. We aim to provide for the first time scientific-based explanations for clinicians to justify the specificity of each therapy for one disease, which can help improve clinical decision-making tasks.

A New Generation of Gene Therapies as the Future of Wet AMD Treatment.

Age-related macular degeneration (AMD) is an eye disease and the most common cause of vision loss in the Western World. In its advanced stage, AMD occurs in two clinically distinguished forms, dry and wet, but only wet AMD is treatable. However, the treatment based on repeated injections with vascular endothelial growth factor A (VEGFA) antagonists may at best stop the disease progression and prevent or delay vision loss but without an improvement of visual dysfunction. Moreover, it is a serious mental and financial burden for patients and may be linked with some complications. The recent first success of intravitreal gene therapy with ADVM-022, which transformed retinal cells to continuous production of aflibercept, a VEGF antagonist, after a single injection, has opened a revolutionary perspective in wet AMD treatment. Promising results obtained so far in other ongoing clinical trials support this perspective. In this narrative/hypothesis review, we present basic information on wet AMD pathogenesis and treatment, the concept of gene therapy in retinal diseases, update evidence on completed and ongoing clinical trials with gene therapy for wet AMD, and perspectives on the progress to the clinic of "one and done" therapy for wet AMD to replace a lifetime of injections. Gene editing targeting the VEGFA gene is also presented as another gene therapy strategy to improve wet AMD management.

Human Pluripotent Stem Cell-Derived Retinal Organoids: A Viable Platform for Investigating the Efficacy of Adeno-Associated Virus Gene Therapy.

With recent advances in adeno-associated virus (AAV)-based gene therapy, efficacy and toxicity screening have become essential for developing gene therapeutic drugs for retinal diseases. Retinal organoids from human pluripotent stem cells (hPSCs) offer a more accessible and reproducible human test platform for evaluating AAV-based gene therapy. In this study, hPSCs were differentiated into retinal organoids composed of various types of retinal cells. The transduction efficiencies of AAV2 and AAV8, which are widely used in clinical trials of inherited retinal diseases, were analyzed using retinal organoids. These results suggest that retinal organoids derived from hPSCs serve as suitable screening platforms owing to their diverse retinal cell types and similarity to the human retina. In summary, we propose an optimal stepwise protocol that includes the generation of retinal organoids and analysis of AAV transduction efficacy, providing a comprehensive approach for evaluating AAV-based gene therapy for retinal diseases.

Genetic testing and gene therapy in retinal diseases: Knowledge and perceptions of optometrists in Australia and New Zealand.

With advances in gene-based therapies for heritable retinal diseases, primary eye care clinicians should be informed on ocular genetics topics. This cross-sectional survey evaluated knowledge, attitudes, and concerns regarding genetic testing and gene therapy for retinal diseases among optometrists in Australia and New Zealand. Survey data included practitioner background, attitudes and practices towards genetic testing for monogenic inherited retinal disease (IRDs) and age-related macular degeneration, and knowledge of ocular genetics and gene therapy. Responses were received from 516 optometrists between 1 April and 31 December 2022. Key perceived barriers to accessing genetic testing were lack of clarity on referral pathways (81%), cost (65%), and lack of treatment options if a genetic cause is identified (50%). Almost all respondents (98%) believed that ophthalmologists should initiate genetic testing for IRDs and fewer understood the role of genetic counsellors and clinical geneticists. This study found that optometrists in Australia and New Zealand have a high level of interest in ocular genetics topics. However, knowledge gaps include referral pathways and awareness of genetic testing and gene therapy outcomes. Addressing perceived barriers to access and promoting sharing of knowledge between interdisciplinary networks can set the foundation for genetic education agendas in primary eye care.

Gene Therapy for Retinal Degenerative Diseases: Progress, Challenges, and Future Directions.

Since long before the first approval of gene therapy for retinal disease, ocular gene therapy has captured the hopes of patients, clinicians, and scientists alike. Indeed, the retina provides a unique system for studying and treating ocular diseases, and it holds the distinction as the first tissue targeted by an approved gene therapy for inherited disorders in the United States. There are many methods for addressing genetic diseases in the eyes using a wide range of potential delivery systems and vectors. However, despite the immense progress over the last several decades, both old and new challenges remain, such as the long-term effects of treatments, immunogenicity, targeting, and manufacturing. This review provides a discussion of the history of ocular gene therapy, the various gene therapy approaches, methods to deliver a gene directly to ocular tissues (including both routes of administration and vectors), challenges to ocular gene therapy, the current clinical trial landscape, and future directions of the field.

Current trends in gene therapy for retinal diseases (Review).

The eye is considered an effective target for genetic therapy, as it has a privileged immune status, it is easily accessed for medication delivery and it is affected by a number of inherited disorders. In particular, the retina is considered for gene therapy due to the fact that it can be visualized with ease, it does not have lymphatic vessels, nor a direct blood network for the outer layers and its cells do not divide after birth, and thus transgene expression is not affected. As gene therapy is currently on a continuously progressive development trend, this emerging field of gene manipulation techniques has yielded promising results. This involves the development of treatments for a number of debilitating and blinding diseases, which were to date considered intractable. However, numerous unanswered questions remain as regards the long-term efficacy and safety profile of these treatments. The present review article discusses the current research status regarding genetic manipulation techniques aimed at addressing visual impairment related to retinal disorders, both inherited and degenerative.

Gene therapy in retinal diseases: A review.

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna—the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.

Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno‐associated virus (rAAV) vector‐based delivery has shown clinical success, and clinical trials based on rAAV‐based optogenetic therapies are currently in progress. Recently, we have developed multi‐characteristic opsin (MCO), which has been shown to effectively re‐photosensitize photoreceptor‐degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO‐I) in live samples and post‐mortem organs following intraocular delivery in wild‐type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO‐I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low‐dose group 13 weeks after injection and in the faeces of the high‐dose group at 3–13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post‐injection. Intravitreal injection of vMCO‐I resulted in few sporadic off‐target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2‐based delivery of MCO‐I for retinal gene therapy is safe.

Optogenetics-Mediated Gene Therapy for Retinal Diseases.

The visual system consists of various types of neurons and a single-nucleotide mutation can sometimes lead to blindness. The phototransduction pathway in the retina starts from the first-order neurons, the photoreceptor cells, and transmits the signals to second-order neurons. Finally, the output signal from third-order neurons, the ganglion cells, is carried to the brain. The photoreceptor cells are the only neurons in the retina that can respond to a light signal; they are hyperpolarised when they receive light, and the ganglion cells carry the signals to the brain following the depolarization. Recently, various types of channelrhodopsins have been found and developed. It is expected that the gene therapies using the cation channel as well as anion channelrhodopsin genes would be effective against diseases that cause severe destruction of visual function, such as the retinitis pigmentosa and age-related macular degeneration. In this review, we mainly describe mVChR1-mediated gene therapy for retinitis pigmentosa and the future application of optogenetic genes in retinal diseases.

Suprachoroidal Delivery of Viral and Nonviral Gene Therapy for Retinal Diseases.

Retinal gene therapy is a rapidly growing field with numerous clinical trials underway, and route of delivery is a critical contributor to its success. Subretinal administration, which involves pars plana vitrectomy in the operating room, offers targeted delivery to retinal-pigment epithelium cells and photoreceptors. Due to the immune-privileged nature of the subretinal space, the risk of an immune reaction against viral capsid antigens is minimized, an advantage of subretinal administration in patients with preexisting neutralizing antibodies. Intravitreal administration, with fewer procedure-related complications, is challenged by potential immune response and incomplete vector penetration through the internal limiting membrane. However, novel vectors, optimized by “directed evolution” may address these issues. Nonsurgical in-office suprachoroidal gene delivery offers the potential for greater surface-area coverage of the posterior segment compared to focal subretinal injection, and is not hindered by the internal limiting membrane. However, the vector must pass through multiple layers to reach the targeted retinal layers, and there is a risk of immune response. This review highlights recent developments, challenges, and future opportunities associated with viral and nonviral suprachoroidal gene delivery for the treatment of chorioretinal diseases. While ocular tolerability and short-term effectiveness of suprachoroidal gene delivery have been demonstrated in preclinical models, durability of gene expression, long-term safety, potential systemic exposure, and effective delivery to the macula require further exploration. Although the safety and efficacy of suprachoroidal gene delivery are yet to be proven in clinical trials, further optimization could facilitate nonsurgical in-office suprachoroidal gene therapy.

Can an in vivo imaging system be used to determine localization and biodistribution of AAV5-mediated gene expression following subretinal and intravitreal delivery in mice?

Recombinant adeno associated viruses (AAV) are the most commonly used vectors in animal model studies of gene therapy for retinal diseases. The ability of a vector to localize and remain in the target tissue, and in this manner to avoid off-target effects beyond the site of delivery, is critical to the efficacy and safety of the treatment. The in vivo imaging system (IVIS) is a non-invasive imaging tool used for detection and quantification of bioluminescence activity in rodents. Our aim was to investigate whether IVIS can detect localization and biodistribution of AAV5 vector in mice following subretinal (SR) and intravitreal (IVT) injections. AAV5 carrying firefly luciferase DNA under control of the ubiquitous cytomegalovirus (CMV) promoter was injected unilaterally IVT or SR (in the central or peripheral retina) of forty-one mice. Luciferase activity was tracked for up to 60 weeks in the longest surviving animals, using repeated (up to 12 times) IVIS bioluminescence imaging. Luciferase presence was also confirmed immunohistochemically (IHC) and by PCR in representative animals. In the SR group, IVIS readings demonstrated luciferase activity in all (32/32) eyes, and luciferase presence was confirmed by IHC (4/4 eyes) and PCR (12/12 eyes). In the IVT group, IVIS readings demonstrated luciferase activity in 7/9 eyes, and luciferase presence was confirmed by PCR in 5/5 eyes and by IHC (2/2 eyes). In two SR-injected animals (one each from the central and peripheral injection sites), PCR detected luciferase presence in the ipsilateral optic nerves, a finding that was not detected by IVIS or IHC. Our results show that when evaluating SR delivery, IVIS has a sensitivity and specificity of 100% compared with the gold standard PCR. When evaluating IVT delivery, IVIS has a sensitivity of 78% and specificity of 100%. These finding confirm the ability of IVIS to detect in-vivo localized expression of AAV following SR delivery in the retina up to 60 weeks post-treatment, using repeated imaging for longitudinal evaluation, without fading of the biological signal, thereby replacing the need for post mortem processing in order to confirm vector expression. However, IVIS is probably not sensitive enough, compared with genome detection, to demonstrate biodistribution to the optic nerve, as it could not detect luciferase activity in ipsilateral optic nerves following SR delivery in mice.

The Role of Gene Therapy in the Treatment of Retinal Diseases: A Review.

Gene therapy represents the therapeutic delivery of nucleic acid polymers into patient cells with the aim of treating an underlying disease. Over the past 2 decades this new therapy has made substantial progress owing to better understanding of the pathobiologic basis of various diseases coupled with growth of gene transfer biotechnologies. The eye, in particular, represents a suitable target for such therapy due to the immune privilege provided by the blood-ocular barrier, the ability to directly visualize, access and locally treat the cells and the minimal amount of vector needed given the size of this organ. It is not surprising therefore that several clinical trials are now ongoing in this field. The purpose of this review was to provide an update on gene therapy for retinal diseases, discussing differences in treatment strategies, vector designs and surgical techniques. Research was performed on PubMed, ClinicalTrials.gov, and Home Genetic Reference. We additionally utilized the internet database for genetics of retinal diseases, the portal for rare diseases and orphan drugs and the NCBI database Online Mendelian Inheritance in Man. No restriction was applied on the language of publications. We present the available results of current active clinical trials for inherited retinal disease such as Leber's congenital amaurosis type 2, choroideremia, Stargardt disease, achromatopsia and juvenile X-linked retinoschisis. We also illustrate a new approach of this therapy for the treatment of much more common ocular diseases such as age-related macular degeneration and diabetic retinopathy. Gene therapy represents an emerging and promising therapeutic approach for the treatment not only of rare inherited retinal diseases but also much more common retinal pathologies.

500. Triple AAV Vectors Expand AAV Cargo Capacity in the Retina

We have recently shown that dual AAV vectors expand AAV cargo capacity in the retina up to 9kb thus allowing their application to the therapy of inherited blinding conditions like Stargardt disease or Usher syndrome type IB (USHIB). However, for several other inherited blinding conditions the size of the coding sequence to be transferred would not fit into dual AAV vectors and would require a triple AAV vector system. We have generated triple AAV vectors encoding a reporter EGFP-DsRed fusion protein under the transcriptional control of the ubiquitous CMV promoter. The expression cassette was split in three parts and packaged in three distinct AAV vectors. In order to favour the directional concatemerization upon co-infection, highly recombinogenic sequences were added to the constructs which would then be eliminated from the mature transcript by the inclusion of flanking splicing signals (Figure 1). Triple AAV 2/2 vectors were used to infect HEK293 cells in vitro and AAV 2/8 vectors in vivo by subretinal administration in C57Bl/6 mice. Direct fluorescence analysis showed EGFP and DsRed colocalization in HEK293 cells; full length protein expression was confirmed by Western Blot analysis of HEK293 cell lysates. Fundoscopy analysis of C57Bl/6 mice up to 2 months after subretinal delivery shows signal compatible with EGFP-DsRed expression. Western-blot analysis of retinal lysates supports the production of the full length protein. Our results suggest that AAV DNA cargo capacity can be increased up to 12kb in the retina by triple AAV vectors. This bodes well for gene therapy of retinal diseases which require a high DNA cargo capacity. View Large Image | Download PowerPoint Slide

270. Nanoparticle-Mediated Rhodopsin cDNA But Not the Intron-Containing DNA Delivery Causes Transgene Silencing in a Rhodopsin Knockout Model

Gene expression is moderated by a multitude of patterns. The cross-talk between the native promoters and all the non-coding endogenous elements, such as transcription factors, enhancers and introns, are tightly controlled in a precise and real-time manner.The use of cDNA transgene often causes transgene silencing as over-expression or under-expression is known to affect protein function. In contrast, large genomic DNA transgene showed positionindependent, copy number-dependent expression of the transgene. Precise control of rhodopsin expression is critical in maintaining viable photoreceptors. In the present study, two forms of rhodopsin DNAs, an intron-containing short form genomic DNA and an intron-less cDNA, were cloned into an S/MAR vector driven by the 500 bp mouse opsin (MOP) promoter. The vectors were further compacted into nanoparticles (NPs) and subretinally injected into a rhodopsin knockout mouse (RKO) model at postnatal day 3. Following injection, DNA methylation and histone status of the specific regions (bacteria plasmid backbone, promoter, rhodopsin gene and S/MAR) in the vectors were evaluated at various time points. Persistent expression and phenotypic improvement were found in RKO mice up to 8 months after NP-mediated intron-containing delivery but not the NPs housing the intron-less cDNA delivery (data submitted for publication under review). In this study, our results further indicated that epigenetic silencing occurred in vector-mediated gene transfer, which were contributed by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene as evaluated by DNA methylation specific PCR followed by DNA sequencing and chromatin immunoprocipitation (ChIP) assay with highly specific antibodies for euchromatin and heterochromatin. No toxicity or inflammation was found in the treated eyes as detected by both PCR for pro-inflammatory cytokines and immunohistochemistry using macrophage marker, F4/80. These results suggest that cDNA of transgene and bacteria backbone interfere with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochratin-associated modification. While the benefits of including genomic elements may vary from gene to gene, our data suggest reconsider the current paradigm using cDNA as the standard in gene delivery cassettes. This paradigm shift has wide-ranging application for ocular gene therapy for retinal diseases or for which a genetic treatment is of interest. Further experiments are currently underway to determine whether inclusion of additional endogenous elements will provide added advantages in terms of physiologically relevant gene expression.

Gene therapy for retinal diseases.

Gene therapy has a growing research potential particularly in the field of ophthalmic and retinal diseases owing to three main characteristics of the eye; accessibility in terms of injections and surgical interventions, its immune-privileged status facilitating the accommodation to the antigenicity of a viral vector, and tight blood-ocular barriers which save other organs from unwanted contamination. Gene therapy has tremendous potential for different ocular diseases. In fact, the perspective of gene therapy in the field of eye research does not confine to exclusive monogenic ophthalmic problems and it has the potential to include gene based pharmacotherapies for non-monogenic problems such as age related macular disease and diabetic retinopathy. The present article has focused on how gene transfer into the eye has been developed and used to treat retinal disorders with no available therapy at present.

Effective delivery of large genes to the retina by dual AAV vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina

Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.

Dawn of ocular gene therapy: implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients' genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease. In this review, we discuss the application of Next Generation Sequencing (NGS) to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases: Leber's Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt's disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinformatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

BackgroundAdeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.Methodology/Principal FindingsWe subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.Conclusion/SignificanceIn the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.