Following the development of technologies in molecular biology,more and more mutant genes that cause retinal degenerative diseases have been found.Meanwhile,many naturally occurring or genetically engineered animal models have showed similar gene mutations and phenotypes as the human inherited retinal diseases,which have led to the development of a variety of therapeutic strategies for those traditionally incurable inherited diseases.Following Leber congenital amaurosis 2 (LCA2) gene therapy clinical trial,more gene therapy clinical trials including retinitis pigmentosa (RP) with MERTK mutation,Stargardt disease with ABCA4 mutation,Usher syndrome with MYO7A mutation and age-related macular degeneration (AMD) are ongoing.Adeno associated virtal (AAV) vectormediated gene replacement therapy that covers the whole retina showed great potential to cure early stage of those patients;while,gene replacement therapy combined with other approaches like treatment with anti-apoptotic agent and/or neurotropic factor,which can extend the therapeutic window in middle to late stages of those patients,is a potentially promising strategy for improving photoreceptor function and significantly slowing the process of retinal degeneration.

Stargardt disease (STGD) is an inherited disorder of retinal pigment epithelium.Three genes have been found to be implicated in STGD including Abca4 (adenosine triphosphate-binding cassette,sub-family A,member 4),Elovl4 (elongation of very long chain fatty acids protein 4) and Prom1 (prominin-1).Target genes can be delivered to the retina by various methods such as lentivirus (LV) vectors,adeno-associated virus (AAV) vectors and non-viral nano-particles.The Abca4-/-,Elovl4-/-and Prom1-/ mice model are used to study the pathogenesis mechanism and treatment of STGD.Retinal function improved significantly upon gene therapy in these models.Based on these works using animal model,phase Ⅰ /Ⅱ a clinical trial of Abca4-associated STGD gene therapy are underway.As a LV vector,equine infectious anemia virus (EIAV) is used to carry the Abca4 gene.These studies will evaluate three dose levels of the EIAV vector for safety,tolerability and biological activity.Moreover,some preclinical attempts to deliver Abca4 via AAV have been made using a modified AAV vectors because of the large size of the ABCA4 cDNA.The good responses in animal models render STGD a very attractive object for human gene therapy after the successful of the phase Ⅰ /Ⅱ clinical trials of Leber's congenital amaurosis.

Background Trans-corneally subretinal injection in rodent model is a useful method for genetic therapy,stem cell transplantation and the study on the ophthalmic research.Standarized operation process is critical for the successful treatment.However,there is no literature to report the detailed procedure and the influence of this technique on morphology and function of retina.Objective This sudy was to introduce a method of trans-corneally subretinal injection and evaluate its influence on the morphology and function of retina.Methods Trans-corneallly subretinal injection was performed on the left eyes of 2-month-old SPF C57BL/6J mice after dilation of pupils.A 301/2G disposable needle was used to puncture the cornea within the pupil area near limbus and avoid touching the lens and irises under eye surgery microscope.Then,a 33G blunt needle was used to insert into the vitreous and toward subretinal space via corneal puncture.Normal saline with 0.1％ fluorescein sodium of 1 μl was slowly injected into the space,and 2.5％ hydroxypropyl methylcellulose was dropped on ocular surface for the observation of the fundus clearly.According to the percentage of the retina filled with subretinally injected solution,the experimental eyes were divided into 80％-100％ area group,50％-70％ area group after injection,and the mice in the pseudo-injected group,in which injection procedure stopped just before the solution was pushed in to the subretinal space did not inject any solution after punctured.The right uninjected eyes of the mice served as normal control group.Four eyes were selected for each group.The structural changes were evaluated by optical coherance tomograpby (OCT) 1 day,2 days,3 days and 5 weeks after injection,and retinal function was assessed by the recored of electroretinography (ERG) 5 weeks after injection.The retinal sepcimens were prepared to examin the morphological changes by hematoxylin and esosin staning.The use of care followd the Regulations for the Administration of Affair Concerning Experimental Animals of Zhejiang Province.Results About 70％ of the injected eyes showed that retinal blebs filled with injected green fluorescein solution occupied 50％ or more retinal area with minimal damages.The focal detachment between neurosensory retinal layer and retinal pigment epithelium (RPE) was exhibited 1 day postinjection,and almost all the retinas retached 2 days after injection.In the fifth week after injection,the amplitudes of ERG b wave were (386.25±37.88),(357.50±41.03),(324.25±53.45) and (410.50±14.88) μV in the sham operation group,50％-70％ area group,80％-100％ area group and normal control group,respectively,showing a significant difference among the 4 groups (F=3.574,P=0.047),and the amplitudes of b wave in the normal control group were higher than those in the 80％-100％ area group (all at P ＜ 0.05).The detachment between retinal neuroepithelium layer and RPE layer,cell proliferation and transposition in the outer nuclear layer were dispalyed under the light microscope in the sham operation group,50％-70％ area group and 80％-100％ area group,and the disordered outer segment of photoreceptors at the injecting area was seen in the 50％-70％ area and 80％-100％ area groups at five weeks after injection.However,retinal sructure and morphology were normal at the non-injection area.Conclusions Trans-corneally subretinal injection is an effective and safe way for subretinal injection.

Background The retinal degeneration 11 (rd11) mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 (Lpcatl) mutation.Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rd1 1 mice,while cone degeneration pattern in rd11 mice is unclcar.Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rd1 1 mice.Methods A total of thirty rd1 1 and C57BL/6J mice at postnatal (P) day 14,28,42 (five in each age group) were sacrificed and retinal wholemounts were prepared.Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts,which were photographed with a fluorescent microscope.Cone opsins were compared between rd1 1 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas.Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina.M-opsin positive fluorescent dots in dorsal/temporal,ventral/temporal,dorsal/nasal and ventral/nasal quadrants of rdl 1 retina at P28 were (414±32),(300± 8),(324 ± 22) and (250± 20)/0.037 mm2,which were lower than the age-matched normal C57BL/6J mice (t =4.114,15.225,7.505,17.990,all at P＜0.05).At the same time the S-opsin positive fluorescent dots in P28 rd11 were (8 ±4),(175 ± 16),(74 ± 13) and (315 ±20)/0.037 mm2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice (t =8.555,17.076,21.637,13.498,all at P＜0.05).With the development of retinal degeneration in rd11 mice,the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina.Conclusions Most of the M-opsin and S-opsins,especially the S-opsins in rd11 mice,degenerate in 6 weeks.Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study.

Inherited retinal diseases (IRDs) are rare and incurable eye diseases.Gene therapy has become a new method for the treatment of IRDs.At present, at least 26 clinical trials of gene therapy involving at least 16 different IRDs genes are in progress or imminent.Such as autosomal recessive retinitis pigmentosa (RP, MERTK mutation), choroideremia (CHM mutation), Stargardt’s disease (ABCA4 mutation), Usher syndrome 1B subtype (Myo7a mutation), X-linked retinoschisis (RS1 mutation), mitochondrial-related Leber hereditary optic neuropathy (ND4 mutation), panchromatic blindness (CNGA3 mutation and CNNGB3 mutation), sex-linked RP (RPGR mutation). Adeno-associated viral (AAV) vector-mediated gene expression is a conventional method for gene transduction, which has showed efficacies in autosomal recessive IRDs caused by small gene mutations, especially for those IRDs, which have the original lesions in retinal pigment epithelial (RPE) cells and photoreceptor cells.Novel progress has been made in animal experiments by gene editing technologies for autosomal dominant IRDs or autosomal recessive IRDs with large gene mutations, which is going to clinical trial.Oligonucleotide therapy based on RNA has also shown good efficacy for the IRDs caused by large genes, which beyond the AAV capacity.These research achievements are attacking more and more attentions in these fields.Although there are still some efficacy and safety issues on the way to treat IRDs, it is reasonable to expect that more and more patients with IRDs will be treated in the near future.

Background Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new,spontaneously arising mouse model for human Leber congenital amaurosis (LCA),and it is helpful for us to explore the pathogenesis and determine the treating target of RP.Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice,a LCA Rpe65rd12 (B6 [A]-Rpe65rd12/J) mouse.Methods The rd12 mice at postnatal (P) 14,P21,P35 and P90 were selected (5 mice for each),and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2,and short wave-length sensitive cone response was recorded using ultraviolet light ([363 ±6] nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning,respectively.Results In P14 rd12 mice,the ERG responses of overall cone cells presented the negative waveform and the latency was delayed,and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75％ in P21 rd12 mice compared with wild-type B6 mice,and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80± 11.39] ms vs.[43.40± 5.60] ms) (t =-8.106,P =0.001).The mean b-wave amplitudes of U Vsensitive cone cells were (59.60± 36.00),(82.40± 12.22) and (68.43 ± 17.63) μV in the wild-type B6 mice,andthose in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen,and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice;while only a few opsin positive-response cells were seen in P14,P21 and P35 rd12 mice.Conclusions In rd12 mice,the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days,and the loss of UV-sensitive cone cells is earlier and more obvious.

Epigenetics pertains to heritable alterations in gene expression when the nucleotide sequence remains unchanged.Epigenetic regulation mechanisms are diverse, among which DNA methylation, histone modification and non-coding RNA (ncRNA) regulation have been studied in depth.Epigenetic regulation is associated with a variety of human diseases.In the occurrence and development of retinal degenerative diseases, many epigenetic regulation processes such as DNA methylation, histone acetylation and ncRNA regulation have changed.DNA methylation is one of the important regulation processes in retinal degeneration.Aberrant DNA methylation patterns are associated with retinitis pigmentosa (RP), age-related macular degeneration (AMD), inflammation and oxidative stress.Histone acetylation is associated with RP, diabetic retinopathy (DR), glaucoma and retinal nerve ischemic injury.NcRNA is associated with RP, AMD, pathological angiogenesis, and DR.In this article, the application of epigenetic regulation in retinal degeneration was reviewed.

Inherited retinal diseases (IRDs), one type of the major eye diseases resulting in blindness, can be caused by more than 270 identified causative genes.The most common form of IRDs is retinitis pigmentosa.There is no generally accepted cure for vision impairment due to IRDs.In recent years, the first gene replacement therapy has been approved for the treatment of autosomal recessive IRDs.Because of the variety of pathogenesis, including gain-of-function and dominant-negative effects in addition to a few loss-of-function mutations, gene replacement therapy of autosomal dominant IRDs is not always effective.The clinical manifestations of autosomal dominant IRDs are extremely complex, and there is no appropriate treatment in clinical practice.The latest progresses in pathogenesis, clinical features, treatment strategies and directions of autosomal dominant IRDs globally were reviewed, and the most common genes causing autosomal dominant IRDs were summarized in this article in order to provide a deeper understanding of autosomal dominant IRDs.