About our work

Former NEI director Dr. Paul Sieving's research program focuses on structural and functional studies of photoreceptors, neurons, and glia in the retina of animal models and humans, in health and disease. The laboratory (STRRMD) is structured to include a broad range of expertise: retinal electrophysiology, genetic models of human blinding diseases, retinal cell biology and biochemistry, molecular biology, and gene therapy. A major impetus of STRRMD research is translational, whereby studies in rodent models of human genetic blinding diseases provide clues for therapeutic intervention. Dr. Sieving leads a highly dynamic research team and has established a unique and strong research program that is well poised to make breakthroughs to bring novel therapies to retinal degeneration patients.

This laboratory recently conducted the first human trial of ciliary neurotrophic factor (CNTF) as possible treatment for human hereditary retinal degenerative diseases, known principally as retinitis pigmentosa [Sieving, 2006]. This phase-1 trial of intraocular implants that release CNTF evolved as a translational project from laboratory work conducted by Dr. Sieving and Dr. Ronald Bush to explore small molecules and proteins that provide narrow protection for RP-class ocular diseases [Bush, 2004].

A major current laboratory translational focus is on gene therapy for X-linked retinoschisis (XLRS). The laboratory is completing pre-clinical studies to show efficacy of gene replacement therapy with a gene-knockout XLRS mouse model. These data, along with gene vector toxicity results, will be presented to FDA in preparation to mounting a human gene therapy trial for XLRS.

Current research

RCS rodent models of retinal degeneration

The STRRMD utilizes the electroretinogram (ERG) to dissect complex retinal processes in health and disease. The laboratory has characterized a novel and prominent corneal-negative ERG response in the Royal College of Surgeons (RCS) rat, a retinal degeneration model with a mutation in the Mertk gene. The retinal cellular origin of this response, named “negative photopic response” (NPR), was dissected by applying agents that disrupt synaptic signaling between specific cell types to identify the contributing neurons. This study implicated the activity of amacrine cells in the generation of the NPR [Machida, 2008]. The lab has also determined the role of K+ channels in the RCS-NPR response by developing a novel technique using antibodies against K+ channels coupled to a short Pep-1 peptide that facilitates delivery across the cell membrane and into the cell. In vivo functional data has demonstrated that Kir2.1 and Kir4.1 channels contribute to the generation of the RCS-NPR component. This was shown using electroretinogram recordings and by single cell patch clamp electrophysiology. The STRRMD has developed neurotrophic factor (LEDGF) gene therapy in the RCS rat using adeno-associated virus (AAV) as the delivery vector. This gene therapy strategy has restored retinal signaling and normalized the ERG response. Similar work could be extended to human patients.

Staining of retinal cells

Figure 1: Using antibodies to probe the origin of electroretinogram (ERG) signals in a rat model of retinitis pigmentosa. Antibodies to Kir 2.1 potassium channels administered to the eye of an RCS rat before recording the ERG reduced the signal coming from the inner retina, but non-specific immunoglobulin (IgG) did not. The Kir 2.1 antibody (stained red) administered in vivo binds to specific inner retinal cells in the same location as when the antibody is applied to retinal sections in vitro. IgG staining is diffuse and non-specific.

Rac1 involvement in ROS morphogenesis, homeostasis, and rod degeneration

Another thread to the research in the laboratory is related to the investigation of the protein Rac1 in rod photoreceptor outer-segment (OS) turnover and homeostasis. In animal models that fail to express rhodopsin, there is a failure of rod OS to develop, leading to rod-cell degeneration. In Drosophila mutants lacking opsin expression, photoreceptor rhabdomeres fail to develop normally but can be rescued by constitutively active Rac1. This suggests that rhodopsin controls the development of rod OS via Rac1 activation. The STRRMD is investigating whether Rac1 plays a similar role in the mouse, and if so, whether the manipulation of Rac1 expression provides a means to rescue photoreceptors in retinal degeneration associated with rhodopsin mutations. Dr. Sieving and his team have created and characterized a Rac1 rod conditional knockdown mouse (Rac1-CKO) and have demonstrated that Rac1 depletion in rods reduced light-induced photoreceptor degeneration [Haruta, 2009]. Recent data show that the molecular pathways downstream of Rac1 might contribute to the neuroprotective response via AP1 and STAT3, and that decreased activation of Rac1 and NADPH-oxidase function contribute to photoreceptor survival following light-induced photoreceptor damage in Rac1-CKO mice.

Diagram of RAC1 photoreceptors

Figure 2: Depleting Rac1 in mouse photoreceptors protects them from damage by light. Staining for Rac1 (brown) in retinas of mice in which the gene in photoreceptors has been knocked down shows that less Rac1 is present in the photoreceptors than in litter mates in which the gene is normal (wild type). When exposed to bright light, the damage and loss of photoreceptors is less in mice with depletion of Rac1.

X-linked retinoschisis (XLRS) disease

Dr. Sieving and his group are investigating the molecular pathology of XLRS disease and are working toward developing gene therapy for this disorder. XLRS is the most common from of macular dystrophy in young males; and there is no effective treatment. All affected males have macular changes, and the majority also present changes in the peripheral retina. The natural history is a slow deterioration in central retinal function. XLRS does not cause outright complete blindness unless there are secondary complications such as retinal detachment or vitreous hemorrhage. The causative gene (retinoschisin, RS1) was identified in 1997, and molecular diagnosis and genetic testing are now available through an affiliated lab. XLRS mutations have been correlated with the disease phenotype. A functional hallmark of XLRS is an electronegative ERG with a markedly reduced b-wave.

Progress in this research includes creation of RS1 null mice [Zeng, 2004] and the characterization of the long-term history of retinal degeneration [Kjellstrom 2007]. They identified synaptic pathology in RS1 knockout mice—which should provide novel insights into the mechanisms underlying RS1 defects [Takada, 2008]. Study showed the effect of retinoschisin deficit during retinal detachment (in collaboration with Dr. Steve Fisher: Luna, 2009); the characterization of a recombinant viral vector (AAV8-RS1promoterhumanRS1 gene) in preparation for clinical gene therapy; and demonstrated structural and functional rescue of photoreceptors by intravitreal RS1 gene transfer in the XLRS mice. Collectively, these studies form the basis for planning translational work toward a phase-1 human clinical trial by intravitreal administration of the human XLRS gene. An exciting part of the research relates to the demonstration that gene therapy can restore normal ERG responses in the mouse model. Although initial work used subretinal injections to deliver the vector, the Sieving lab has now successfully demonstrated that intravitreal injections using AAV2 vectors are equally effective. Moreover after transfection, high levels of retinoschisin protein expression could be demonstrated in photoreceptors—a surprising finding since previous publications, including Dr. Sieving’s own work, have shown that these vectors normally do not penetrate the outer retina in wild type animals. The demonstration that intravitreal administration of vector is effective in XLRS is likely to have major implications for the ease and safety of gene therapy for human ocular disease in the future. The Sieving laboratory has developed a human clinical trial protocol and has had preliminary discussions with the FDA regarding a phase-1 trial of gene replacement therapy.

OCT images of retina restoration

Figure 3: This figure demonstrated that the retina structure restoration after gene therapy. Left panel showed OCT images (Optical Coherence Tomography) taken from mouse retina; right panel showed retinoschisin protein expressing in mouse retina. a. Images were taken from wild type mouse. b. and c. Images were taken from RS1-null mouse: b. gene therapy treated eye of RS1-null mouse, there was no cavities and split in the inner nuclear layer (INL), and the structure of retina was well organized, but the outer nuclear layer (ONL) was still thinner than the retina of wild type mouse; c. Untreated eye of RS1-null mouse, there were severe cavities in the inner nuclear layer (INL).

Tools and approaches

The laboratory works at multiple levels, from cultured cells to whole animal ocular physiology, biochemistry and molecular biology and genetics. Ronald Bush, Ph.D., is the principal staff scientist and directs the work using electroretinogram recordings and rhodopsin photochemistry for retinal functional study and structural analysis using morphometric techniques and immunohistochemistry. He has particular expertise and has published extensively on retinal light damage and photostasis in rats, models of rod photoreceptor degeneration and plasticity.

Selected publications

2019

Heymann JB Vijayasarathy CHuang RKDearborn ADSieving PA.   Cryo-EM of retinoschisin branched networks suggests an intercellular adhesive scaffold in the retina.  J Cell Biol. pii: jcb.201806148. doi: 10.1083/jcb.201806148. [Epub ahead of print] (2019 Jan 10).

Song H, Bush RA, Zeng Y, Qian H, Wu Z, Sieving PA. Trans-ocular electric current in vivo enhances AAV-mediated retinal gene transduction after intravitreal vector administration. Mol Ther Meth Clin Dev. 13:77-85 doi: 10.1016/j.omtm.2018.12.006. (2019).

2018

Cukras CA, Wiley HE, Jeffrey BG, Sen HN, Turriff A, Zeng Y, Vijayasarathy C, Marangoni D, Ziccardi L, Kjellstrom S, Park TK, Hiriyanna S, Wright JF, Colosi P, Wu X, Bush RA, Wei LL, Sieving PA. Retinal AAV8-RS1Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery. Mol Ther. 26(9):2282-2294 doi: 10.1016/j.ymthe.2018.05.025. (2018).  

2017

Marangoni D, Yong Z, Kjellström S, Vijayasarathy C, A Sieving P, Bush RA. Rearing Light Intensity Affects Inner Retinal Pathology in a Mouse Model of X-Linked Retinoschisis but Does Not Alter Gene Therapy Outcome. Invest Ophthalmol Vis Sci. 58(3):1656-1664. doi: 10.1167/iovs.16-21016 (2017)

2016

Bush RA, Zeng Y, Colosi P, Kjellstrom S, Hiriyanna S, Vijayasarathy C, Santos M, Li J, Wu Z, Sieving PA. Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function. Hum Gene Ther. 27(5):376-89 doi: 10.1089/hum.2015.142. (2016)

Song H, Vijayasarathy C, Zeng Y, Marangoni D, Bush RA, Wu Z, Sieving PA. NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice. Invest Ophthalmol Vis Sci. 57(6):2864-75. doi: 10.1167/iovs.15-18974 (2016)

Zeng Y, Petralia RS, Vijayasarathy C, Wu Z, Hiriyanna S, Song H, Wang YX, Sieving PA, Bush RA. Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography. Invest Ophthalmol Vis Sci. 57(9):OCT277-87. doi: 10.1167/iovs.15-18920. (2016)

Marangoni D, Bush RA, Zeng Y, Wei LL, Ziccardi L, Vijayasarathy C, Bartoe JT, Palyada K, Santos M, Hiriyanna S, Wu Z, Colosi P, Sieving PA. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice. Mol Ther Methods Clin Dev. 5:16011. doi: 10.1038/mtm.2016. (2016)

Tolun G, Vijayasarathy C, Huang R, Zeng Y, Li Y, Steven AC, Sieving PA, Heymann JB. Paired octamer rings of retinoschisin suggest a junctional model for cell-cell adhesion in the retina. Proc Natl Acad Sci. 113(19):5287-92. doi: 10.1073/pnas.1519048113. (2016)

More information

The Section for Translational Research on Retinal and Macular Degeneration (STRRMD), a laboratory engaged in eye and vision research at the National Institutes of Health (NIH), is administered by the Division of Intramural Research, National Institute on Deafness and Other Communication Disorders (DIR/NIDCD/NIH). For practical considerations of web browsing and research, the STRRMD appears here on the web pages of the Division of Intramural Research, National Eye Institute.

Translational Research on Retinal and Macular Degeneration key staff

Last updated: August 2019