A number of recent polls in the U.S claim that the fear of going blind is ranked higher than that of any other health issue, including cancer. And yet, over 20 million Americans are living with retinal diseases for which the only prognosis is severe or complete vision loss. Age-related Macular Degeneration (both “wet” and “dry” forms) and Diabetic Retinopathy are the most common causes of vision loss in the U.S., accounting for approximately 18 million sufferers. Another approximately 200,000 individuals are born with genetic predispositions to degeneration of the retina and progressive loss of vision throughout life. For 90% of these individuals, little or no treatment exists to slow the progression of blindness.
The most successful treatment for retinal disease to date has been an injection of a therapeutic protein into the vitreous of patients. Specifically, intravitreal injection of an antibody against Vascular Endothelial Growth Factor (VEGF) has revolutionized the treatment of one of the most common causes of blindness in the elderly, namely, “wet” Age-related Macular Degeneration (AMD). The unprecedented clinical success of VEGF antibody to inhibit the growth of “leaky” blood vessels (a process known as neovascularization) into the retina and their concomitant destruction of retinal tissue has been achieved despite a lack of knowledge regarding the primary cause of this devastating disease. Targeting the neovascularization pathway by intravitreal injection of VEGF antibody has resulted in a treatment impacting approximately 2 million patients facing vision loss, including individuals with “wet” AMD, Diabetic Macular Edema and Diabetic Retinopathy. Prior to injection of VEGF antibody, these individuals underwent painful laser coagulation of blood vessels to prevent leakage, a procedure that caused irreparable damage to surrounding healthy tissue.
Despite the success of the intravitreal delivery of therapeutic protein for the treatment of neovascular and edema-related retinal disease, there has been no development of this approach for the treatment of severe loss of vision in the approximately 18 million patients inflicted with other retinal diseases. This has been the case for a number of reasons: (i) protein therapy for the retina requires frequent intravitreal injection, a technique that, although it can be performed in the ophthalmologist’s office, can be uncomfortable and distressing; (ii) significant advances in determining the molecular basis of retinal disease has led to the investment of resources in the development of gene-specific therapies that can be delivered using gene transfer technologies; and (iii) the structure of the retina, in particular, the location of the light-sensing neurons (photoreceptors), poses a significant challenge for the delivery of proteins that require entry into photoreceptor cells to elicit a therapeutic effect.
In our study published in Experimental Eye Research in 2018, we have made significant progress in addressing the latter two obstacles preventing the development of intravitreal protein therapy for retinal disease. The first obstacle is being addressed by significant improvements in the safety and efficacy of intravitreal injection, likely due to an increase in the number of conditions for which this technique is relevant. Between 2001 and 2012, there has been an approximately 600-fold increase in the number of intravitreal injections being administered in the United States.
Retinal diseases that result in damage to photoreceptors and subsequent vision loss are some of the most heterogeneous genetic disorders known, involving over 60 different genes and more than 200 different mutations. Even with the current advances in molecular tools, the development and testing of specific treatments for each of these diseases are well beyond the scope of current resources. Despite the considerable heterogeneity observed in the spectrum of retinal disease, it is known that loss of photoreceptors due to cell death is the ultimate cause of vision loss. Progressive loss of photoreceptors results in an irreversible reorganization of the structure of the retina, rendering the tissue refractory to future treatment.
From a variety of animal models of environmentally- and genetically-induced retinal degeneration, we know that loss of photoreceptors occurs due to an integrated network of cell death pathways, including apoptosis, necroptosis, and autophagy. In our published study, we developed an approach for intravitreal delivery of a protein known to function as a regulator of apoptosis, necroptosis, and autophagy – an X-linked inhibitor of apoptosis protein (XIAP). XIAP is an intracellularly-acting protein that requires entry into cells to inhibit cell death.
Significant limitations to protein delivery to photoreceptors in retina, to date, have been either the requirement for injection of the protein into the subretinal space by inducing retinal detachment, a surgical procedure fraught with complications, or inefficient delivery to the appropriate intracellular location due to sequestering of the protein by endosomes – an integral component of the cellular uptake pathway. By exploiting the endosomal-independent intracellular trafficking pathway of the protein, nucleolin, a protein fortuitously observed by us to be abundantly present on the surface of photoreceptor cells, we have shown efficient delivery of proteins to photoreceptor cells in the mouse retina following intravitreal injection. For delivery of the cell death inhibitor protein, XIAP, into photoreceptor cells, we employed a short DNA sequence of unique structure, a G-quartet oligonucleotide or aptamer. The aptamer, called AS1411, has been shown by us and others to efficiently bind nucleolin on the surface of cells. The aptamer has also been determined to be safe following intravenous injection into humans for the treatment of cancer. Nucleolin is typically present on the surface of proliferating cells, such as those observed in tumors, as well as cells involved in the growth of new blood vessels. Photoreceptor cells, being neurons, are highly differentiated, non-mitotic cells, i.e. they do not proliferate, so it was an unusual finding to observe nucleolin on the surface of mouse photoreceptor cells. Additionally, we have confirmed the presence of nucleolin on both human and non-human primate photoreceptors, essential to the development of nucleolin-targeted delivery of protein for the clinic.
In our study entitled G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection, we linked XIAP to the aptamer, AS1411, via an intermediate protein called traptavidin to generate the therapeutic complex, AS1411-TRAP-XIAP. The use of traptavidin permits the linkage of up to three XIAP protein molecules to one molecule of AS1411, potentially increasing the potency of the therapeutic complex. In addition, employing traptavidin allows us to avail of the stable, yet versatile, biotin-streptavidin chemistry for conjugation of XIAP with AS1411. This is important as one of our goals in this study was to develop a platform technology that could be extended to the delivery of a variety of proteins. The streptavidin-biotin linkage has been shown to effectively deliver antibody-directed therapeutic radionuclides in clinical studies. In addition, biotinylation is typically not observed to adversely affect protein function.
We employed traptavidin, a slightly altered form of streptavidin, as we determined in our study that unlike the streptavidin-biotin linkage, the traptavidin-biotin linkage was more stable following intravitreal injection in the mouse retina and provided significantly improved delivery of XIAP to photoreceptor cells. In fact, intravitreal injection of an AS1411-conjugated marker protein – green fluorescent protein (GFP) – indicated efficient delivery to a variety of retinal neurons, including those of the inner retina (closest to the vitreous).
To test the therapeutic efficacy of the AS1411-TRAP-XIAP, we employed a chemically-induced mouse model of retinal degeneration. Injection of N-methyl-D-aspartate (NMDA) into the retina causes excessive activation of NMDA receptors on the surface of retinal cells, including photoreceptors, and the induction of cell death in multiple retinal cell types. When NMDA-treated mice were injected in the vitreous with AS1411-TRAP-XIAP, we observed an approximately 87% reduction in overall cell death in the retina relative to NMDA-treated mice injected with a control molecule. Following injection of AS1411-TRAP-XIAP, detection techniques indicated virtual elimination of cell death in photoreceptors (located in the outer retina) and significant inhibition of cell death in inner neurons. Quantitation of the activation of the protein, caspase-3/7 (a reliable indicator of the induction of apoptosis), showed an approximately 72% reduction of caspase 3/7 activation in the retinas of NMDA-treated mice injected with AS1411-TRAP-XIAP relative to that of NMDA-treated mice injected with the control molecule.
Our study did not indicate any signs of toxicity associated with intravitreal injection of AS1411-TRAP-XIAP or AS1411-TRAP-GFP. The study, however, was short-term aimed at providing proof of concept. Streptavidin has been shown to generate an immune response and, although we employ traptavidin, the high degree of structural similarity between streptavidin and traptavidin could be a limiting factor. However, due to the versatility and efficiency of the streptavidin-biotin linkage for delivery of biomolecules, considerable effort has been successfully invested in generating non-immunogenic forms of streptavidin. Given the similarity between traptavidin and streptavidin structure, the non-immunogenic structure of streptavidin could likely be applied to traptavidin.
Although techniques for intravitreal injection are being actively improved, patient non-compliance remains an issue. To bypass the need for frequent intravitreal injections, a variety of intraocular implants that act as a reservoir of therapeutic protein have been developed. However, intraocular implants which have yet to acquire the stamp of approval for use in the clinic, remain limited to delivering proteins that function at the extracellular level and do not require entry into the cell. Another approach to reducing the frequency of intravitreal injections is to increase the stability of the therapeutic complex in the vitreous, the cell, or both. The duration of VEGF antibody in the vitreous has been extended through addition to the antibody of a short peptide sequence that allows binding of the antibody to hyaluronic acid, an abundant constituent of the vitreous. The stability of therapeutic proteins following cell entry could reasonably be extended through manipulation of the protein structure to limit targeting to protein degradation pathways. Much is known regarding the regulation of XIAP stability in the cell and the stability of XIAP has been altered by the amino acid exchange.
Intravitreal delivery of anti-apoptotic proteins for the treatment of retinal disease won’t address the underlying genetic defect, but it could significantly add to the “seeing” years of millions of patients worldwide currently facing devastating vision loss. In addition, it could maintain the integrity of the retina of these patients for future gene-specific treatments as they become available. Blindness is next only to cancer as one of the most feared of diagnoses; the psychological impact of vision loss is as grim and harsh as the physical impairment. Given the success of intravitreal injection in the clinic, in addition to our knowledge of common pathways in retinal disease, the time is ripe for the development of protein therapy for retinal disease. Our data showing the use of AS1411-mediated intravitreal delivery of XIAP for the protection of photoreceptors and inner retinal neurons from NMDA-induced cell death in mouse retina is a significant step towards the reality of intravitreal protein therapy in the clinic.
These findings are described in the article entitled G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection, recently published in the journal Experimental Eye Research. This work was conducted by Deepa Talreja, Siobhan M. Cashman, Bhanu Dasari, Binit Kumar, and Rajendra Kumar-Singh from the Tufts University School of Medicine.