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The inherited childhood blindness caused by mutations in NPHP5, a form of Leber congenital amaurosis, results in abnormal development, dysfunction and degeneration of photoreceptors. A naturally occurring NPHP5 mutation in dogs results in a phenotype that very nearly duplicates the human retinopathy in terms of the photoreceptors involved, spatial distribution of degeneration and the natural history of vision loss. We show that AAV-mediated NPHP5 gene augmentation of mutant canine retinas at the time of active degeneration and peak cell death stably restores photoreceptor structure, function, and vision with either the canine or human NPHP5 transgenes. Mutant cone photoreceptors, which failed to form outer segments during development, reform this structure after treatment. Degenerating rod photoreceptor outer segments are stabilized and develop normal structure. This process begins within 8 weeks following treatment, and remains stable throughout the 6 month post treatment period. In both photoreceptor cell classes, mislocalization of rod and cone opsins is minimized or reversed. Retinal function and functional vision are restored. Efficacy of gene therapy in this large animal ciliopathy model of Leber congenital amaurosis provides a path for translation to human treatment.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood–brain barrier. Modifications of adeno-associated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y444 + 733F) and a triple-mutant (Y444 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y–F + T–V) compared to AAV8 (double Y–F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y–F + T–V) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Purpose/Objective(s) Single-fraction palliative spine radiation is an attractive option for patients with poor prognosis because of its convenience and effectiveness in terms of pain relief. However, the associated large doses to normal tissues can lead to toxicities that negatively impact quality of life. Proton therapy offers a dosimetric solution to this problem but has not historically been used for palliative treatment because of inherent cost and time needed for planning. The purpose of this study was to assess a process for delivering cost-effective and efficient palliative proton therapy by using diagnostic scan-based planning (DSBP) and prefabricated treatment delivery devices. Materials/Methods We designed and characterized a reusable proton aperture system capable of adjusting to multiple field sizes for spine treatment. Next, we retrospectively identified 10 patients treated with proton therapy for thoracic malignancies in 2019 who also had a diagnostic CT of the thorax within 4 months of simulation. For each case, one physician contoured T6-T9 as the target volume on both the diagnostic and simulation scans. Using the reusable proton aperture system, we generated passive-scatter proton plans on the diagnostic scans using a single PA beam with no custom range compensator to treat T6-T9 to 8 Gy x 1. The plans were then transferred to the simulation scans to compare target coverage and doses to normal tissues, followed by robustness analysis with 5mm X, Y, and Z shifts. Finally, we generated AP/PA photon plans on the simulation scans to compare doses to normal tissues between proton and photon plans. Results were compared using the Wilcoxon signed-rank test. Results Median D95% on the diagnostic scan plans was 101% (range, 100%-102%) of the prescription dose using the same 12L x 7W aperture for each case. Median Dmax was 107% (range, 105-108%). When transferred to simulation scans, coverage remained excellent and hot spots acceptable for all cases with a median D95% of 101% (range, 100%-102%) and a median Dmax of 106% (range, 105%-106%). Heart and esophagus mean and max doses did not vary significantly between the diagnostic scan and the simulation scan proton plans (P > 0.2 for all). Robustness analysis with 5mm X, Y, and Z shifts showed that coverage remained acceptable with D95% > 98% for all cases. Compared to the proton plans, mean heart dose was markedly higher for the AP/PA photon plans (median 4.7 Gy v. 0.4 Gy, P 0.05). Conclusion Palliative proton DSBP is technically feasible and robust with superior sparing of normal tissues compared to AP/PA photon plans. By eliminating the need for simulation and custom devices, the cost-benefit ratio for palliative proton therapy may become more acceptable, particularly in a bundled payment environment.

Leber congenital amaurosis type 9 (LCA9) is an autosomal recessive, early onset retinal neurodegenerative disease caused by mutations in the gene encoding the nuclear NAD + synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1 and its role in NAD + homeostasis, LCA9 patients do not manifest pathologies other than retinal degeneration. To investigate the mechanism of degeneration, we examined retinas of developing and adult mice with conditional or tissue-specific NMNAT1 loss. Widespread NMNAT1 depletion in adult mice resulted in loss of photoreceptors, indicating these cells are exquisitely vulnerable to NMNAT1 loss. NMNAT1 is required within the photoreceptor, as conditional deletion of NMNAT1 in photoreceptors but not retinal pigment epithelial cells is sufficient to cause photoreceptor neurodegeneration and vision loss. Moreover, delivery of NMNAT1 into eyes of adult mice lacking NMNAT1 using a modified AAV8 vector containing a photoreceptor-specific promoter rescued the retinal degeneration phenotype and partially restored vision. Finally, we defined the molecular mechanism driving photoreceptor cell death. Loss of NMNAT1 activates SARM1, an inducible NADase best known as the central executioner of axon degeneration. SARM1 is required for the photoreceptor death and vision loss that occurs following NMNAT1 deletion. This surprising finding demonstrates that the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, and establishes a commonality of mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway that is active in the setting of dysregulated NAD + metabolism and identifies SARM1 as a therapeutic candidate for the treatment of retinal degeneration.

Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures &gt, 4 &deg, C as detrimental factors.

Despite efforts to use adeno-associated viral (AAV) vector–mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/−, KrasG12D+/−/Pten+/−, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45–70%) compared with WT-AAV8 (7%; p

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a - / - mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

Contains fulltext : 203169.pdf (Publisher’s version ) (Open Access) Age-related macular degeneration (AMD) is a progressive disease of the retinal pigment epithelium (RPE) and the retina leading to loss of central vision. Polymorphisms in genes involved in lipid metabolism, including the ATP-binding cassette transporter A1 (ABCA1), have been associated with AMD risk. However, the significance of retinal lipid handling for AMD pathogenesis remains elusive. Here, we study the contribution of lipid efflux in the RPE by generating a mouse model lacking ABCA1 and its partner ABCG1 specifically in this layer. Mutant mice show lipid accumulation in the RPE, reduced RPE and retinal function, retinal inflammation and RPE/photoreceptor degeneration. Data from human cell lines indicate that the ABCA1 AMD risk-conferring allele decreases ABCA1 expression, identifying the potential molecular cause that underlies the genetic risk for AMD. Our results highlight the essential homeostatic role for lipid efflux in the RPE and suggest a pathogenic contribution of reduced ABCA1 function to AMD.

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative "subILM" injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.).

Recent studies have implicated thyroid hormone (TH) signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we found that antithyroid treatment preserves cones. This work investigates the significance of targeting intracellular TH components locally in the retina. The cellular TH level is mainly regulated by deiodinase iodothyronine (DIO)-2 and -3. DIO2 converts thyroxine (T4) to triiodothyronine (T3), which binds to the TH receptor, whereas DIO3 degrades T3 and T4. We examined cone survival after overexpression of DIO3 and inhibition of DIO2 and demonstrated the benefits of these manipulations. Subretinal delivery of AAV5-IRBP/GNAT2-DIO3, which directs expression of human DIO3 specifically in cones, increased cone density by 30–40% in a Rpe65−/− mouse model of Lebers congenital amaurosis (LCA) and in a Cpfl1 mouse with Pde6c defect model of achromatopsia, compared with their respective untreated controls. Intravitreal and topical delivery of the DIO2 inhibitor iopanoic acid also significantly improved cone survival in the LCA model mice. Moreover, the expression levels of DIO2 and Slc16a2 were significantly higher in the diseased retinas, suggesting locally elevated TH signaling. We show that targeting DIOs protects cones, and intracellular inhibition of TH components locally in the retina may represent a novel strategy for retinal degeneration management.—Yang, F., Ma, H., Belcher, J., Butler, M. R., Redmond, T. M., Boye, S. L., Hauswirth, W. W., Ding, X.-Q. Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration.

Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to

Dysregulation of the complement system is implicated in neurodegeneration, including human and animal glaucoma. Optic nerve and retinal damage in glaucoma is preceded by local complement upregulation and activation, but whether targeting this early innate immune response could have therapeutic benefit remains undefined. Because complement signals through three pathways that intersect at complement C3 activation, here we targeted this step to restore complement balance in the glaucomatous retina, and to determine its contribution to degeneration onset and/or progression. To achieve this, we combined adeno-associated viral retinal gene therapy with the targeted C3 inhibitor CR2-Crry. We show that intravitreal injection of AAV2.CR2-Crry produced sustained Crry overexpression in the retina, and reduced deposition of the activation product complement C3d on retinal ganglion cells and the inner retina of DBA/2J mice. This resulted in neuroprotection of retinal ganglion cell axons and somata despite continued intraocular pressure elevation, suggesting a direct restriction of neurodegeneration onset and progression, and significant delay to terminal disease stages. Our study uncovers a damaging effect of complement C3 or downstream complement activation in glaucoma and establishes AAV2.CR2-Crry as a viable therapeutic strategy to target pathogenic C3-mediated complement activation in the glaucomatous retina.

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. Gene replacement and gene editing therapies have shown great promise in treating several of these diseases, however, most degenerations cannot yet be approached by these therapeutic options. In addition, age-related macular degeneration, the most common retinal degenerative disease, is a multifactorial disorder, suggesting multiple approaches are needed. An attractive option is to use gene-independent neuroprotective therapies to treat a broad-spectrum of diseases either as a primary or adjunct therapy. While this strategy has had success, the inability to control transgene expression has been a major limitation. To address this problem, we developed a drug-tunable neuroprotective factor named Retinal Protective Factor 2 (RPF2). We show that RPF2 is a potent neuroprotective factor for photoreceptors against multiple causes of degeneration. We further show that RPF2 expression is tunable by drug concentration and is reversible with drug withdrawal. In addition, our data suggest that the modular design can be used to regulate the expression of other AAV-based transgenes, including transcription factors, anti-inflammatory factors, and anti-angiogenic factors, which would facilitate the development of many new therapies.

Leber congenital amaurosis (LCA) is a devastating pediatric retinal degenerative disease, accounting for 20% of blindness in children attending schools for the blind. Mutations in the RPE65 gene, which encodes the retinal pigment epithelium-specific isomerohydrolase RPE65, account for 16% of all LCA cases. Recent findings have linked cone photoreceptor viability to thyroid hormone (TH) signaling. TH signaling regulates cell proliferation, differentiation, and metabolism. At the cellular level, TH action is regulated by the two iodothyronine deiodinases, DIO2 and DIO3. DIO2 converts the prohormone thyroxine (T4) to the bioactive hormone triiodothyronine (T3), and DIO3 inactivates T3 and T4. The present work investigates the effects of overexpression of DIO3 to suppress TH signaling and thereby modulate cone death/survival. Subretinal delivery of AAV5-IRBP/GNAT2-hDIO3 induced robust expression of DIO3 in the mouse retina and significantly reduced the number of TUNEL-positive cells in the cone-dominant LCA model Rpe65 −/− /Nrl −/− mice. Our work shows that suppressing TH signaling by overexpression of DIO3 preserves cones, supporting that suppressing TH signaling locally in the retina may represent a treatment strategy for LCA management.

Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P&nbsp

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~ 4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.

Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.

Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS.

Purpose To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). Methods Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. Results Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. Conclusions NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw−/−) expressing only S-opsin as a model for human BCM. We show that recombinant M-opsin delivered by AAV5 vectors rescues M-cone function in Opn1mw−/− mice. We also show that AAV delivered M-opsin localizes in the dorsal cone outer segments, and co-localizes with S-opsin in the ventral retina. Our study demonstrates that cones without M-opsin remain viable and respond to gene augmentation therapy, thereby providing proof-of-concept for cone function restoration in BCM patients.

Despite mutations in the rod phosphodiesterase 6-alpha (PDE6A) gene being well-recognized as a cause of human retinitis pigmentosa, no definitive treatments have been developed to treat this blinding disease. We performed a trial of retinal gene augmentation in the Pde6a mutant dog using Pde6a delivery by capsid-mutant adeno-associated virus serotype 8, previously shown to have a rapid onset of transgene expression in the canine retina. Subretinal injections were performed in 10 dogs at 29–44 days of age, and electroretinography and vision testing were performed to assess functional outcome. Retinal structure was assessed using color fundus photography, spectral domain optical coherence tomography, and histology. Immunohistochemistry was performed to examine transgene expression and expression of other retinal genes. Treatment resulted in improvement in dim light vision and evidence of rod function on electroretinographic examination. Photoreceptor layer thickness in the treated area was preserved compared with the contralateral control vector treated or uninjected eye. Improved rod and cone photoreceptor survival, rhodopsin localization, cyclic GMP levels and bipolar cell dendrite distribution was observed in treated areas. Some adverse effects including foci of retinal separation, foci of retinal degeneration and rosette formation were identified in both AAV-Pde6a and control vector injected regions. This is the first description of successful gene augmentation for Pde6a retinitis pigmentosa in a large animal model. Further studies will be necessary to optimize visual outcomes and minimize complications before translation to human studies.

Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers. Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations. Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique. Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target retinal ganglion cells and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally- delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species.

RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a ‘proof of concept’ for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Achromatopsia is a genetic disorder of cones, and one of the most common forms is a channelopathy caused by mutations in the β-subunit, CNGB3, of the cone cyclic nucleotide-gated (CNG) channel. Recombinant adeno-associated virus of serotype 5 (rAAV5)-mediated gene transfer of human CNGB3 cDNA to mutant dog cones results in functional and structural rescue in dogs 1 year. We now test a new therapeutic concept by combining gene therapy with the administration of ciliary neurotrophic factor (CNTF). Intravitreal CNTF causes transient dedifferentiation of photoreceptors, a process called deconstruction, whereby visual cells become immature with short outer segments, and decreased retinal function and gene expression that subsequently return to normal. Cone function was successfully rescued in all mutant dogs treated between 14 and 42 months of age with this strategy. CNTF-mediated deconstruction and regeneration of the photoreceptor outer segments prepares the mutant cones optimally for gene augmentation therapy.

Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator ( RPGR ) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5–vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

Purpose To evaluate differences in target motion during prostate irradiation in the prone versus supine position using electromagnetic tracking to measure prostate mobility. Materials/methods Twenty patients received prostate radiotherapy in the supine position utilizing the Calypso Localization System® for prostate positioning and monitoring. For each patient, 10 treatment fractions were followed by a session in which the patient was repositioned prone, and prostate mobility was tracked. The fraction of time that the prostate was displaced by >3, 5, 7, and 10mm was calculated for each patient, for both positions (400 tracking sessions). Results Clear patterns of respiratory motion were seen in the prone tracks due to the influence of increased abdominal motion. Averaged over all patients, the prostate was displaced >3 and 5mm for 37.8% and 10.1% of the total tracking time in the prone position, respectively. In the supine position, the prostate was displaced >3 and 5mm for 12.6% and 2.9%, respectively. With both patient setups, inferior and posterior drifts of the prostate position were observed. Averaged over all prone tracking sessions, the prostate was displaced >3mm in the posterior and inferior directions for 11.7% and 9.5% of the total time, respectively. Conclusions With real-time tracking of the prostate, it is possible to study the effects of different setup positions on the prostate mobility. The percentage of time the prostate moved >3 and 5mm was increased by a factor of three in the prone versus supine position. For larger displacements (>7mm) no difference in prostate mobility was observed between prone and supine positions. To reduce rectal toxicity, radiotherapy in the prone position may be a suitable alternative provided respiratory motion is accounted for during treatment. Acute and late toxicity results remain to be evaluated for both patient positions.

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single-stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) and AAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10-14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1-2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5-7 and in neuroretinal wholemounts around PID 7-10; ssAAV5-mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors (Natkunarajah et al., 2008; Yokoi et al., 2007). scAAV5 vectors may be more useful than standard, single-stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells.

Purpose To analyze changes in parotid gland dose resulting from anatomic changes throughout a course of radiotherapy in a cohort of head-and-neck cancer patients. Methods and Materials The study population consisted of 10 head-and-neck cancer patients treated definitively with intensity-modulated radiotherapy on a helical tomotherapy unit. A total of 330 daily megavoltage computed tomography images were retrospectively processed through a deformable image registration algorithm to be registered to the planning kilovoltage computed tomography images. The process resulted in deformed parotid contours and voxel mappings for both daily and accumulated dose–volume histogram calculations. The daily and cumulative dose deviations from the original treatment plan were analyzed. Correlations between dosimetric variations and anatomic changes were investigated. Results The daily parotid mean dose of the 10 patients differed from the plan dose by an average of 15%. At the end of the treatment, 3 of the 10 patients were estimated to have received a greater than 10% higher mean parotid dose than in the original plan (range, 13–42%), whereas the remaining 7 patients received doses that differed by less than 10% (range, −6–8%). The dose difference was correlated with a migration of the parotids toward the high-dose region. Conclusions The use of deformable image registration techniques and daily megavoltage computed tomography imaging makes it possible to calculate daily and accumulated dose–volume histograms. Significant dose variations were observed as result of interfractional anatomic changes. These techniques enable the implementation of dose-adaptive radiotherapy.

Purpose To compare different image-guidance strategies in the alignment of prostate cancer patients. Using data from patients treated using daily image guidance, the remaining setup errors for several different strategies were retrospectively calculated. Methods and Materials The alignment data from 74 patients treated with helical tomotherapy were analyzed, resulting in a data set of 2,252 fractions during which a megavoltage computed tomography image was used for image guidance with intraprostatic metallic fiducials. Given the daily positional adjustments, a variety of protocols, differing in imaging frequency and method, were retrospectively studied. The residual setup errors were determined for each protocol. Results As expected, the systematic errors were effectively reduced with imaging. However, the random errors were unaffected. Even when image guidance was performed every other day with a running mean of the previous displacements, residual setup errors >5 mm occurred in 24% of all fractions. This frequency increased to about 40% if setup errors >3 mm were scored. Conclusion Setup errors increased with decreasing frequency of image guidance. However, residual errors were still significant at the 5-mm level, even with imaging was performed every other day. This suggests that localizations must be performed daily in the set up of prostate cancer patients during a course of external beam radiotherapy.

Prostate motion during external-beam radiotherapy can affect outcomes in patients with localized prostate cancer. Prostate motion and deformation are currently being characterized with different techniques. There is significant individual variation among patients with respect to the observed motion and its dosimetric consequences. There is also significant difference in the accuracy of different localization methods currently used to adjust for prostate motion. The motion of the prostate gland can itself affect the accuracy of different localization methods. The dosimetric impact on target areas and organs at risk should be studied for different localization techniques, treatment plan margins, and treatment schedules. Such assessments will be increasingly important with smaller treatment margins, smaller fraction numbers, and higher radiation doses. Understanding and managing the consequences of anatomic variations within the lower pelvis should be a priority in designing and implementing future clinical trials.

To report and describe implantation techniques and stability of metallic fiducials in lung lesions to be treated with external beam radiotherapy.Patients undergoing radiation therapy for small early-stage lung cancer underwent implantation with small metallic markers. Implantation was either transcutaneous under computed tomographic (CT) or fluoroscopic guidance or transbronchial with the superDimension/Bronchus system (radiofrequency signal-based bronchoscopy guidance related to CT images).Implantation was performed transcutaneously in 15 patients and transbronchially in 8 patients. Pneumothorax occurred with eight of the 15 transcutaneous implants, six of which required chest tube placement. None of the patients who underwent transbronchial implantation developed pneumothorax. Successfully inserted markers were all usable during gated image-guided radiotherapy. Marker stability was determined by observing the variation in gross target volume (GTV) centroid relative to the marker on repeated CT scans. Average three-dimensional variation in the GTV center relative to the marker was 2.6 +/- 1.3 (SD) mm, and the largest variation along any anatomic axis for any patient was5 mm. Average GTV volume decrease during the observation period was 34% +/- 23%. Gross tumor volumes do not appear to shrink uniformly about the center of the tumor, but rather the tumor shapes deform substantially throughout treatment.Transbronchial marker placement is less invasive than transcutaneous placement, which is associated with high pneumothorax rates. Although marker geometry can be affected by tumor shrinkage, implanted markers are stable within tumors throughout the treatment duration regardless of implantation method.

Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.

The enteric nervous system (ENS) is a division of the autonomic nervous system that coordinates the control of gastrointestinal (GI) function. Several diseases are associated with abnormalities of the ENS, for example, GI dysfunction is one of the most common non-motor complaints associated with Parkinson's disease (PD). GI pathology affects virtually every level of the GI tract, primarily presenting as decreased colonic motility. Recent studies have demonstrated that the protein, α-synuclein (α-syn), forms large inclusion within neurons of the ENS, suggesting that α-syn pathology within the ENS may play a role in the etiology of GI dysfunction in PD. To determine if α-syn expression within neurons of the ENS is sufficient to produce GI dysfunction, we aimed to overexpress human wildtype α-syn within the ENS using direct injections of adeno associated virus (AAV) to the descending colon. However, as there have been no focused investigations into the targeted transduction of the ENS using AAV, we first examined the efficiency and tropism of transduction using several AAV serotypes. Rats received direct injections (6 × 5ml) of either AAV 1, 2, 5, 6, 8 or 9 expressing green fluorescent protein (GFP) into the descending colon. Transduction occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction, with AAV9 primarily transducing neurons, and AAV6 transducing neurons as well as enteric glia. Transduction efficiency scaled with titer and time, to plateau at 4-weeks post injection at a titer of 6.25×1012 viral genomes (vg)/ml. Following characterization of AAV transduction in the ENS, it was determined that AAV9 demonstrated the highest levels of neuronal transduction, and was selected to deliver the α-syn transgene to the ENS. Adult male rats received direct injections (6 × 5ml at 1×1013vg/ml) of AAV9 expressing either human wildtype α-syn or a GFP control transgene into the descending colon. Four weeks post-surgery, colonic motility was assayed by quantifying the time necessary to transit and excrete a bead through 5cm of the descending colon. Animals that received AAV-α-syn had significantly impaired colonic motility, with a mean colonic transit time of 171.5 ± 43.4 minutes compared to animals that received AAV-GFP with a mean colonic transit time of 15 ± 6.8 minutes. Here we present a thorough characterization of the transduction profile of AAV in the ENS following direct injection to the descending colon. Further, using AAV to deliver human α-syn to neurons of the ENS, colonic motility was significantly decreased.

Background Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye. Methodology/Principle Findings AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient. Conclusions/Significance This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.

Purpose: To report on the initial clinical use of a commercially available system to deliver gated treatment using implanted fiducials, in-room kV X-rays, and an infrared camera tracking system. Methods and Materials: ExacTrac Adaptive Gating from BrainLab is a localization system using infrared cameras and X-rays. Gating signals are the patient’s breathing pattern obtained from infrared reflectors on the patient. kV X-rays of an implanted fiducial are synchronized to the breathing pattern. After localization and shift of the patient to isocenter, the breathing pattern is used to gate the radiation. Feasibility tests included localization accuracy, radiation output constancy, and dose distributions with gating. Clinical experience is reported on treatment of patients with small lung lesions. Results: Localization accuracy of a moving target with gating was 1.7 mm. Dose constancy measurements showed insignificant change in output with gating. Improvements of dose distributions on moving targets improved with gating. Eleven patients with lung lesions were implanted with 20 mm × 0.7 mm gold coil (Visicoil). The implanted fiducial was used to localize and treat the patients with gating. Treatment planning and repeat computed tomographic scans showed that the change in center of gross target volume (GTV) to implanted marker averaged 2.47 mm due in part to asymmetric tumor shrinkage. Conclusion: ExacTrac Adaptive Gating has been used to treat lung lesions. Initial system evaluation verified its accuracy and usability. Implanted fiducials are visible in X-rays and did not migrate.

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose-response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65-associated LCA.

Purpose: The gross tumor volume (GTV) for many lung cancer patients can decrease during the course of radiation therapy. As the tumor reduces in size during treatment, the margin added around the GTV effectively becomes larger, which can result in the excessive irradiation of normal lung tissue. The specific goal of this study is to evaluate the feasibility of using image-guided adaptive radiation therapy to adjust the planning target volume weekly based on the previous week's CT image sets that were used for image-guided patient setup. Methods and Materials: Megavoltage computed tomography (MVCT) images of the GTV were acquired daily on a helical tomotherapy system. These images were used to position the patient and to measure reduction in GTV volume. A planning study was conducted to determine the amount of lung-sparing that could have been achieved if adaptive therapy had been used. Treatment plans were created in which the target volumes were reduced after tumor reduction was measured. Results: A total of 158 MVCT imaging sessions were performed on 7 lung patients. The GTV was reduced by 60–80% during the course of treatment. The tumor reduction in the first 60 days of treatment can be modeled using the second-order polynomial R = 0.0002 t 2 − 0.0219 t + 1.0, where R is the percent reduction in GTV, and t is the number of elapsed days. Based on these treatment planning studies, the absolute volume of ipsilateral lung receiving 20 Gy can be reduced between 17% and 23% (21% mean) by adapting the treatment delivery. The benefits of adaptive therapy are the greatest for tumor volumes ≥25 cm 3 and are directly dependent on GTV reduction during treatment. Conclusions: Megavoltage CT-based image guidance can be used to position lung cancer patients daily. This has the potential to decrease margins associated with daily setup error. Furthermore, the adaptive therapy technique described in this article can decrease the volume of healthy lung tissue receiving above 20 Gy. However, further study is needed to determine whether adaptive therapy could result in the underdosing of microscopic extension.

In-stent restenosis is commonly observed in coronary arteries after intervention. Intravascular brachytherapy has been found effective in reducing the recurrence of restenosis after stent placement. Conventional dosing models for brachytherapy with beta (/spl beta/) radiation neglect vessel geometry as well as the position of the delivery catheter. This paper demonstrates in computer simulations on phantoms and on in vivo patient data that the estimated dose distribution varies substantially in curved vessels. In simulated phantoms of 50-mm length with a shape corresponding to a 60/spl deg/-180/spl deg/ segment of a respectively sized torus, the average dose in 2-mm depth was decreased by 2.70%-7.48% at the outer curvature and increased by 2.95%-9.70% at the inner curvature as compared with a straight phantom. In vivo data were represented in a geometrically correct three-dimensional model that was derived by fusion of intravascular ultrasound (IVUS) and biplane angiography. These data were compared with a simplified tubular model reflecting common assumptions of conventional dosing schemes. The simplified model yielded significantly lower estimates of the delivered radiation and the dose variability as compared with a geometrically correct model (p < 0.001). The estimated dose in ten vessel segments of eight patients was on average 8.76% lower at the lumen/plaque and 6.52% lower at the media/adventitia interfaces (simplified tubular model relative to geometrically correct model). The differences in dose estimates between the two models were significantly higher in the right coronary artery as compared with the left coronary artery (p < 0.001).

Purpose Intensity-modulated radiotherapy (IMRT) has been shown to reduce the radiation dose to small bowel in pelvic RT in gynecology patients. Prone positioning has also been used to decrease small bowel dose by displacement of small bowel from the RT field in these patients. The purpose of this study was to determine whether the combination of both IMRT and prone positioning on a belly board can reduce small bowel dose further in gynecologic cancer patients undergoing pelvic RT. Methods and materials IMRT plans for pelvic RT were computed in 16 patients with gynecologic cancer who had undergone planning CT scans in both the supine and the prone positions on a belly board. For the gross tumor volume, the uterus, cervix, and tumor (or postoperative region) were traced. The clinical target volume was defined as the vessels and lymph nodes from the obturator level to the aortic bifurcation, presacral region, and upper 4 cm of the vagina, in addition to gross tumor volume. The planning target volume was defined as a 2-cm margin in addition to the gross tumor volume and upper 4 cm of the vagina, and 1.5 cm for lymph nodes and vessels. Normal tissue regions of interest included small bowel, large bowel, and bladder. IMRT plans using ( 1 ) the limited arc technique (180° arc length) and ( 2 ) the extended arc technique (340° arc length) were computed. Dose–volume histograms for normal tissue structures and target were compared between the supine and prone IMRT plans using the paired t test. Results Prone positioning on a belly board decreased the small bowel dose in gynecologic pelvic IMRT, and the magnitude of improvement depended on the specific IMRT technique used. With the limited arc technique, prone positioning significantly decreased the irradiated small bowel volume at the 25–50-Gy dose levels compared with supine positioning. Small bowel volumes receiving ≥45 Gy decreased from 19% to 12.5% ( p = 0.005) with prone positioning. With the extended arc technique, the decrease in irradiated small bowel volume was less marked, but remained detectable in the 35–45-Gy dose levels. Small bowel volumes receiving ≥45 Gy decreased from 13.6% to 10.1% ( p = 0.03) with prone positioning. The effect of prone positioning on large bowel and bladder was variable. Large bowel volumes receiving ≥45 Gy increased with prone positioning from 16.5% to 20.6% ( p = 0.02) in the limited arc technique and was unaffected in the extended arc technique. Conclusion These preliminary data suggest that prone positioning on a belly board can reduce the small bowel dose further in gynecology patients treated with pelvic RT, and that the dose reduction depends on the IMRT technique used.

Purpose To rescue visual loss and optic neuropathy in experimental autoimmune encephalomyelitis (EAE). Methods Encephalomyelitis was induced in mice that received intravitreal injections of AAV2-mtHSP70Flag or AAV2-Cox8-mCherry. Additional mice were injected with AAV2-Cox8-mCherry, but not sensitized for EAE. Visual function was assessed by pattern electroretinograms (PERG) at 1, 3, and 6 months post injection (MPI). Optical coherence tomography (OCT) evaluated the thickness of the inner plexiform layer + nerve fiber layers at 1, 3, and 6 MPI. Retinas and optic nerves (ONs) of mice euthanized 6 MPI were processed for light and electron microscopy. Expression of mtHSP70Flag in the retina and ONs was evaluated by RT-PCR, immunofluorescence, and Western blotting. The activities of respiratory complexes I and III, as well as mitochondrial protein import were quantitated. Results Expression: immunofluorescence revealed punctate and perinuclear expression of mtHSP70Flag that colocalized with mitochondrial porin in thy1.2 labeled retinal ganglion cells (RGCs). Immunoblotting and RT-PCR confirmed mtHSP70Flag expression in the retina and ON. Rescue: treatment with mtHSP70Flag resulted in a 44% increase in PERG amplitude and less delays in latency relative to the EAE-mCherry group that also showed progressive inner retinal thinning. At 6 MPI, the almost 50% loss of RGCs and optic nerve axons in EAE mice was suppressed by mtHSP70Flag. In addition, retinas of EAE-mtHSP70Flag mice showed nearly complete rescue of complex I and III activities that was reduced by one-third in the EAE-mCherry retinas. Lastly, reductions in import of COX8-mCherry into mitochondria of mice sensitized for EAE improved by 30% with mtHSP70Flag gene therapy. Conclusions Mitochondrial HSP70 ameliorates mitochondrial dysfunction that culminates in irreversible visual loss and atrophy of the optic nerve in EAE suggesting that it may be useful to prevent irreversible disability in patients with optic neuritis and multiple sclerosis (MS).

At the University of Florida, head and neck cancer often is irradiated using parallel opposed lateral fields (with inferior borders slanted superiorly) and an anterior low neck field. A common criticism is that overlap may occur at the match-line junction of the three fields, resulting in an increased risk of radiation myelitis. One setup for treatment of the oropharynx and two for the larynx were irradiated in an anthropomorphic head and neck phantom made of tissue-equivalent polyacrylamide gel with a two-dimensional thermoluminescent dosimeter array in its sagittal midplane. The results showed that no excess radiation dose was measured at the junction of the three fields. The “spinal cord dose,” as percentage of dose to the central axis of the primary field, was as follows: oropharynx setup, 15% to 100%; larynx setup with midline tracheal block, 10% to 90%; larynx setup without tracheal block, 10% to 90%. In conclusion, the University of Florida three-field technique for head and neck cancer produces no measured increase in dose at field junctions.