Knut Stieger

Detection of Intact rAAV Particles up to 6 Years After Successful Gene Transfer in the Retina of Dogs and Primates

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

Immune responses to gene product of inducible promoters.

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling long-term transgene regulation in future clinical developments of gene transfer.

Adeno-Associated Virus Mediated Gene Therapy for Retinal Degenerative Diseases

Retinal gene therapy holds great promise for the treatment of inherited and noninherited blinding diseases such as retinitis pigmentosa and age-related macular degeneration. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because it mediates long-term transgene expression in a variety of retinal cell types and elicits minimal immune responses. Inherited retinal diseases are nonlethal and have a wide level of genetic heterogeneity. Many of the genes have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial.In this chapter, we focus on AAV-mediated specific gene therapy for inherited retinal degenerative diseases, describing the disease targets, the preclinical studies in animal models and the recent success of the LCA-RPE65 clinical trials.

Automated segmentation of retinal blood vessels in spectral domain optical coherence tomography scans.

The correct segmentation of blood vessels in optical coherence tomography (OCT) images may be an important requirement for the analysis of intra-retinal layer thickness in human retinal diseases. We developed a shape model based procedure for the automatic segmentation of retinal blood vessels in spectral domain (SD)-OCT scans acquired with the Spectralis OCT system. The segmentation procedure is based on a statistical shape model that has been created through manual segmentation of vessels in a training phase. The actual segmentation procedure is performed after the approximate vessel position has been defined by a shadowgraph that assigns the lateral vessel positions. The active shape model method is subsequently used to segment blood vessel contours in axial direction. The automated segmentation results were validated against the manual segmentation of the same vessels by three expert readers. Manual and automated segmentations of 168 blood vessels from 34 B-scans were analyzed with respect to the deviations in the mean Euclidean distance and surface area. The mean Euclidean distance between the automatically and manually segmented contours (on average 4.0 pixels respectively 20 µm against all three experts) was within the range of the manually marked contours among the three readers (approximately 3.8 pixels respectively 18 µm for all experts). The area deviations between the automated and manual segmentation also lie within the range of the area deviations among the 3 clinical experts. Intra reader variability for the experts was between 0.9 and 0.94. We conclude that the automated segmentation approach is able to segment blood vessels with comparable accuracy as expert readers and will provide a useful tool in vessel analysis of whole C-scans, and in particular in multicenter trials.

Chromatic Pupillometry Dissects Function Of The Three Different Light Sensitive Retinal Cell Populations In RPE65 Deficiency

PURPOSE The aim of the study was to objectively characterize the function of rods, cones, and intrinsic photosensitive retinal ganglion cells (ipRGCs) in patients with RPE65 mutations by using two published protocols for chromatic pupillometry, and to correlate the data with the clinical phenotype. METHODS The study group comprised 11 patients with RPE65 mutations, and for control purposes, 32 healthy probands and 2 achromats. A custom-made binocular chromatic pupillometer (Bino I) connected to a ColorDome Ganzfeld stimulator was used to assess changes in pupil diameter in response to red (640 nm) and blue (462 nm) light stimuli. Light intensities, stimulus duration, and background varied depending on the protocol used. Results were compared to the clinical phenotype, that is, visual field (Goldmann perimetry), best corrected visual acuity, and full-field stimulus testing (FST). RESULTS No significant differences in any of the pupil response parameters were observed in intraday or intervisit variability tests. Pupil responses to rod-weighted stimulation were significantly diminished in all RPE65 patients. Pupil responses to cone-weighted stimuli differed among RPE65 patients and did not always correlate with residual visual field and cone sensitivity loss in FST. Pupil responses to ipRGC-weighted answers were slightly but significantly diminished, and the postillumination pupil response was significantly increased. CONCLUSIONS Chromatic pupillometry represents a highly sensitive and objective test to quantify the function of rods, cones, and ipRGCs in patients with RPE65 mutations.

RPGR: Role in the photoreceptor cilium, human retinal disease, and gene therapy

Cilia are specialized dynamic organelles extending from the surface of almost all mammalian cells. Since proteins and protein precursors are transported across the ciliary compartments via intraflagellar transport (IFT), mutations in genes encoding proteins that participate in IFT can cause a spectrum of different ciliopathies. Photoreceptors of the mammalian retina contain ciliary structures that connect the inner (IS) with the outer segments (OS). This structure, the connecting cilium (CC), serves as the only junction between OS and IS, the correct passage of proteins through the CC is crucial for the functioning and maintenance of the cells. Therefore, any impairment of the IFT leads to severe malfunction of photoreceptors, and may induce apoptosis ultimately leading to the degeneration of the retina. The Retinitis Pigmentosa GTPase Regulator (RPGR), which is located in the CC, participates in the IFT and interacts with a variety of proteins, including RPGRIP-1, CEP290, NPM, SMC1 and 3 and IFT88. However, the function of RPGR through its interaction with these proteins is not yet entirely understood. Mutations in the RPGR gene lead to X-linked Retinitis pigmentosa (XLRP), one of the most severe and early onset forms of RP. Gene therapy is considered a potential therapeutic option and is currently under investigation in several animal models of XLRP. However, some of the currently available mouse models are only partially suitable for the development of therapeutic strategies and the quest for more appropriate small animal models is still an issue.

The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies

Over the last decade, huge progress has been made in the understanding of the molecular mechanisms underlying inherited retinal dystrophy (IRD), as well as in the development and implementation of novel therapies, especially in the field of gene therapy. The use of mutant animal models, either naturally occurring or generated by genetic modification, have contributed greatly to our knowledge on IRD. Yet, these mutant animal models do not always mimic the retinal phenotype that is observed in humans with mutations in the orthologous gene, often due to species-specific characteristics of the retina, and/or diverse functions of the gene products in different species. In this manuscript, we compare general and ocular characteristics of a series of widely used vertebrate animal models, i.e. zebrafish, chicken, rodents, cats, dogs, sheep, pigs and monkeys, in terms of genetic architecture and sequence homology, methods to modify genomes, anatomy of the eye, and structural details of the retina. Furthermore, we present an overview of mutant vertebrate animal models that have been used to study or develop treatments for the various genetic subtypes of IRD, and correlate the suitability of these models to the specific characteristics of each animal. Herewith, we provide tools that will help to select the most suitable animal model for specific research questions on IRDs in the future, and thereby assist in an optimal use of animals and resources to further increase our understanding of inherited retinal dystrophies, and develop novel treatments for these disorders.

Automated Segmentation of Pathological Cavities in Optical Coherence Tomography Scans

PURPOSE To develop and evaluate a method for automated segmentation and quantitative analysis of pathological cavities in the retina visualized by spectral-domain optical coherence tomography (SD-OCT) scans. METHODS The algorithm is based on the segmentation of the gray-level intensities within a B-scan by a k-means cluster analysis and subsequent classification by a k-nearest neighbor algorithm. Accuracy was evaluated against three clinical experts using 130 bullous cavities identified on eight SD-OCT B-scans of three patients with wet age-related macular degeneration (AMD) and five patients with X-linked retinoschisis, as well as on one volume scan of a patient with X-linked retinoschisis. The algorithm calculated the surface area of the cavities for the B-scans and the volume of all cavities for the volume scan. In order to validate the applicability of the algorithm in clinical use, we analyzed 31 volume scans taken over the course of 4 years for one AMD patient with a serous retinal detachment. RESULTS Discrepancies in area measurements between the segmentation results of the algorithm and the experts were within the range of the area deviations among the experts. Volumes interpolated from the B-scan series of the volume scan were comparable among experts and algorithm (0.249 mm³ for the algorithm, 0.271 mm³ for expert 1, 0.239 mm³ for expert 2, and 0.262 mm³ for expert 3). Volume changes of the serous retinal detachment were quantifiable. CONCLUSIONS The segmentation algorithm represents a method for the automated analysis of large numbers of volume scans during routine diagnostics and in clinical trials.

Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.

RETINAL VASCULAR DEVELOPMENT WITH 0.312 MG INTRAVITREAL BEVACIZUMAB TO TREAT SEVERE POSTERIOR RETINOPATHY OF PREMATURITY: A Longitudinal Fluorescein Angiographic Study.

Purpose: To report the outcome of intravitreal 0.312 mg bevacizumab (IVB) monotherapy in acute retinopathy of prematurity (ROP) and to describe the vascular development over time. Methods: Seventeen prematurely born infants were treated with IVB (0.312 mg in 0.025 mL per eye) because of acute ROP in posterior Zone II or Zone I, including aggressive posterior ROP. Infants were examined by fluorescein angiography (FA) using RetCam II or III (Clarity Medical Systems Inc) before IVB (n = 21 eyes), within 6 weeks (n = 23 eyes), 8 to 13 weeks (n = 22 eyes), and up to 45 months (n = 10 eyes). Results: Acute ROP regressed in 19 out of 27 analyzed eyes (70%), including 100% and 80% of posterior Zone II and Zone I eyes, respectively, but only 25% of aggressive posterior ROP eyes. Early recurrences (11%, all aggressive posterior ROP) and late reactivations (18%) were observed within 1 week and at 9 to 12 weeks, respectively. All eyes showed leakage at the junction of the vascularized zone and capillary malformation on FA before treatment. Vessel branching abnormalities and circumferential vessel formation were typical FA features after treatment. Vascular outgrowth after one IVB became complete in 87.5% of eyes for which FA was available up to at least 9 weeks after IVB. Conclusion: A single dose of 0.312 mg bevacizumab was efficient to induce regression of ROP in posterior Zone II and most of Zone I cases, but not in aggressive posterior ROP. FA describes vascular abnormalities, the importance of which warrants further investigation.