Publications by authors named "Stylianos Michalakis"

105 Publications

CNGB1-related rod-cone dystrophy: a mutation review and update.

Hum Mutat 2021 Apr 12. Epub 2021 Apr 12.

Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Institut de la Vision, Paris, France.

CNGB1 (Cyclic nucleotide-gated channel β 1) encodes the 240-kDa β subunit of the rod photoreceptor cyclic nucleotide-gated ion channel. Disease-causing sequence variants in CNGB1 lead to autosomal recessive rod-cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at non-canonical positions), 10 small deletions, 1 small insertion, 1 small insertion-deletion, 7 small duplications and 1 gross deletion. According to the ACMG classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1-related RP cases which, overall, are in line with previous findings suggesting that this form of RP has a long-term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials. This article is protected by copyright. All rights reserved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.24205DOI Listing
April 2021

Gene Therapy for Inherited Retinal Disorders: Update on Clinical Trials.

Klin Monbl Augenheilkd 2021 Mar 30;238(3):272-281. Epub 2021 Mar 30.

Department of Ophthalmology, University Hospital, LMU Munich, München, Germany.

Within the last decade, continuous advances in molecular biological techniques have made it possible to develop causative therapies for inherited retinal disorders (IRDs). Some of the most promising options are gene-specific approaches using adeno-associated virus-based vectors to express a healthy copy of the disease-causing gene in affected cells of a patient. This concept of gene supplementation therapy is already advocated for the treatment of retinal dystrophy in RPE65-linked Leber's congenital amaurosis (LCA) patients. While the concept of gene supplementation therapy can be applied to treat autosomal recessive and X-linked forms of IRD, it is not sufficient for autosomal dominant IRDs, where the pathogenic gene product needs to be removed. Therefore, for autosomal dominant IRDs, alternative approaches that utilize CRISPR/Cas9 or antisense oligonucleotides to edit or deplete the mutant allele or gene product are needed. In recent years, research retinal gene therapy has intensified and promising approaches for various forms of IRD are currently in preclinical and clinical development. This review article provides an overview of current clinical trials for the treatment of IRDs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/a-1384-0818DOI Listing
March 2021

Optimized adeno-associated virus vectors for efficient transduction of human retinal organoids.

Hum Gene Ther 2021 Mar 23. Epub 2021 Mar 23.

German Center for Neurodegenerative Diseases Dresden, Dresden, Germany.

The most widely used vectors for gene delivery in the retina are recombinant adeno-associated virus (rAAV) vectors. They have proven to be safe and effective in retinal gene therapy studies aimed to treat inherited retinal dystrophies, albeit with various limitations in transduction efficiency. Novel variants with modified capsid sequences have been engineered to improve transduction and overcome limitations of naturally occurring variants. Although preclinical evaluation of rAAV vectors based on such novel capsids is mostly done in animal models, the use of human induced pluripotent stem cell (hiPSC)-derived organoids offers an accessible, abundant, and human testing platform for rAAV evaluation. In this study, we tested the novel capsids AAV9.GL and AAV9.NN for their tropism and transduction efficiency in hiPSC-derived human retinal organoids (HRO) with all major neuronal and glial cell types in a laminated structure. These variants are based on the AAV9 capsid and were engineered to display specific surface-exposed peptide sequences, previously shown to improve the retinal transduction properties in the context of AAV2. To this end, HROs were transduced with increasing concentrations of rAAV9, rAAV9.GL or rAAV9.NN carrying a self-complementary genome with a CMV-eGFP cassette and were monitored for eGFP expression. The rAAV vectors transduced HROs in a dose-dependent manner, with rAAV9.NN achieving the highest efficiency and fastest onset kinetics, leading to detectable eGFP signal in photoreceptors, some interneurons and Müller glia already at 2 days post-transduction. The potency enhancing effect of the NN peptide-insert was replicated when using the corresponding AAV2-based version (rAAV2.NN). Taken together, we report the application of an HRO system for screening novel AAV vectors and introduce novel vector candidates with enhanced transduction efficiency for human retinal cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2020.321DOI Listing
March 2021

A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting.

Mol Ther Methods Clin Dev 2021 Mar 26;20:587-600. Epub 2021 Jan 26.

Department of Ophthalmology, Ludwig-Maximilians-University Munich, 80336 Munich, Germany.

Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895692PMC
March 2021

Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice.

Int J Mol Sci 2021 Feb 26;22(5). Epub 2021 Feb 26.

Department of Ophthalmology, Ludwig-Maximilians-Universität München, Mathildenstraße 8, 80336 Munich, Germany.

Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or -mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectroscopy-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify NAcetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22052345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956175PMC
February 2021

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.

EMBO Mol Med 2021 Apr 22;13(4):e13392. Epub 2021 Feb 22.

Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3 mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.202013392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033523PMC
April 2021

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the -Deficient Mouse Model of Achromatopsia.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-University, 81377 Munich, Germany.

Mutations in the gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in KO mice. Virus-mediated knockdown or genetic ablation of in KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793084PMC
December 2020

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Sci Adv 2020 Aug 19;6(34):eaba5614. Epub 2020 Aug 19.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin () in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aba5614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438099PMC
August 2020

COVID-19: Ophthalmological Aspects of the SARS-CoV 2 Global Pandemic.

Klin Monbl Augenheilkd 2020 May 6;237(5):675-680. Epub 2020 May 6.

Augenklinik der LMU, Klinikum der Universität München, München, Germany.

Purpose: To perform a systematic analysis of articles on the ophthalmological implications of the global COVID-19 pandemic.

Methods: PubMed.gov was searched for relevant articles using the keywords "COVID-19", "coronavirus", and "SARS-CoV-2" in conjunction with "ophthalmology" and "eye". Moreover, official recommendations of ophthalmological societies were systematically reviewed, with a focus on the American Academy of Ophthalmology (AAO) and the Royal College of Ophthalmologists (RCOphth).

Results: As of April 16, 2020, in total, 21 peer-reviewed articles on the ophthalmological aspects of COVID-19 were identified. Of these, 12 (57.1%) were from Asia, 6 (28.6%) from the United States of America, and 3 (14.3%) from Europe. There were 5 (23.8%) original studies, 10 (47.6%) letters, 3 (14.2%) case reports, and 3 (14.2%) reviews. These articles could be classified into the topics "Modes and prevention of (ocular) transmission", "Ophthalmological manifestations of COVID-19", "Clinical guidance concerning ophthalmological practice during the COVID-19 pandemic", and "Practical recommendations for clinical infrastructure". Practical recommendations could be extracted from official statements of the AAO and the RCOphth.

Conclusion: Within a short period, a growing body of articles has started to elucidate the ophthalmological implications of COVID-19. As the eye can represent a route of infection (actively via tears and passively via the nasoacrimal duct), ophthalmological care has to undergo substantial modifications during this pandemic. In the eye, COVID-19 can manifest as keratoconjunctivitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/a-1164-9381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295305PMC
May 2020

Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial.

JAMA Ophthalmol 2020 06;138(6):643-651

Institute for Ophthalmic Research, Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany.

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available.

Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia.

Design, Setting, And Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018.

Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017).

Main Outcomes And Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment.

Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples).

Conclusions And Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains.

Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamaophthalmol.2020.1032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193523PMC
June 2020

Neuropathic and cAMP-induced pain behavior is ameliorated in mice lacking CNGB1.

Neuropharmacology 2020 07 6;171:108087. Epub 2020 Apr 6.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438, Frankfurt am Main, Germany.

Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2020.108087DOI Listing
July 2020

Synthesis of Galactosyl-Queuosine and Distribution of Hypermodified Q-Nucleosides in Mouse Tissues.

Angew Chem Int Ed Engl 2020 07 21;59(30):12352-12356. Epub 2020 Apr 21.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNA , tRNA , tRNA , and tRNA . It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNA and tRNA of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl-queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q-family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q-nucleoside family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202002295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384130PMC
July 2020

Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1.

FASEB J 2020 02 27;34(2):2465-2482. Epub 2019 Dec 27.

German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.

The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (Aβ) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201902347RDOI Listing
February 2020

Advancing Gene Therapy for PDE6A Retinitis Pigmentosa.

Adv Exp Med Biol 2019 ;1185:103-107

Center for Integrated Protein Science Munich (CIPSM) at the Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.

Mutations in the gene encoding the phosphodiesterase 6 alpha subunit (PDE6A) account for 3-4% of autosomal recessive retinitis pigmentosa (RP), and currently no treatment is available. There are four animal models for PDE6A-RP: a dog with a frameshift truncating mutation (p.Asn616ThrfsTer39) and three mouse models with missense mutations (Val685Met, Asp562Trp, and Asp670Gly) showing a range of phenotype severities. Initial proof-of-concept gene augmentation studies in the Asp670Gly mouse model and dog model used a subretinally delivered adeno-associated virus serotype 8 with a 733 tyrosine capsid mutation delivering species-specific Pde6a cDNAs. These restored some rod-mediated function and preserved retinal structure. Subsequently, a translatable vector (AAV8 with a human rhodopsin promoter and human PDE6A cDNA) was tested in the dog and the Asp670Gly mouse model. In the dog, there was restoration of rod function, a robust rod-mediated ERG, and introduction of dim-light vision. Treatment improved morphology of the photoreceptor layer, and the retina was preserved in the treated region. In the Asp670Gly mouse, therapy also preserved photoreceptors with cone survival being reflected by maintenance of cone-mediated ERG responses. These studies are an important step toward a translatable therapy for PDE6A-RP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-030-27378-1_17DOI Listing
February 2020

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.

J Pharm Sci 2020 01 19;109(1):854-862. Epub 2019 Oct 19.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany; Center for Integrated Protein Science Munich (CiPSM), Munich, Germany; Department of Ophthalmology, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (T), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the T of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid T. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2019.10.031DOI Listing
January 2020

Morpho-Rheological Fingerprinting of Rod Photoreceptors Using Real-Time Deformability Cytometry.

Cytometry A 2019 11 20;95(11):1145-1157. Epub 2019 May 20.

CRTD/Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany.

Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900160PMC
November 2019

Safety and Toxicology of Ocular Gene Therapy with Recombinant AAV Vector rAAV.hCNGA3 in Nonhuman Primates.

Hum Gene Ther Clin Dev 2019 06;30(2):50-56

1 University Eye Hospital Tübingen, University of Tübingen, Tübingen, Germany.

The purpose of this study was to examine the toxicity and side effects of a recombinant adeno-associated virus 8 (AAV8) vector, aimed to treat cyclic nucleotide gated channel alpha 3 ()-linked achromatopsia, after a single subretinal administration in cynomolgus macaques. Animals were followed in two studies: a 13-week study with 22 animals and a 28-day study with 12 animals. Both groups were divided into subgroups receiving either vehicle only, a low (1 × 10 vector genomes (vg)), or a high dose (1 × 10 vg) of rAAV.hCNGA3. In the 13-week study, an extra group received single high-dose intravitreal injections. Here we present the group results of the histological examinations carried out after necropsy from the 28-day study, the retinal functional (electroretinography) in the 13-week study, and clinical observations from both studies. Treatment-related adverse effects were not found, and parameter changes were mostly related to the surgical procedure. The treatment of achromatopsia with rAAV.hCNGA3 is therefore deemed safe to apply to humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/humc.2018.188DOI Listing
June 2019

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

Genes (Basel) 2019 01 5;10(1). Epub 2019 Jan 5.

Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, 81377 Munich, Germany.

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes10010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356711PMC
January 2019

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

J Clin Invest 2018 12 12;128(12):5663-5675. Epub 2018 Nov 12.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI96098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264655PMC
December 2018

Optimized Subretinal Injection Technique for Gene Therapy Approaches.

Methods Mol Biol 2019 ;1834:405-412

Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls Universität Tübingen, Tübingen, Germany.

Gene therapy for inherited eye diseases requires local viral vector delivery by intraocular injection. Since large animal models are lacking for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The method described here aims to provide the details relevant to perform a transscleral pars plana virus-mediated gene transfer to achieve an optimized therapeutic effect in the small mouse eye.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8669-9_26DOI Listing
April 2019

In Vitro Evaluation of AAV Vectors for Retinal Gene Therapy.

Methods Mol Biol 2019 ;1834:383-390

Department of Pharmacy-Center for Drug Research, Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

Gene therapy holds promise for treating previously untreatable retinal disorders. The most promising approaches use gene transfer vectors derived from adeno-associated virus (AAV) to supplement a gene function in the affected cell type. One example is gene therapy for achromatopsia which affects daylight vision. In this case, recombinant AAV (rAAV) vectors are being developed to specifically target cone photoreceptors. Development of rAAV vectors could be facilitated by the use of in vitro models. In this chapter we provide a protocol which utilizes mouse 661W cells, an in vitro model of cone photoreceptors for evaluation of the transduction efficacy of rAAV vectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8669-9_24DOI Listing
April 2019

Development of Methodology and Study Protocol: Safety and Efficacy of a Single Subretinal Injection of rAAV.hCNGA3 in Patients with CNGA3-Linked Achromatopsia Investigated in an Exploratory Dose-Escalation Trial.

Hum Gene Ther Clin Dev 2018 09;29(3):121-131

3 STZ eyetrial at the Centre for Ophthalmology , Tuebingen, Germany .

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/humc.2018.088DOI Listing
September 2018

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

J Cell Sci 2018 08 17;131(16). Epub 2018 Aug 17.

Animal Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina and via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.218487DOI Listing
August 2018

Optogenetic Control of Neural Circuits in the Mongolian Gerbil.

Front Cell Neurosci 2018 24;12:111. Epub 2018 Apr 24.

Division of Neurobiology, Department Biology II, Biocenter, Ludwig Maximilian University of Munich, Munich, Germany.

The Mongolian gerbil () is widely used as a model organism for the human auditory system. Its hearing range is very similar to ours and it uses the same mechanisms for sound localization. The auditory circuits underlying these functions have been characterized. However, important mechanistic details are still under debate. To elucidate these issues, precise and reversible optogenetic manipulation of neuronal activity in this complex circuitry is required. However, genetic and genomic resources for the Mongolian gerbil are poorly developed. Here, we demonstrate a reliable gene delivery system using an AAV8(Y337F)-pseudotyped recombinant adeno-associated virus (AAV) 2-based vector in which the pan-neural human synapsin (hSyn) promoter drives neuron-specific expression of CatCH (Ca-permeable channelrhodopsin) or NpHR3.0 ( halorhodopsin). After stereotactic injection into the gerbil's auditory brainstem (medial nucleus of the trapezoid body, dorsal nucleus of the lateral lemniscus) and midbrain [inferior colliculus (IC)], we characterized CatCH- and/or NpHR3.0-transduced neurons in acute brain slices by means of whole-cell patch-clamp recordings. As the response properties of optogenetic tools strongly depend on neuronal biophysics, this parameterization is crucial for their application. In a proof-of-principle experiment in anesthetized gerbils, we observed strong suppression of sound-evoked neural responses in the dorsal nucleus of the lateral lemniscus (DNLL) and IC upon light activation of NpHR3.0. The successful validation of gene delivery and optogenetic tools in the Mongolian gerbil paves the way for future studies of the auditory circuits in this model system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2018.00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928259PMC
April 2018

Humoral Immune Response After Intravitreal But Not After Subretinal AAV8 in Primates and Patients.

Invest Ophthalmol Vis Sci 2018 04;59(5):1910-1915

University Eye Hospital, University of Tübingen, Tübingen, Germany.

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients.

Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients.

Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg.

Conclusions: No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.17-22494DOI Listing
April 2018

Hif1a inactivation rescues photoreceptor degeneration induced by a chronic hypoxia-like stress.

Cell Death Differ 2018 12 17;25(12):2071-2085. Epub 2018 Apr 17.

Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zürich, Zürich, Switzerland.

Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-018-0094-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261999PMC
December 2018

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Center for Integrated Protein Science Munich (CIPSM), Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr, 5-13, 81377 Munich, Germany.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19030749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877610PMC
March 2018

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice.

Front Mol Neurosci 2017 9;10:436. Epub 2018 Jan 9.

Center for Integrated Protein Science and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians University, Munich, Germany.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3 mice show normal visual, photic, and non-photic circadian function. In addition, HCN3 mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2017.00436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767300PMC
January 2018

Early Microglia Activation Precedes Photoreceptor Degeneration in a Mouse Model of CNGB1-Linked Retinitis Pigmentosa.

Front Immunol 2017 5;8:1930. Epub 2018 Jan 5.

Center for Integrated Protein Science Munich CiPSM and Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.

Retinitis pigmentosa (RP) denotes a family of inherited blinding eye diseases characterized by progressive degeneration of rod and cone photoreceptors in the retina. In most cases, a rod-specific genetic defect results in early functional loss and degeneration of rods, which is followed by degeneration of cones and loss of daylight vision at later stages. Microglial cells, the immune cells of the central nervous system, are activated in retinas of RP patients and in several RP mouse models. However, it is still a matter of debate whether activated microglial cells may be responsible for the amplification of the typical degenerative processes. Here, we used mice, which represent a slow degenerative mouse model of RP, to investigate the extent of microglia activation in retinal degeneration. With a combination of FACS analysis, immunohistochemistry and gene expression analysis we established that microglia in the retina were already activated in an early, predegenerative stage of the disease. The evidence available so far suggests that early retinal microglia activation represents a first step in RP, which might initiate or accelerate photoreceptor degeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.01930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760536PMC
January 2018

Gene Therapy Successfully Delays Degeneration in a Mouse Model of -Linked Retinitis Pigmentosa (RP43).

Hum Gene Ther 2017 Dec;28(12):1180-1188

Divisions of Ocular Neurodegeneration, Eberhard Karls University, Tuebingen, Germany.

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the -mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed by means of electroretinography. The degree of morphological rescue was investigated both using optical coherence tomography and by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2017.156DOI Listing
December 2017