Publications by authors named "Hildegard Büning"

112 Publications

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.
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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.
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http://dx.doi.org/10.1016/j.omtm.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895692PMC
March 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.
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http://dx.doi.org/10.15252/emmm.202013392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033523PMC
April 2021

Gene and Cell Therapy for Inherited and Acquired Immune Deficiency.

Hum Gene Ther 2021 Jan;32(1-2):1-3

Great Ormond Street Institute of Child Health, University College London, London, UK.

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http://dx.doi.org/10.1089/hum.2021.29147.hbuDOI Listing
January 2021

Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy.

Expert Opin Biol Ther 2020 Dec 30:1-18. Epub 2020 Dec 30.

Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.

Gene therapy clinical trials with adeno-associated virus (AAV) vectors report impressive clinical efficacy data. Nevertheless, challenges have become apparent, such as the need for high vector doses and the induction of anti-AAV immune responses that cause the loss of vector-transduced hepatocytes. This fostered research focusing on development of next-generation AAV vectors capable of dealing with these hurdles. While both the viral vector genome and the capsid are subjects to engineering, this review focuses on the latter. Specifically, we summarize the principles of capsid engineering strategies, and describe developments and applications of engineered capsid variants for liver-directed gene therapy. Capsid engineering is a promising strategy to significantly improve efficacy of the AAV vector system in clinical application. Reduction in vector dose will further improve vector safety, lower the risk of host immune responses and the cost of manufacturing. Capsid engineering is also expected to result in AAV vectors applicable to patients with preexisting immunity toward natural AAV serotypes.
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http://dx.doi.org/10.1080/14712598.2021.1865303DOI Listing
December 2020

Use of Cell and Genome Modification Technologies to Generate Improved "Off-the-Shelf" CAR T and CAR NK Cells.

Front Immunol 2020 7;11:1965. Epub 2020 Aug 7.

Institute of Experimental Hematology, Hannover Medical School, Hanover, Germany.

The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modification of autologous and allogenic immune cells with chimeric antigen receptors (CAR) designed to target specific antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modification and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce "off-the-shelf" CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modification technologies have accelerated the generation of designer cell therapy products, including development of "off-the-shelf" CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal "off-the-shelf" CAR T and NK cell therapeutics.
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http://dx.doi.org/10.3389/fimmu.2020.01965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438733PMC
April 2021

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives.

Cancers (Basel) 2020 Jul 14;12(7). Epub 2020 Jul 14.

Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.

Adeno-associated virus (AAV) vectors have gained tremendous attention as in vivo delivery systems in gene therapy for inherited monogenetic diseases. First market approvals, excellent safety data, availability of large-scale production protocols, and the possibility to tailor the vector towards optimized and cell-type specific gene transfer offers to move from (ultra) rare to common diseases. Cancer, a major health burden for which novel therapeutic options are urgently needed, represents such a target. We here provide an up-to-date overview of the strategies which are currently developed for the use of AAV vectors in cancer gene therapy and discuss the perspectives for the future translation of these pre-clinical approaches into the clinic.
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http://dx.doi.org/10.3390/cancers12071889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409174PMC
July 2020

Controlled Functional Zonation of Hepatocytes by Engineering of Wnt Signaling.

ACS Synth Biol 2020 07 6;9(7):1638-1649. Epub 2020 Jul 6.

Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.

Key liver functions, including protein synthesis, carbohydrate metabolism, and detoxification, are performed by specific populations of hepatocytes that are defined by their relative positions within the liver lobules. On a molecular level, the functional heterogeneity with periportal and pericentral phenotypes, so-called metabolic liver zonation, is mainly established by a gradient of canonical Wnt signaling activity. Since the relevant physiological cues are missing in liver models, they fail to reflect the functional heterogeneity and thus lack many liver functions. We synthetically re-engineered Wnt signaling in murine and human hepatocytes using a doxycycline-dependent cassette for externally controlled digital expression of stabilized β-catenin. Thereby, we achieved adjustable mosaic-like activation of Wnt signaling in -cultured hepatocytes that was resistant to negative-feedback loops. This allowed the establishment of long-term-stable periportal-like and pericentral-like phenotypes that mimic the heterogeneity observed . The -zonated hepatocytes show differential expression of drug-metabolizing enzymes and associated differential toxicity and higher levels of autophagy. Furthermore, recombinant adeno-associated virus and hepatitis C virus preferentially transduce the pericentral-like zonation phenotype, suggesting a bias of these viruses that has been unappreciated to date. These tightly controlled -like systems will be important for studies evaluating aspects of liver zonation and for the assessment of drug toxicity for mouse and man.
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http://dx.doi.org/10.1021/acssynbio.9b00435DOI Listing
July 2020

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Nat Commun 2020 01 31;11(1):644. Epub 2020 Jan 31.

IONIS Pharmaceuticals, Carlsbad, CA, 92010, USA.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.
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http://dx.doi.org/10.1038/s41467-020-14323-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994702PMC
January 2020

AAV Entry: Filling in the Blanks.

Mol Ther 2020 02 28;28(2):346-347. Epub 2020 Jan 28.

Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2020.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002894PMC
February 2020

Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

iScience 2020 Jan 12;23(1):100764. Epub 2019 Dec 12.

Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany. Electronic address:

Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.
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http://dx.doi.org/10.1016/j.isci.2019.100764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941859PMC
January 2020

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells.

Gut 2020 06 25;69(6):1104-1115. Epub 2019 Nov 25.

Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany

Objective: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases.

Design: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of promoter.

Results: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver.

Conclusion: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.
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http://dx.doi.org/10.1136/gutjnl-2019-318812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282557PMC
June 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.
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http://dx.doi.org/10.1016/j.xphs.2019.10.031DOI Listing
January 2020

Breaking the Barriers of Genetic and Metabolic Disorders.

Hum Gene Ther 2019 10;30(10):1177-1179

Genethon, Evry, France.

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http://dx.doi.org/10.1089/hum.2019.29093.hbuDOI Listing
October 2019

Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes.

Stem Cell Reports 2019 10 19;13(4):590-598. Epub 2019 Sep 19.

Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47 subunit of the oxidase complex carry the deletion c.75_76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach.
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http://dx.doi.org/10.1016/j.stemcr.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829751PMC
October 2019

GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry.

Mol Ther Methods Clin Dev 2019 Sep 23;14:252-260. Epub 2019 Jul 23.

Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany.

Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.
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http://dx.doi.org/10.1016/j.omtm.2019.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706527PMC
September 2019

Engineering and Manufacturing Adeno-Associated Viral (AAV) Vectors.

Hum Gene Ther Methods 2019 08;30(4):121

Hannover Medical School.

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http://dx.doi.org/10.1089/hgtb.2019.29000.cfpDOI Listing
August 2019

Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity.

Sci Rep 2019 03 6;9(1):3631. Epub 2019 Mar 6.

Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8 T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.
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http://dx.doi.org/10.1038/s41598-019-40071-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403382PMC
March 2019

Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors.

Mol Ther Methods Clin Dev 2019 Mar 26;12:248-265. Epub 2019 Jan 26.

Division of Cellular and Molecular Therapy, Departments of Pediatrics and Molecular Genetics & Microbiology, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA.

In the past decade, recombinant vectors based on a non-pathogenic parvovirus, the adeno-associated virus (AAV), have taken center stage as a gene delivery vehicle for the potential gene therapy for a number of human diseases. To date, the safety of AAV vectors in 176 phase I, II, and III clinical trials and their efficacy in at least eight human diseases are now firmly documented. Despite these remarkable achievements, it has also become abundantly clear that the full potential of first generation AAV vectors composed of naturally occurring capsids is not likely to be realized, since the wild-type AAV did not evolve for the purpose of therapeutic gene delivery. In this article, we provide a brief historical account of the progress that has been made in the development of capsid-modified next-generation AAV vectors to ensure both the safety and efficacy of these vectors in targeting a wide variety of human diseases.
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http://dx.doi.org/10.1016/j.omtm.2019.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378346PMC
March 2019

Rational Design of Gene Therapy Vectors.

Mol Ther Methods Clin Dev 2019 Mar 16;12:246-247. Epub 2019 Feb 16.

Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL, USA.

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http://dx.doi.org/10.1016/j.omtm.2019.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378344PMC
March 2019

Characterization of a Novel Third-Generation Anti-CD24-CAR against Ovarian Cancer.

Int J Mol Sci 2019 Feb 3;20(3). Epub 2019 Feb 3.

Institute for Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.

Novel therapeutic approaches against ovarian cancer (OC) are urgently needed because of its high rate of recurrence even after extensive surgery and multi-agent chemotherapy. We aimed to develop a novel anti-CD24 chimeric antigen receptor (CAR) as an immunotherapeutic approach against OC cells and cancer stem cells (CSC). CSC represents a subpopulation of the tumor characterized by enhanced chemoresistance as well as the increased capability of self-renewal and metastasis. We designed a codon-optimized third-generation CAR containing the highly active single chain variable fragment (scFv) "SWA11" against CD24. We equipped the human NK-cell line NK-92 with the anti-CD24 CAR and an anti-CD19 control CAR using lentiviral transduction. Engineered NK-92 cells showed high cytotoxic activity against CD24-positive OC cell lines (SKOV3, OVCAR3). This effect was restricted to CD24-expressing cells as shown after lentiviral transduction of CD24-negative cell lines (A2780, HEK-293T) with CD24 transmembrane proteins. Additionally, NK-92 cells equipped with our novel anti-CD24 CAR were highly effective against patient-derived primary ovarian cancer cells. The activation of NK cells was shown by specific IFNγ secretion upon antigen stimulation. To further reduce possible off-target effects in vivo, we applied a dual-CAR approach using an anti-CD24-CD28-41BB fusion protein linked via a 2A sequence to an anti-mesothelin-CD3ζ-CAR. The dual-CAR was simultaneously active against CD24 and mesothelin expressing cells. Our novel anti-CD24-CAR showed a highly cytotoxic effect against OC cell lines and primary OC cells and will be evaluated in future in vivo trials as a promising immunotherapeutic approach against OC.
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http://dx.doi.org/10.3390/ijms20030660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387114PMC
February 2019

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Viruses 2018 04 15;10(4). Epub 2018 Apr 15.

Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt, Germany.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.
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http://dx.doi.org/10.3390/v10040197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923491PMC
April 2018

Characterization of AAV vector particle stability at the single-capsid level.

J Biol Phys 2018 06 14;44(2):181-194. Epub 2018 Apr 14.

Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, Laboratoire de Physique, F-69342, Lyon, France.

Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.
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http://dx.doi.org/10.1007/s10867-018-9488-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928021PMC
June 2018

Key to Delivery: The (Epi-)genome Editing Vector Toolbox.

Methods Mol Biol 2018 ;1767:147-166

Laboratory for Infection Biology & Gene Transfer, Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.

Curing a genetic disease by repairing the underlying genetic defect is a fascinating concept that has been addressed so far by gene compensation therapy. For this, a functional copy of the gene in question together with elements controlling its expression is produced as a vector and introduced ex vivo into the patient's own cells that subsequently are reinfused. Alternatively, vectors are administered directly in vivo. Although this strategy resulted in impressive therapeutic benefits for patients, the ultimate goal of gene therapy, i.e., a cure by repairing the actual genetic or epigenetic defect, remained an unresolved task. With the advent of designer DNA-binding domains, this goal is coming into reach. These domains are either combined with nucleases and used as molecular precision scissors for introducing DNA breaks at defined sites in the cell's genome preparing for position-selective DNA repair, or they are used as programmable DNA-binding units for positioning epigenome-modifying domains to predefined target sequences. However, for reaching its full potential, these components need to be delivered into cells in an efficient and safe manner. Here, we summarize current viral and non-viral delivery approaches applicable for genome and epigenome editing and discuss their respective advantages and limitations.
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http://dx.doi.org/10.1007/978-1-4939-7774-1_7DOI Listing
February 2019

Analyzing the Genotoxicity of Retroviral Vectors in Hematopoietic Cell Gene Therapy.

Mol Ther Methods Clin Dev 2018 Mar 5;8:21-30. Epub 2017 Oct 5.

Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.

Retroviral vectors, including those derived from gammaretroviruses and lentiviruses, have found their way into the clinical arena and demonstrated remarkable efficacy for the treatment of immunodeficiencies, leukodystrophies, and globinopathies. Despite these successes, gene therapy unfortunately also has had to face severe adverse events in the form of leukemias and myelodysplastic syndromes, related to the semi-random vector integration into the host cell genome that caused deregulation of neighboring proto-oncogenes. Although improvements in vector design clearly lowered the risk of this insertional mutagenesis, analysis of potential genotoxicity and the consequences of vector integration remain important parameters for basic and translational research and most importantly for the clinic. Here, we review current assays to analyze biodistribution and genotoxicity in the pre-clinical setting and describe tools to monitor vector integration sites in vector-treated patients as a biosafety readout.
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http://dx.doi.org/10.1016/j.omtm.2017.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684499PMC
March 2018

Celebrating 25 Years of the European Society of Gene and Cell Therapy.

Hum Gene Ther 2017 11;28(11):939

2 Vice President, ESGCT .

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http://dx.doi.org/10.1089/hum.2017.29054.rraDOI Listing
November 2017

Gene and Cell Therapy in Germany.

Hum Gene Ther 2017 10;28(10):781

3 Scientific Secretary, DG-GT e.V.

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http://dx.doi.org/10.1089/hum.2017.29057.cvkDOI Listing
October 2017