Gill, Deborah

Hyde, SC

Van Haasteren, J

Molecular Therapy

English

Oxford University Research Archive

ASGCT 2019 – Abstract Joost van Haasteren  Title: Genome Editing Of The Liver For Treatment Of Alpha-1 Antitrypsin Deficiency Using Homology-Independent Targeted Integration (HITI)    Authors: Joost van Haasteren1, Stephen C. Hyde1, Deborah R. Gill1  Affiliation: 1 Gene Medicine Research Group, Nuffield Division of Clinical Laboratory Science, Radcliffe Department of Medicine, University of Oxford  Abstract (3500 characters excluding spaces):   Homology directed repair (HDR) is often selected for correction of genomic mutations for therapeutic benefit due to its favourable repair fidelity, but activity of the main repair pathway, non-homologous end joining (NHEJ) is several-fold higher in most systems. Using NHEJ, Homology-Independent Targeted Integration (HITI) allows for precise integration of a chosen sequence by cutting both the genome and donor sequence. This approach is applicable for diseases such as Alpha-1 Antitrypsin (AAT) deficiency in which genome editing could allow for expression of functional AAT whilst simultaneously reducing expression of the highly prevalent, toxic PI*Z form of the gene. Here, we assess the feasibility of HITI genome editing for AAT deficiency in liver cell lines and human liver organoids.  A fluorescent reporter 293T cell line containing an mCherry cDNA flanked by two defined murine saCas9 gRNA target sites provided a platform to test four permutations of HITI dependent genomic integration. Each permutation could support a different therapeutic application, using mCherry as a proxy for the target mutant gene. One permutation proved optimal for the treatment of the PI*Z mutant version of AAT. In this permutation, the integration event is targeted to the 5` UTR region of AAT and the integrated cDNA (reporter or therapeutic) is followed by a transcription blocker to prevent expression of the downstream PI*Z allele. Transfection of Huh-7 liver carcinoma cells with a 1:2 molar ratio of SaCas9/HITI Donor plasmid was effective, both in expressing the integrated cDNA of mNeonGreen from the AAT promoter as well as in preventing endogenous AAT expression in ~12% of total cells despite only ~30% transfection efficiency (n=4). A ddPCR copy number variation assay at the 5`-integration junction confirmed this observation. Sequencing showed that HITI integration was almost seamless (~90% in 78 clones from 2 different loci) with any mutations limited to 1 or 2bp indels, implying that NHEJ is less error-prone than expected. Analysis of a pilot capture-seq experiment proved that the majority of the integration events occurred at the intended integration site.   This proof-of-concept in a cell line is currently being translated to more relevant models, including human and murine liver organoids. Human liver samples were obtained, with consent, from liver resection surgery and healthy tissue, as assessed by a pathologist, was processed into liver organoids. Organoids were transfected with plasmids or transduced with recombinant adeno-associated virus (rAAV) as a single cell suspension to allow access to the basolateral membrane of the hepatocytes. Transduction of mouse organoids with rAAV of various serotypes showed that rAAV capsid 6.2 was the most efficient with 45% and 39% of organoids EGFP-positive 2 days post-transduction (n=2 independent experiments). Human liver organoids cultured as immature, actively dividing cells were differentiated into mature liver organoids as evidenced by AAT expression, providing a human liver model for ongoing genome editing studies.  We conclude that HITI is capable of efficiently integrating a DNA sequence into a targeted location in the genome in a variety of therapeutically applicable permutations, one of which can provide potential correction of AAT deficiency regardless of the patients’ mutation. Human liver organoids provide a physiologically and genetically relevant in vitro model of the human liver, acting as an appropriate stepping stone to in vivo studies in mouse disease models.        

Genome editing of the liver for treatment of alpha-1 antitrypsin deficiency using homology-independent targeted integration (HITI)

Munday, RJ

Gill, D

Oxford University Research Archive (ORA)

High levels of native eGFP detected following F/HN lentiviral vector transduction of a variety of lung models

Meyer-Berg, HCM

Title: Gene therapy for surfactant protein B deficiency using recombinant AAV   Authors: Helena Meyer-Berg1, Stephen C. Hyde1, Deborah R. Gill1  Affiliation: 1 Gene Medicine Group, Nuffield Division of Clinical Laboratory Science, Radcliffe Department of Medicine, University of Oxford  Surfactant Protein B (SP-B) deficiency is a rare, autosomal, recessive genetic disease of the lung that causes alveolar collapse and severe respiratory distress in neonates. The disease is invariably fatal during the first months of life. Surfactant replacement therapy in these full-term infants has been shown to be ineffective. The only treatment option is lung transplantation, which is rarely attempted as the mean age of death is 3 months. At an estimate, 1 in 1 million live births in North America and Europe are affected. To improve prognosis, we wish to develop gene therapy for SP-B deficiency using recombinant adeno-associated viral (rAAV) vectors.  Effective gene therapy for SP-B deficiency needs to target alveolar type II (ATII) cells in the lung parenchyma as they are the only cells that can produce mature SP-B. We selected AAV serotypes 5, 6.2 and 9 to compare transduction of ATII cells using eGFP as a reporter transgene. Mice (n=2-4) were dosed intranasally with 1E11 genome copies (GC). After 28 days, fixed-frozen sections of the treated lungs were analysed for cell transduction using confocal microscopy and automated imaging of whole lung sections. Immunostaining (with anti-pro-surfactant protein C antibody) was used to identify ATII cells. Recombinant AAV2/5 achieved the highest transduction rate of total murine lung cells (mean 3.06%, SD=0.96) which was significantly different from naive (Kruskal-Wallis test with Dunn’s post-hoc test, P= 0.0234). Mean transduction rates with serotypes 9 and 6.2 were lower (0.32%, SD=0.16 and 0.34%, SD=0.20, respectively). The rAAV2/5 vector also achieved the highest mean transduction rate in the target ATII cells which was significantly different from naive (19.4%, SD=4.6, p=0.014 with Kruskal-Wallis test with Dunn’s post-hoc test, P=0.0141) compared with rAAV2/6.2 and rAAV2/9 (4.0%, SD=1.8 and 4.5%, SD= 1.0 respectively). Furthermore, serotype 5 was the most specific for transduction of ATII cells with no other transduced cell types observed.  Serotypes were further investigated in preliminary studies using human precision cut lung slices (PCLS) of surgical lung resections. Fresh human lung tissue was cut in 500 µm sections and treated ex-vivo with 1E11 GC of rAAV2/5 expressing eGFP as reporter transgene. On day 12, PCLS (n=4) were processed in fixed-frozen sections and analysed for native eGFP fluorescence using confocal microscopy. Although serotype 5 showed the highest transduction rate in the murine lung, transduction was not observed in any of the 2-3 analysed sections per human lung slice. This stresses the importance of choosing appropriate model systems for the investigation of gene therapies. Further studies are ongoing to compare rAAV2/5 with other serotypes in human models of the lung.  

Gene therapy for surfactant protein B deficiency using recombinant AAV

Final- Submitted  High Levels of Native eGFP Detected Following F/HN Lentiviral Vector Transduction of a Variety of Lung Models. Rosie J Munday1, Stephen C Hyde1 and Deborah R Gill1. 1Gene Medicine Research Group, Radcliffe Department of Medicine (NDCLS), University of Oxford, Oxford OX3 9DU, UK.  Lentiviral vector pseudotyped with F and HN coat proteins from Sendai virus has been shown to successfully target a variety of cell types in the murine lung. Transduced cells expressing eGFP were identified using immunohistochemistry, an indirect staining method, showing approximately 15% of airway epithelial cells as eGFP-positive following delivery of 8E8 transducing units (TU) of rSIV.F/HN.hCEF.eGFP (Alton et al. 2017, Thorax 72:137). In this study, we use native eGFP fluorescence to identify eGFP-expressing cells directly, following transduction of murine lungs in-vivo and human, ferret and pig lung tissue ex-vivo.  Method: Mice were dosed intranasally with either 1E8 or 3E8 TU of rSIV.F/HN.hCEF.eGFP (n=4 per dose). In addition, precision cut lung slices (PCLS) of 500m were prepared from human, ferret and pig lungs and treated ex-vivo with 3E8 TU of rSIV.F/HN expressing eGFP or secreted reporter Gaussia luciferase (GLuc) under the control of the hCEF promoter. The PCLS were imaged during culturing for eGFP fluorescence and supernatant was assayed for secreted GLuc activity over a period in excess of 30 days. Lung tissue was fixed, cryoprotected and cryosectioned on day 7 or 28 following treatment. By imaging the cryosections using the EVOS FL Auto 2 imaging system (Thermo Fisher), the entire lung lobe (or PCLS) could be captured by ‘stitching’ together multiple fields of view. EGFP expression from the mouse lungs was quantified using Image J software to calculate the percentage area of epithelium or parenchymal tissue above a threshold fluorescence (autofluorescence) level for regions of interest.  Results: Approximately 40% of the mouse airway epithelium in both treated groups of mice (n=4 per dose) exceeded the fluorescence threshold, compared with epithelia from naïve mice where the median percentage area above threshold was zero (ANOVA P<0.0001). For PCLS cultured ex-vivo, high levels of secreted GLuc activity were detected in supernatant from treated PCLS (n=4-8 per species) compared with naïve slices for all species (t-test of area under the curve p<0.05). High levels of eGFP fluorescence were visualised in ex-vivo treated human, ferret and pig lung slices in culture. Studies are underway to characterise the transduced cells in each animal species, by combining native eGFP detection with cell type-specific immune staining of cryosections. These studies will evaluate the relative transduction rates and cell types in each animal species to aid selection of the most appropriate system to model human lung transduction.  

ORA - Oxford University Research Archive

https://ora.ox.ac.uk/objects/uuid:89c21e04-7713-4393-bf03-63532a862135/files/rpv63g0299

Genome editing of the liver for treatment of alpha-1 antitrypsin deficiency using homology-independent targeted integration (HITI)

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