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Unlocking Precision Gene Therapy

Periodic Reporting for period 2 - UPGRADE (Unlocking Precision Gene Therapy)

Reporting period: 2020-07-01 to 2021-12-31

UPGRADE main objectives are to: i) improve efficiency and safety of novel and emerging genome and epigenome editing technologies; ii) precisely assess and enhance specificity of genome editing; iii) tailor delivery of the novel genome editing technologies for in vivo application; iv) characterize and circumvent the immunogenicity of the AMPs; v) translate these novel AMPs into novel treatment paradigms applicable to to prototypic diseases, some of which affect ; vi) successfully manage, disseminate and exploit the results obtained by the consortium.
Overall, UPGRADE will exploit and further develop disruptive new technologies for precision gene and epigenome editing and stringently characterize their specificity and cellular responses. It will then combine these improved technologies with advanced gene delivery approaches to generate prototypes of advanced medicinal products (AMP). The safety and efficacy profile of each AMP will be stringently validated in disease models paradigmatic for unmet medical need and potential long-term cure, if the limitations of current gene therapy strategies are overcome. These AMPs will represent versatile products portable to the treatment of several other diseases because of related pathogenesis or correction strategies.
Objective 1: To improve efficiency and safety of novel and emerging genome and epigenome editing technologies; Objective 2: To precisely assess and enhance specificity of genome editing:
We have identified Epigenetic editing (aka Epi-editing) reagents capable to induce efficient and long-term stable silencing of the Pcsk9 gene in both human and mouse cell lines. We have adapted the Epi-editing platform to the molecular requirements necessary for its non-viral delivery to the liver. We have assessed the off-target profile of the Pcsk9 Epi-editing platform, showing its high degree of specificity.
We have both trained personnel supported by this grant in the practice of our GUIDE-seq and CIRCLE-seq assays at both the experimental and computational analysis levels. We envision replacing CIRCLE-seq with the new ONE-seq assay recently developed and validated. ONE-seq is at least as sensitive as CIRCLE-seq for detecting gene editing nuclease off-target sites and is more sensitive than Digenome-seq for detecting base editor off-target sites.
We have devoted considerable efforts to alter the DNA binding specificity of site-specific recombinases (SSRs), employing both directed molecular evolution and rational design. Proof-of-concept of the SSR therapeutic potential has been shown. We are aiming to optimize these designer recombinase platforms accelerating the development of novel Designer-SSRs towards loci of interest and develop an assay that allows the detection of potential off-targets experimentally. For all of these points considerable progress has been achieved and some results from these objectives have already been published.
Objective 3: To tailor delivery of the novel genome editing technologies for in vivo application; Objective 4: To characterize and circumvent the immunogenicity:
The integrated accomplishment of objectives will unlock the full potential of the novel therapeutic modalities developed in this project. We will deploy them by targeting either stem cells ex vivo followed by transplantation into conditioned hosts or long-lived differentiated tissues in vivo, such as retina, liver, heart and skeletal muscle. Studies will be conducted in disease entities for which we have already extensive pre-clinical and clinical experience, and for which there are still unmet medical needs. These studies will set the stage to broaden applications of the newly developed AMPs to diseases that per se or cumulatively are of high prevalence, such as retinal and muscle degeneration, and genetic or acquired disorders affecting the hematopoietic system and liver metabolism.
Objective 5: To translate these novel AMPs into novel treatment paradigms applicable to diseases affecting large patient populations:
We designed three different strategies for the selection of edited cells and achieved proof-of-principle for the enrichment of edited cells in vitro. We identified molecular enhancers (p53 inhibitor and adenoviral protein E4orf6/7) improving editing efficiency and tolerability, as well as clonality of edited HSPCs. We set up models (ossicles and NSGW41 mice) for stringent evaluation of non-genotoxic engraftment of engineered HSPCs. We identified two candidates (CD47 and CXCR4) whose overexpression improved engraftment of HSPCs. We are generating a novel mouse model of familial hypercholesterolemia, which harbour one of the most frequent human gain-of-function mutation in the murine Pcsk9 gene.
Objective 6: To successfully manage, disseminate and exploit the results:
Overall, the outcome of the project will be novel AMPs stringently validated in preclinical disease models and enabling knowledge to set the stage for their pr ompt translation into gene therapy trials for diseases affecting large patient populations. The project management team continuously works to ensure effective governance of the Consortium, dissemination and exploitation of its scientific output.
Targeted engineering of the human genome opens up an enormous number of new research opportunities and therapeutic possibilities. We are just at the beginning of the “age of genome editing” and we have already witnessed the amazing potential of these technologies to transform research, making possible to design and execute an unprecedented arrays of experimental studies, and attract from their very first steps amazing interest and resources from the biotechnological and pharmaceutical sectors as well as from less risk-adverse venture capital. We thus expect that a fallout of the technological advances brought forward by UPGRADE will be to foster new fundamental research interrogating gene function and regulation also in previously intractable cells and tissues, and to trigger new investments in the biotechnological space aimed at capturing and exploiting their novel R&D potential. Even more importantly, UPGRADE will provide proof-of-principle of several new therapeutic strategies developed and validated in paradigmatic disease models that could be expanded upon suitable adaptation to the treatment of many more disease entities. UPGRADE will launch its investigation starting right from where the promise of gene and cell therapy has already brought some fruits, with the aim of overcoming the hurdles that currently limit the harvest only to the lowest hanging fruits. Whereas the high costs of developing and manufacturing “live” drugs and highly personalized AMPs might still constrain their sustainable commercialization, UPGRADE will contribute in several aspects to ease the burden and overcome some of the barriers in the path towards making gene therapy a widespread reality.