Genome Editing: Moving Toward a New Era of Innovation, Development, and Approval.

Therapeutic genome editing is currently reshaping and transforming the development of advanced therapies as more ex vivo and in vivo gene editing-based technologies are used to treat a broad range of debilitating and complex disorders. With first-generation gene editing modalities (notably those based on ZFNs, TALENs and CRISPR/Cas9), comes a new second-generation of gene editing-based therapeutics including base editing, prime editing and other nuclease-free genome editing modalities. Such ground-breaking innovative products warrant careful considerations from a product development and regulatory perspective, that take into account not only the common development considerations that apply to standard gene and cell therapy products, but also other specific considerations linked with the technology being used. This article sheds light into specific considerations for developing safe and effective in vivo and ex vivo genome editing medicines that will continue to push barriers even further for the cell and gene therapy field.

Convergence or divergence: next frontiers toward globalization of current- and next-generation cell and gene therapies.

The last decades have seen a massive transformation in the field of advanced therapies, culminating in the marketing approval of different cutting-edge gene, cell- and tissue engineering-based therapies across different regions of the world. Although this success is promising, the global clinical development pathway of such therapies is often hindered by unique manufacturing, preclinical and clinical regulatory challenges; with different expectations, sometimes linked with divergence in opinions between international regulatory authorities. Such technologies call for a science-based approach and an early regulatory dialogue to set the key elements of quality, safety and efficacy for the next generation cell and gene therapies that can be harmonized across different regional jurisdictions, hence speeding up patient access to innovative therapies across the globe.

The Genetically Modified Organism Medicinal Framework in Europe, United States, and Japan: Underlying Scientific Principles and Considerations Toward the Development of Gene Therapy and Genetically Modified Cell-Based Products.

In vivo viral gene therapy and somatic cell therapy products (whether autologous, allogeneic, or xenogeneic) that have been subjected to an ex vivo gene transfer procedure will normally be classified as genetically modified organisms (GMOs) in Europe, not just the gene transfer vectors used in their construction. These products are, therefore, expected to fulfill certain environmental requirements with regard to the biosafety aspects of their clinical use (which may be subject to review by government departments responsible for environmental affairs). In the European Union (EU), clinical trials using GMOs generally require three levels of review (in addition to local review processes), which are often performed by separate national agencies. In this study, the principles under which certain EU member states control use of the GMOs in clinical trials under the definitions of either "contained use" or "deliberate release" will be discussed and evaluated from a scientific and a regulatory perspective, with comparisons with non-EU expectations as described by the U.S. Food and Drug Administration and the Japanese living modified organisms (LMOs) regulations. For the latter, an understanding of the criteria under which LMOs exemptions apply, notably with respect to the nature of the viral construct used, the manufacturing process, and demonstration that there is no detectable residual replication-competent virus in the final gene-modified cells, is of paramount importance. Building on the existing European, U.S., and Japanese experience with GMOs/LMOs within the context of experimental gene and cell therapies, a through reflection on, and harmonization of, the current global GMO framework is needed to avoid unnecessary delays in clinical development and to ensure a smooth and a rapid access by patients to innovative life-saving therapies.

Ex vivo and in vivo genome editing: a regulatory scientific framework from early development to clinical implementation.

Recent advances in human genome science have paved the way to a new class of human gene therapies based on gene editing, with the potential to provide a long-lasting curative strategy for many debilitating and complex disorders, for which there is an unmet medical need. Therapeutic genome editing encompasses both ex vivo and in vivo gene correction modalities, for which similar and also application-specific considerations apply, which dictate the overall strategy to be followed from a scientific, clinical and regulatory perspective. Here, the major regulatory barriers to successful clinical implementation are discussed, together with the key issues to be considered for generating safe (minimizing risks of tumorigenesis and off-target effects) and effective gene editing-based medicines for application in regenerative medicine.

Progress on gene therapy, cell therapy, and pharmacological strategies toward the treatment of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a muscle-specific, late-onset degenerative disorder whereby muscles of the eyes (causing ptosis), throat (leading to dysphagia), and limbs (causing proximal limb weakness) are mostly affected. The disease is characterized by a mutation in the poly(A)-binding protein nuclear-1 (PABPN1) gene, resulting in a short GCG expansion in the polyalanine tract of PABPN1 protein. Accumulation of filamentous intranuclear inclusions in affected skeletal muscle cells constitutes the pathological hallmark of OPMD. This review highlights the current translational research advances in the treatment of OPMD. In vitro and in vivo disease models are described. Conventional and experimental therapeutic approaches are discussed with emphasis on novel molecular therapies including the use of intrabodies, gene therapy, and myoblast transfer therapy.

Tumour necrosis factor-alpha increases extravasation of virus particles into tumour tissue by activating the Rho A/Rho kinase pathway.

Tumour Necrosis Factor alpha (TNF) is a pleiotropic pro-inflammatory cytokine with known vascular permeabilising activity. It is employed during isolated limb perfusion to enhance delivery of chemotherapeutic drugs into tumour tissue. The use of conditionally-replicating lytic viruses, so called 'oncolytic virotherapy', provides a new approach to cancer treatment that is currently limited by the low efficiency of extravasation of viral particles into tumours. We report here evidence that TNF significantly enhances the delivery of virus particles through the endothelial layer to allow access to tumour cells both in vitro and in vivo. Intravenous administration of TNF resulted in a 3- to 6-fold increase in EL4 tumour uptake of Evans Blue/Albumin, adenovirus and long-circulating polymer coated adenovirus. Interestingly, endothelial permeabilisation could be suppressed in vitro and in vivo by Y-27632, a Rho kinase inhibitor, without inhibiting viral infection. These data indicate that TNF can enhance the delivery of virus particles into tumours through a Rho A/Rho kinase dependent mechanism and may be a valuable strategy for increasing the delivery of oncolytic viruses and other therapeutic agents.

E-selectin is a viable route of infection for polymer-coated adenovirus retargeting in TNF-α-activated human umbilical vein endothelial cells.

BACKGROUND: E-selectin is an attractive endothelial cell surface marker in inflammation and cancer. PURPOSE: We sought to investigate retargeting of adenovirus via E-selectin as a viable pathway of infection in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs). METHODS: E1, E3-deleted Ad5 expressing cytomegalovirus immediate-early (CMV IE) promoter-driven luciferase (Adluc) was coated with an amino-reactive multivalent hydrophilic polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to generate pHPMA-adenovirus (pcAdluc). This was then retargeted by covalent attachment of a mouse antihuman E-selectin monoclonal antibody (MHES mAb), purified from the H18/7 hybridoma cell line (MHESpcAdluc). RESULTS: MHESpcAdluc was efficiently taken up into HUVECs, generating a high level of transduction in TNF-α-treated E-selectin positive cells but not in untreated receptor-negative cells. Specific retargeting of MHESpcAdluc was demonstrated through reduced transduction of stimulated HUVEC when incubated in the presence of free E-selectin antibodies. DISCUSSION AND CONCLUSION: Our results suggest that E-selectin could be a valuable target for gene transfer strategies internalizing polymer-coated modified adenovirus particles through a viable receptor-mediated endocytosis pathway, generating adequate levels of transgene expression per virus genome copy without compromising the specific activity of the parental virus.

Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins.

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. Tumour-associated endothelium offers an additional mechanism for enhanced viral uptake into tumours which is accessible for systemic gene delivery. Building on expertise in using polymer 'stealthed' viruses for targeting in vivo, adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to ablate normal infection pathways. Direct linkage of a monoclonal antibody against E-selectin (MHES) demonstrated E-selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells. A two-component targeting system using protein G was developed, to provide optimal antibody orientation. We report an enhancement in transduction of TNF-α-activated endothelium in vitro and ex vivo in a human umbilical vein cord model using the MHES antibody. Similarly a virus retargeted using a chimeric P-selectin Glycoprotein Ligand-1-Fc fusion (PSGL-1) protein showed better circulation kinetics and significant uptake into HepG2 xenografts following systemic administration in mice, with 36-fold higher genome copies, compared with non-modified virus. Immunohistochemistry staining of tumour sections from mice treated with PSGL-1-retargeted virus showed a co-localisation of firefly luciferase with CD31 suggesting selective endothelial targeting. Employment of optimal viral modification using protein G will enable exploration and comparison of alternative targeting ligands targeting tumour-associated endothelium.

Cancer gene therapy with targeted adenoviruses.

BACKGROUND: Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. OBJECTIVE: This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. METHODS: Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. RESULTS/CONCLUSION: Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.