Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. Recent advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Preclinical and clinical successes in AAV-mediated gene replacement, gene silencing and gene editing have helped AAV gain popularity as the ideal therapeutic vector, with two AAV-based therapeutics gaining regulatory approval in Europe or the United States. Continued study of AAV biology and increased understanding of the associated therapeutic challenges and limitations will build the foundation for future clinical success.

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Adeno-associated virus vector as a platform for gene therapy delivery

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.

Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

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Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

BACKGROUND Nearly five decades ago, visionary scientists hypothesized that genetic modification by exogenous DNA might be an effective treatment for inherited human diseases. This “gene therapy” strategy offered the theoretical advantage that a durable and possibly curative clinical benefit would be achieved by a single treatment. Although the journey from concept to clinical application has been long and tortuous, gene therapy is now bringing new treatment options to multiple fields of medicine. We review critical discoveries leading to the development of successful gene therapies, focusing on direct in vivo administration of viral vectors, adoptive transfer of genetically engineered T cells or hematopoietic stem cells, and emerging genome editing technologies. ADVANCES The development of gene delivery vectors such as replication-defective retro viruses and adeno-associated virus (AAV), coupled with encouraging results in preclinical disease models, led to the initiation of clinical trials in the early 1990s. Unfortunately, these early trials exposed serious therapy-related toxicities, including inflammatory responses to the vectors and malignancies caused by vector-mediated insertional activation of proto-oncogenes. These setbacks fueled more basic research in virology, immunology, cell biology, model development, and target disease, which ultimately led to successful clinical translation of gene therapies in the 2000s. Lentiviral vectors improved efficiency of gene transfer to nondividing cells. In early-phase clinical trials, these safer and more efficient vectors were used for transduction of autologous hematopoietic stem cells, leading to clinical benefit in patients with immunodeficiencies, hemoglobinopathies, and metabolic and storage disorders. T cells engineered to express CD19-specific chimeric antigen receptors were shown to have potent antitumor activity in patients with lymphoid malignancies. In vivo delivery of therapeutic AAV vectors to the retina, liver, and nervous system resulted in clinical improvement in patients with congenital blindness, hemophilia B, and spinal muscular atrophy, respectively. In the United States, Food and Drug Administration (FDA) approvals of the first gene therapy products occurred in 2017, including chimeric antigen receptor (CAR)–T cells to treat B cell malignancies and AAV vectors for in vivo treatment of congenital blindness. Promising clinical trial results in neuromuscular diseases and hemophilia will likely result in additional approvals in the near future. In recent years, genome editing technologies have been developed that are based on engineered or bacterial nucleases. In contrast to viral vectors, which can mediate only gene addition, genome editing approaches offer a precise scalpel for gene addition, gene ablation, and gene “correction.” Genome editing can be performed on cells ex vivo or the editing machinery can be delivered in vivo to effect in situ genome editing. Translation of these technologies to patient care is in its infancy in comparison to viral gene addition therapies, but multiple clinical genome editing trials are expected to open over the next decade. OUTLOOK Building on decades of scientific, clinical, and manufacturing advances, gene therapies have begun to improve the lives of patients with cancer and a variety of inherited genetic diseases. Partnerships with biotechnology and pharmaceutical companies with expertise in manufacturing and scale-up will be required for these therapies to have a broad impact on human disease. Many challenges remain, including understanding and preventing genotoxicity from integrating vectors or off-target genome editing, improving gene transfer or editing efficiency to levels necessary for treatment of many target diseases, preventing immune responses that limit in vivo administration of vectors or genome editing complexes, and overcoming manufacturing and regulatory hurdles. Importantly, a societal consensus must be reached on the ethics of germline genome editing in light of rapid scientific advances that have made this a real, rather than hypothetical, issue. Finally, payers and gene therapy clinicians and companies will need to work together to design and test new payment models to facilitate delivery of expensive but potentially curative therapies to patients in need. The ability of gene therapies to provide durable benefits to human health, exemplified by the scientific advances and clinical successes over the past several years, justifies continued optimism and increasing efforts toward making these therapies part of our standard treatment armamentarium for human disease.

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Gene therapy comes of age.

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9–based genome editing as a potential therapy to treat DMD.

X. Pan

Papers

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy