Next-generation muscle-directed gene therapy by in silico vector design.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Haemophilia gene therapy: From trailblazer to gamechanger.

Haemophilia is an attractive disease target for gene therapy that fostered the development of the field at large. The delivery of the clotting factor genes into the patients' cells could be accomplished using different types of gene delivery vehicles or vectors. Adeno-associated viral vectors (AAV) and lentiviral vectors represent some of the most promising gene delivery technologies that allow for a relatively efficient delivery of the therapeutic FVIII and FIX transgenes into the relevant target cells. To reduce the risks associated with insertional mutagenesis due to random vector integration, gene-editing approaches have also been considered based primarily on zinc finger nuclease (ZFN) and CRISPR/Cas. However, comprehensive analysis of off-target effects is still required. It is particularly encouraging that relatively stable therapeutic FVIII or FIX expression levels were reached in severe haemophilia patients in recent clinical trials after liver-directed AAV gene therapy. This success could be ascribed in part to improvements in vector design. In particular, clotting factor levels could be increased by codon optimization of coagulation factor transgenes. Alternatively, incorporation of a hyperactive gain-of-function R338L mutation (FIX Padua) in the FIX gene improved the overall efficacy. However, some patients still show transient liver toxicity, especially at high vector doses, possibly due to inflammatory immune responses, requiring the need for transient immunosuppression. The exact immune mechanisms are not fully understood, but may at least in some patients involve an AAV-capsid specific T cell response. Moreover, there is a need to identify the key factors that contribute to the interpatient variability in therapeutic efficacy and safety after gene therapy.

Gene therapy for hemophilia.

Hemophilia A and B are X-linked monogenic disorders resulting from deficiencies of factor VIII and FIX, respectively. Purified clotting factor concentrates are currently intravenously administered to treat hemophilia, but this treatment is non-curative. Therefore, gene-based therapies for hemophilia have been developed to achieve sustained high levels of clotting factor expression to correct the clinical phenotype. Over the past two decades, different types of viral and non-viral gene delivery systems have been explored for hemophilia gene therapy research with a variety of target cells, particularly hepatocytes, hematopoietic stem cells, skeletal muscle cells, and endothelial cells. Lentiviral and adeno-associated virus (AAV)-based vectors are among the most promising vectors for hemophilia gene therapy. In preclinical hemophilia A and B animal models, the bleeding phenotype was corrected with these vectors. Some of these promising preclinical results prompted clinical translation to patients suffering from a severe hemophilic phenotype. These patients receiving gene therapy with AAV vectors showed long-term expression of therapeutic FIX levels, which is a major step forwards in this field. Nevertheless, the levels were insufficient to prevent trauma or injury-induced bleeding episodes. Another challenge that remains is the possible immune destruction of gene-modified cells by effector T cells, which are directed against the AAV vector antigens. It is therefore important to continuously improve the current gene therapy approaches to ultimately establish a real cure for hemophilia.