Table 1.

Comparison of ex vivo and in vivo viral-based DMD editing strategies under investigation

✓ Pros × Cons	Viral-based DMD editing
	Ex vivo	In vivo
Background	× Knowledge about the grafting of different types of myogenic cells into recipient human muscles is scarce	✓ Builds upon an increasing amount of knowledge about the in vivo administration of viral vectors into recipient human muscles (for example, microdystrophin-encoding rAAVs)
Production	✓ Potentially less dependent on large-scale production of viral vectors × Reliant on the upscaling of cell culture systems × The required numbers of certain myogenic cell types might not be achievable owing to senescence (for example, myoblasts) × The current protocols do not permit culturing bona fide skeletal muscle stem cells (that is, satellite cells) in vitro	✓ Independent from the upscaling of cell culture systems × More reliant on large-scale production of viral vectors
Delivery	✓ Well-defined genetic modification environment that enables careful monitoring of procedures, events, and outcomes ✓ Lower stringency for monitoring the biodistribution (for example, gonads and shedding of vector elements) × Protocols for effective myogenic cell engraftment, migration, and differentiation need to be improved (for example, via signaling gradients and cell-autonomous reprogramming of iPSCs) × Local and locoregional administration of myogenic cells might be difficult to apply to a broad range of muscle groups × Protocols for the systemic delivery and tissue homing of myogenic cells need to be developed	✓ Direct exposure to gene-editing tools facilitates in situ correction of differentiated striated muscle tissues ✓ Possible in situ transduction of resident tissue-specific stem cells might generate a long-term source of gene-edited muscle progenitor cells ✓ Expanding range of viral vector pseudotypes enables the investigation of different transduction patterns—for example, tropism for affected tissues—while avoiding APCs. Such transductional targeting can be combined with transcriptional targeting (that is, use of tissue-specific promoters) × Local and locoregional administration of viral vector particles might be difficult to apply to a broad range of muscle groups × Protocols for the systemic delivery of viral vectors to affected tissues need to be improved   Higher stringency for monitoring the biodistribution (for example, gonads and shedding of vector elements)
Strategy	✓ Relies mostly on targeting replicating cells that are amenable to gene-editing approaches based on NHEJ as well as HR	× Relies mostly on targeting post-mitotic cells, which are less amenable to HR-based gene-editing principles
Immunology	✓ Minimizes the exposure of the patient to immunogenic components of viral vectors and gene-editing tools ✓ Possibly compatible with the re-administration of gene-edited autologous cells ✓ Avoids the blocking of viral vector particles by neutralizing antibodies present in the majority of the human population	× Patient exposure to immunogenic components of vector particles and gene-editing tools. Possible mounting of cellular responses to transduced cells displaying foreign epitopes × Anti-vector neutralizing antibodies in the majority of the human population. Serotype cross-neutralizing activity might render vector pseudotyping and vector re-administration ineffective

In vivo approaches entail the direct administration of gene-editing viral vectors to the patient. Ex vivo approaches encompass the in vitro transduction of patient-derived cells (for example, myogenic stem or progenitor cells) with gene-editing viral vectors, which is followed by cell culture and autologous transplantation back into the patient. Both treatment modalities can, in principle, be applied either locally or systemically. APCs antigen-presenting cells, HR homologous recombination, iPSCs induced pluripotent stem cells, NHEJ non-homologous end joining, rAAVs recombinant adeno-associated viruses