Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3-6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures-micronuclei and chromosome bridges-that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

Key Points Cas9 editing of the γ-globin gene promoters in hematopoietic stem cells (HSCs) increases red cell HbF by ≤40%. No deleterious effects on hematopoiesis or off-target mutations were detected 16 weeks after xenotransplantation of edited HSCs.

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors in response to several limitations inherent to the clinical design: 1) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; 2) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and 3) a possibly faster depletion of pathogenic myofibers in this patient population. Immune responses triggered by the first vector administration, and to subsequent ones, represent a major obstacle for successful gene transfer in young patient population. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or ERT treatment for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. This study presents a case of Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate the immune responses. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

Summary Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation.

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Enzyme and gene replacement strategies have developed into viable therapeutic approaches for the treatment of Pompe disease (acid α-glucosidase (GAA) deficiency). Unfortunately, the introduction of GAA and viral vectors encoding the enzyme can lead to detrimental immune responses that attenuate treatment benefits and can impact patient safety. Preclinical and clinical experience in addressing humoral responses toward enzyme and gene therapy for Pompe disease have provided greater understanding of the immunological consequences of the provided therapy. B- and T-cell modulation has been shown to be effective in preventing infusion-associated reactions during enzyme replacement therapy in patients and has shown similar success in the context of gene therapy. Additional techniques to induce humoral tolerance for Pompe disease have been the targeted expression or delivery of GAA to discrete cell types or tissues such as the gut-associated lymphoid tissues, red blood cells, hematopoietic stem cells, and the liver. Research into overcoming preexisting immunity through immunomodulation and gene transfer are becoming increasingly important to achieve long-term efficacy. This review highlights the advances in therapies as well as the improved understanding of the molecular mechanisms involved in the humoral immune response with emphasis on methods employed to overcome responses associated with enzyme and gene therapies for Pompe disease.

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa−/−) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.

Enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (GAA), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of Gaa mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.