Can early treatment of twitcher mice with high dose AAVrh10-GALC eliminate the need for BMT?

Abstract

Introduction: Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone. Methods: Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 1013 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes. Results: When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice. Conclusion: Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT.

Substrate Reduction Therapy for Krabbe Disease: Exploring the Repurposing of the Antibiotic D-Cycloserine

“…For instance, HSCT has been combined with SRT and/or gene therapy. The combinations consistently yield better outcomes than the matched individual therapies ( 22 , 24 , 25 , 36 ). Other approaches such as ERT, chaperone therapy, and anti-inflammatory therapy also have the potential to contribute as part of combination or standalone therapies.…”

“…The obstacles include achieving insufficient levels of the corrected enzyme, the blood-brain barrier limiting the delivery of corrected enzyme or viral vector to the CNS, the development of various adverse side effects including some of which are severe, etc. Despite these obstacles, there has been steady progress toward the goal of developing therapies that have increased efficacy, e.g., lengthening of lifespan ( 22 – 25 ).…”

“…HSCT (inclusive of bone marrow transplantation) was shown in 1984 to increase the lifespan of twitcher mice ( 26 ). A key mechanism accounting for this benefit is the delivery of functional enzyme by the progeny of transplanted cells ( 25 , 27 , 28 ). Additional mechanisms include reducing inflammation ( 29 ).…”

“…Interestingly, neither HSCT nor L-cycloserine by themselves have been found to cause hepatocellular carcinoma in treated mice ( 24 ). Other pretreatment regimens for HSCT (e.g., busulfan, cyclophosphamide) can result in serious adverse effects such as infertility and graft vs. host disease ( 25 ).…”

Advances in the Diagnosis and Treatment of Krabbe Disease

“…In addition to greatly extending the lives of treated mice, there was maintenance of body weight and strength, normal myelination in the brain and sciatic nerves, and elimination of the tremor associated with twi mice. More recent studies in twi mice have shown that BMT may not be required if high dose AAVrh10-GALC is given iv early in life [59]. This treatment eliminates some of the side effects of the drugs needed before BMT.…”