Pathophysiology and Treatment of Murine Globoid Cell Leukodystrophy

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a rapidly progressing, invariably fatal pediatric disorder first described in 1916. Krabbe disease is caused by a deficiency in the lysosomal enzyme, galactosylceramidase (GALC), and is characterized clinically by failure to thrive, limb stiffness, seizures, developmental regression, and death by 2-4 years of age. Galactosylceramidase degrades the cytotoxic glycolipid, galactosylsphingosine (psychosine). In the absence of GALC activity, psychosine accumulates primarily in oligodendrocytes and Schwann cells, resulting in profound demyelination. In 1972, psychosine was hypothesized to be responsible for the clinical signs associated with Krabbe disease. However, the ‘Psychosine Hypothesis’ has never been tested, due to the inability to dissociate GALC deficiency from psychosine accumulation. This is due, in part, to a limited understanding of psychosine biosynthesis. Two studies published in 1960 and 1973 provided complementary evidence suggesting that psychosine is synthesized via an anabolic pathway. However, neither the cDNA nor the enzyme catalyzing that reaction has been identified. In the first part of this dissertation, we overturn those studies and show that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction effectively dissociates GALC deficiency from psychosine accumulation, allowing us to test and confirm the ‘Psychosine Hypothesis. ’ These data also identify ACDase as a potential target for substrate reduction therapy (SRT). We show that pharmacological inhibition of ACDase activity significantly prolongs the lifespan of the Twitcher (Twi) mouse, a GALC-deficient model that faithfully mimics the biochemical, histological, and clinicobehavioral features of Krabbe disease. These data clarify our understanding of psychosine synthesis, confirm the long-held ‘Psychosine Hypothesis,’ and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease. Although Krabbe disease is a monogenic disorder, it is remarkably refractory to treatment, and single modality therapies are minimally effective. However, combining CNS-directed, adenoassociated virus (AAV) 2/5-mediated gene therapy, hematopoietic stem cell transplantation, and SRT greatly increases efficacy in the Twi mouse. In the second part of this dissertation, we incorporated a newer generation vector, AAV2/9, into this combination therapy regimen. This single change significantly increased the lifespan of Twi mice. Importantly, it also dramatically improved, and in some cases, normalized, the clinicobehavioral deficits that remained uncorrected in mice treated with the AAV2/5-combination therapy. Unfortunately, nearly all of the AAV2/9- combination-treated Twi mice and all combination-treated wild type control mice died from hepatocellular carcinoma (HCC). Integration site analysis confirmed AAV sequence incorporation into the mouse genome. These data demonstrate the value of targeting multiple pathogenic mechanisms for complex metabolic diseases, but highlight the potential risks associated with these approaches. Taken together, the data presented in this dissertation greatly advance the field of Krabbe research by increasing our fundamental understanding of Krabbe pathogenesis, and by making significant progress in the treatment of this fatal disease. Modification of the combination therapy regimen to include a safe, ACDase-inhibiting SRT to target psychosine synthesis, as well as a liver de-targeted gene therapy to minimize HCC penetrance will likely bring us even closer to a cure

Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications

Enhanced efficacy and increased long-term toxicity of CNS-directed, AAV-based combination therapy for Krabbe disease

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects: BMT-Lentiviral Vectors Therapy for Krabbe's Disease

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)‐induced central nervous system damage. However, all HSPCT‐treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor‐derived cells and 2) insufficient uptake of donor cell‐secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation‐independent mannose 6‐phosphate‐receptor (CI‐MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu‐mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell‐specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT‐mediated correction of GALC deficiency in target cells expressing low levels of CI‐MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose‐6‐phosphate‐independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc

Caspase-9: A Candidate Susceptibility Factor for Murine Alkylator-induced Leukemia

Mentor: Timothy A. Graubert From the Washington University Undergraduate Research Digest: WUURD, Volume 5, Issue 2, Spring 2010. Published by the Office of Undergraduate Research. Henry Biggs, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator, Co-editor, and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

Caspase-9: A Candidate Susceptibility Factor in Murine Alkylator-induced Therapy-related Acute Myeloid Leukemia

From the Washington University Senior Honors Thesis Abstracts (WUSHTA), Volume 2, Spring 2010. Published by the Office of Undergraduate Research. Henry Biggs, Director, Office of Undergraduate Research / Associate Dean, College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator / Assistant Dean in the College of Arts & Sciences; E. Holly Tasker, Editor. Mentor: Timothy A. Grauber