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Columbus Children's Research Institute - Ohio State University, Dr. Haiyan Fu

AAV-mediated Gene Therapy for Neurological Disease of MPS III
Using a Knock-out Mouse Model

Mucopolysaccharidosis (MPS) IIIB (Sanfilippo B) is an inherited lysosomal storage disorder. This genetic defect leads to the absence of a lysosomal enzyme, a-N-acetylglucosaminidase (NaGlu), which results in the accumulation of a glycosaminoglycan (GAG) in cells. We have conducted gene therapy studies on MPS IIIB in a mouse model, with the goal of developing treatments for the neurological disorders of this disease, and saving the lives of the afflicted children.

We have developed effective methods for delivering reagents which we hope to use to treat the neurological disorders in MPS IIIB patients. As part of our research, we have made an adeno-associated viral (AAV) vector for use as a vehicle to carry genes into cells. This AAV vector contains the normal human NaGlu gene, which is defective in MPS IIIB patients. The vector can produce NaGlu (the enzyme missing in MPS IIIB patients) and leads to the breakdown of the accumulated GAG in cultured MPS IIIB cells and the MPS IIIB mouse brain.

However, the most critical challenge in developing treatment for the CNS diseases in MPS is how to deliver therapeutic reagents (AAV vector) to the whole CNS or broad areas of the brain, and not to just a localized area. The major obstacle is the presence of the blood-brain barrier (BBB), which prevents large molecules, such as AAV vector and NaGlu enzyme, from entering the CNS tissues. It is worth noting that the BBB is closed at birth in humans, but not closed until 21 days of age in mice. Therefore, it has been important to test any prospective treatment in adult mice in order to be applicable to even the youngest of patients, whose BBB is already well formed. Our research efforts have therefore been focused on two major aspects of gene therapy for the treatment of MPS IIIB (Sanfilippo B).

First, we have been developing methods to achieve widespread distribution of the AAV gene delivery vectors in the CNS tissues of adult mice. Second, we have been assessing the therapeutic impacts of AAV vector carrying the NaGlu gene on CNS disorders in the MPS IIIB mouse model.

In our previous studies, we developed two non-surgical approaches to deliver AAV vectors into the CNS of adult mice. We established an intravenous (IV) injection procedure that achieved a global distribution of AAV vector throughout mouse CNS, by pretreatment using an IV infusion of mannitol to temporarily disrupt the blood-brain barrier (BBB). We also demonstrated a broad spread of AAV vector in the CNS by an intracisternal (IC) injection, delivering AAV vector into the cerebral spinal fluid space. These results offered us more effective means to deliver AAV vectors into the CNS for MPS IIIB therapy.

Further, we used the above procedures in AAV gene therapy studies in adult MPS IIIB mice. The mice were treated with AAV2-hNaGlu vector by an IV injection, an IC injection, or a combination of IV and IC injection. The AAV vector resulted in the production of NaGlu enzyme in the brains of all the IC and IV+IC-injected MPS IIIB mice. The NaGlu enzyme subsequently decreased pathological lysosomal storage in these treated MPS IIIB mouse brains. The IV vector injection lead to the clearance of lysosomal storage in liver, and partial correction of storage in other somatic tissues.

Most importantly, these treatments significantly prolonged the lifespan of MPS IIIB mice to 9.2-15.9 months (IV injected), 10.3-21.5 months (IC injected), and 11.1-19.5 months (IV+IC injected), while non-treated MPS IIIB mice only lived 7.9-11.0 months. Normal mice live approximately 2.5 years. Furthermore, IC and IV+IC injection of AAV vector also significantly improved the behavioral performance, especially learning ability, of MPS IIIB mice. These results suggest that the AAV gene therapy procedures in this study greatly slowed down the progress of the CNS disease in MPS IIIB mice, though they did not effect a complete cure. The data gained in these studies showed the great potential for using AAV gene therapy to treat MPS IIIB.

In addition, we considered the relevant factors for future human application in designing the animal experiments in our gene therapy studies. As mentioned above, both IV and IC injection are non-surgical and routine medical procedures used in humans. The IV infusion of mannitol is also a routine medical practice. We calculated the amount of mannitol and the volumes of injections based on dosages commonly used in patients. Furthermore, the therapeutic effect of these treatments is long-term, we may not need to repeatedly conduct the treatment for MPS IIIB patients. Currently, the IC injection and the combined IV + IC injection of AAV vector are more efficient than IV only for treating MPS IIIB. We are continuing our efforts to improve the efficacy of AAV gene therapy for the CNS disorders of MPS IIIB, by optimizing the vector injection approaches as well as testing them in larger animals, and enhancing the enzymatic functions of AAV-expressed NaGlu to maximize its therapeutic effect.

Although we are continuing our research to improve the therapeutic efficacy of AAV gene therapy for MPS IIIB, we feel that we have a therapy in hand that provides meaningful benefits in our MPS IIIB animal model. We feel that the time has come to consider translating our AAV gene therapy procedure from mouse studies into human clinical applications. We are planning to apply for an IND approval from the FDA for a clinical trial in MPS IIIB patients, using the AAV gene therapy vectors and procedures.

At the same time, we are continuing our efforts toward optimizing the efficacy of AAV gene therapy in the treatment of neurological diseases using the mouse model. To achieve theses goals, public support is essential. In addition, it is important to note that many neurological diseases share the same requirement for the delivery of therapeutic materials to the CNS tissues. Although our first concern is finding a cure for MPS IIIB, the delivery procedures that we have developed may also have great potential in treating many other neurological diseases.