Treatment of Neurological Dysfunction in MPS VII and Batten Disease by Transplantation of Lentivirally Transduced Neuronal Stem Cells Cultured from Hematopoietic Tissue.
Authors: Soper Brian W. , Lessard Mark D. , Alley Travis L. , Proctor Jennifer L. , Mourino Anthony J. , Barker Jane E. 
Published: April 2020

The subventricular zone (SVZ) of lateral ventricles in brain contains neuronal stem cells (NSC) that form neurospheres when cultured with EGF and/or bFGF. Progeny from transplanted NSC migrate throughout the brain and replace multiple differentiated cells. Our goal is to develop this phenomenon into a cellular/gene therapeutic approach for treatment of neurological disease. Progeny of normal or gene transduced NSC can replace defective host cells or act as enzyme delivery vehicles. One model for testing this approach is the Mucopolysaccharidosis Type VII (MPS VII) mouse that is deficient in β-glucuronidase (GUS) expression, causing lysosomal storage disease (Sly Syndrome). Undegraded substrates accumulate in brain, causing cognitive dysfunction. A second model is deficient in palmitoyl-protein thioesterase 1 (PPT1), causing neurodegeneration in humans and mice known as infantile neuronal ceroid lipofuscinosis, or Battens Disease. By 24–30wks of age, mice develop motor abnormalities and seizures. Obstacles to NSC therapy include suitable source of donor cells and immunological rejection. Since mesenchymal stem cells (MSC) derived from bone marrow and cord blood have neuronal differentiation capacity, we believe these tissues can generate NSC. If so, self bone marrow or cord blood derived NSC could be transduced with a lentivirus that restores enzyme expression, solving both obstacles. We cultured 15d fetal liver (the murine equivalent of cord blood) from eGFP transgenic mice in neurosphere medium containing EGF and bFGF +/− Noggin/Fc. Noggin is an antagonist for bone morphogenic protein, is expressed in ependymal cells of the SVZ, upregulates neurogenesis, and inhibits bone formation. Growth factors were added days 1–5, and alternating days thereafter. Half the media was replaced every 4d. At 14d, the +Noggin culture had 2–3x the confluency of the -Noggin culture and contained spindle-shaped cells resembling MSC. After another 14d with EGF and bFGF alone, the +Noggin cells formed neurospheres. Neurospheres reformed after trituration, indicating self-renewal capacity. The neurospheres were Nestin+, a marker for NSC, and upon differentiation with serum, β-NGF, BDNF, and NT-3, stained positive for neurons (MAP2) and astrocytes (GFAP). Next, we transplanted 250,000 normal eGFP fetal liver derived NSC (FL NSC) into the lateral ventricles of PPT1−/ − neonatal recipients. Brains 18–54 days post transplant revealed cells in the SVZ of the lateral, dorsolateral, and third ventricles. Donor cells migrated away from the ventricles, into the hippocampal fimbria, under the corpus callosum, and into the rostral migratory stream. Later time points revealed increased migration away from ventricles. We also tested transduction of NSC from MPS VII fetal brain using a lentiviral SIN vector driving human GUS from a PGK promoter. Transduction efficiency was 88–90%. Transplantation of 250,000 transduced NSC into neonatal MPS VII recipients revealed a similar pattern of transplantation as described above for FL NSC. Donor GUS+ cells were detected 4mo post transplant, indicating long-term gene expression and donor cell survival. In conclusion, NSC are efficiently transduced with lentivirus, can be cultured from hematopoietic tissue, and engraft long-term following neonatal injection. These results demonstrate a method to circumvent both donor cell availability and immune barriers to transplantation, providing hope for patients with devastating neurological disorders.