Modified neural stem cells for glycosylation-independent treatment of MPS IIIB

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 DESCRIPTION (provided by applicant): The world dreams of curing every disease with stem cells. We all know this is unlikely. But the main barrier to using stem cells to regenerate tissues and treat diseases is that stem cells that are not genetically identical to the recipient's cells ae prone to immune rejection. An innovative solution to overcome this barrier uses the patient's own somatic cells to generate stem cells called iPSCs (induced pluripotent stem cells). Once generated, these iPSCs can be administered to the patient without fear of rejection because iPSCs are genetically identical to all other cells in the patient. However, administering iPSCs is not a solution for most inherited diseases because the generated iPSCs also contain the disease-causing genetic defect. We have assembled a team with expertise in stem cell biology, neurotherapeutics, translational science, and neuropharmacology to solve this problem. We propose to use a new technology called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) to repair the faulty gene in the recipient's stem cells. CRISPR-Cas9 is the most efficient gene correction system to date. Our approach will use CRISPR-Cas9 to correct the genetic defect in iPSCs isolated from patients. The corrected stem cells will be transplanted back to the patient to provide cells that have a functioning copy of the gene. We propose to test this concept using neural stem cells to treat the inherited childhood neurological disease mucopolysaccharidosis type IIIB (MPS IIIB). Gene-corrected, autologous stem cell therapy is ideally suited to diseases like MPS IIIB, which are due to lack of a secreted protein (in this case, NAGLU [alpha-N-acetylglucosaminidase]), because a very small number of corrected stem cells can have a large therapeutic effect. We will also employ glycosylation-independent lysosomal targeting using a peptide derived from IGF2 that improves the uptake of secreted NAGLU into the brain. In the R21 phase of this proposal, we will perform in vitro studies to determine how effectively our corrected stem cells secrete normal NAGLU and NAGLU-IGF2 that can be taken up by MPS IIIB cells. We will also perform in vivo studies to determine how well the neural stem cells distribute throughout the brain at three different doses, in neonatal and adult animals. In the R33 phase of the proposal, we will put these concepts together for in vivo studies of the effect of corrected neural stem cell on MPS IIIB disease. We will assess biochemical, neuropathological, and neurobehavioral outcomes. The goal is to create a permanent treatment for this devastating, untreatable disease, and to build a platform for treating other genetic diseases that are caused by the lack of a functioning, secreted protein.