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In Alzheimer's disease, progressive supranuclear palsy, and a number of other neurodegenerative diseases, the microtubule associated protein tau aggregates to form intracellular neurofibrillary tangles and glial tangles, abnormal structures that are part of disease pathogenesis. Disorders with aggregated tau are called tauopathies. Presently, there are no disease-modifying treatments for this disease class. Tau is encoded by the MAPT gene. We propose that reducing MAPT expression and thus the amount of tau protein made could prevent aggregation, and potentially be an approach to treat tauopathies. We tested 31 morpholinos, complementary to the sense strand of the MAPT gene to identify oligonucleotides that can downregulate MAPT expression and reduce the amount of tau protein produced. Oligonucleotides were tested in human neuroblastoma cell lines SH-SY5Y and IMR32. We identified several morpholinos that reduced MAPT mRNA expression up to 50% and tau protein levels up to ~80%. The two most potent oligonucleotides spanned the 3′ boundary of exons 1 and 5, masking the 5′-splice sites of these exons. Both morpholinos induced skipping of the targeted exons. These in vitro findings were confirmed in mice transgenic for the entire human MAPT gene and that express human tau protein. These studies demonstrate the feasibility of using modified oligonucleotides to alter tau expression.

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and protooncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near protooncogene TSS before (n ‫؍‬ 157) and after (n ‫؍‬ 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of IntroductionHematopoietic stem cell (HSC) gene therapy has advanced to the point at which engraftment of gene-modified cells in large animal models has reached clinically therapeutic levels (Ͼ 10%-15%) for a variety of diseases (for review, see Neff et al 1 ). In addition, several significant clinical gene therapy trials have clearly demonstrated the potential of retrovirus gene-modified cells as an effective cure for the genetic diseases of X-linked severe combined immunodeficiency (SCID-X1), 2 adenosine deaminase-deficient severe combined immunodeficiency, 3 and X-linked chronic granulomatous disease (X-CGD). 4 These large animal and clinical studies are aided by either the use of ablative radiation or an inherent growth/survival advantage of the gene-modified cells after transplantation. For many other single-gene genetic diseases (ie, ␤-thalassemias and pyruvate kinase deficiency), acquired diseases (ie, glioblastoma and acute myeloid leukemia), or infectious diseases (ie, AIDS) in which retrovirus gene therapy is appropriate, a potential limiting curative factor is gene-marking levels below therapeutic thresholds due to the inability to use ablative conditioning and no substantial growth advantage/ in vivo selection of gene-modified cells (for reviews, see Neff et al 1 and Trobridge et al 5 ). This requires alternate in vivo selection strategies after transplantation to increase the percentage of gene-modified cells to therapeutic levels. To realize the full clinical potential of retrovirus gene therapy, safe in vivo selection strategies using drug resistance gene therapy will be critical to propel these and other promising gene transfer applications.The basic premise of drug resistance gene therapy is to stably deliver a transgene that confers resistance to a cytotoxic drug via several different mechanisms (for reviews, see Neff et al, 1 Trobridge et al, 5 and Sorrentino 6 ). Several selectable markers have been extensively evaluated because of their ability to protect cells from cytotoxic agents, including d...

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