Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins. Keywords: aggresome, Huntington's disease, p62, polyglutamine, proteasome inhibitor, RNA interference. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with midlife onset that gives rise to progressive, selective neural cell death in the striatum associated with choreic movement and dementia (Vonsattel and DiFiglia 1998). The disease is associated with an unstable expansion of CAG repeats within the coding region of the gene encoding the protein huntingtin. Whereas wildtype chromosomes with a stable CAG repeat possess 6-34 repeats, more than 36 repeats result in an unstable, expanded, disease-associated allele. The mutation in huntingtin produces an expanded stretch of glutamine residues, and the mutant huntingtin aggregates with ubiquitylation, forming neuronal nuclear aggregates or inclusions and dystrophic neuritic inclusions in the HD cortex and striatum. Many observations have suggested that abnormal accumulation of the mutant protein is involved in pathogenesis of various neurodegenerative disorders, including polyglutamine diseases such as HD, by conferring a toxic gain of function (Zoghbi and Orr 2000).How inclusions contribute to altered cell function is not well understood but they could have a variety of effects on the regulation of gene transcription, protein interactions, and protein transport within the nucleus and cytoplasm. Various proteins, for example, ubiquitin, molecular chaperones, and components of the proteasome, co-localize with mutant proteins in the inclusions, which may result in the modification of important cellular functions. Recent studies suggest that cells avoid accumulating potentially toxic aggregates by using molecular chaperones to suppress aggregate formation and by using proteasomes to degrade misfolded proteins. This hypothesis is supported by the fact that chaperone Addres...

Background Allogeneic bone marrow transplant (BMT) is the only curative method for a number of monogenic blood disorders, including various forms of hemoglobinopathies and severe combined immunodeficiencies. Aside from the significant hurdle of finding an identical HLA-matched related donor, allogeneic BMT recipients require chronic immunosuppression to mitigate the significant risk of GVHD and are at greater risk for graft failure. Autologous gene modified HSPC potentially provide a much safer alternative to allogeneic BMT and abrogate the need for finding HLA-matched donors. Here, we report the development of a highly efficient process for generating gene modified human HSPC at clinical-scale (>150 x 10^6 cells for 2x10^6/kg) using clinical-grade equipment. Methods Healthy donors were administered Neupogen® (10mg/kg/day) for 4-5 consecutive days and then apheresed. Enrichment of CD34+ cells was performed with the Miltenyi CliniMACS® system. Genome editing was achieved via the introduction of mRNA encoding two engineered zinc finger nucleases (ZFN) using a scalable electroporation device. Cells were harvested and cryo-preserved with a controlled-rate freezer. Cell recovery and viability, gene modification efficiency, stem cell pluripotency and engraftment potential were evaluated by in vitro assays and in a humanized NSG mouse model. Results Enrichment of CD34+ cells from mobilized leukopak products was highly efficient with the Miltenyi CliniMACS® system (median recovery = 327 million CD34+ cells per 10L mobilized leukopak). The positively selected fractions were >98% CD34+ by FACS analysis. Two large scale electroporation devices (BTX AgilePulse Max® and MaxCyte GT®) were evaluated. Each device is capable of electroporating up to 300 million cells. The optimal transfection conditions for both devices were first identified by using a GFP mRNA to evaluate transfection efficiency by flow cytometry, which resulted in the identification of conditions (voltage and duration) that yielded gene transfer efficiencies of >90%. Compatibility of this protocol with driving endogenous gene modification was evaluated using mRNA encoding ZFNs that target various endogenous gene loci. Highly efficient levels of genome editing were observed in CD34+ HSPC each transfected with a different pair of ZFNs (median = 53%, 43%, 45% and 42% modified alleles at four distinct disease-relevant loci). At the optimal mRNA dose for each ZFN pair, cell viability post electroporation was >80%, comparable to untransfected controls. Process suitability was evaluated by in vitro colony forming cell assay. No significant differences in colony formation were observed between gene modified and untransfected control samples. The capacity of electroporated HSPC to engraft and support multi-lineage development of human hematopoietic cells was evaluated in NSG mice, and no differences were observed between the ZFN-treated and untransfected control cells. In addition, high levels of gene modification (19-28%) were detected in bulk human cells from the blood and tissues of engrafted mice, and in various sorted cell types (bone marrow CD34 and differentiated B and T cells). Conclusion We have developed a scalable process capable of deriving >300 million gene-modified CD34+ HSPC. This process supports high levels of ZFN-driven genome editing, is well tolerated, and causes no discernable defect in the hematopoietic potential of these cells to develop into multiple cell lineages, with high gene editing levels maintained in the differentiated progeny of the HSPC. These results support the use of gene modified autologus HSPC for the treatment of monogenic blood disorders. Disclosures: Lee: Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Ya-Li:Sangamo BioSciences: Employment. Kim:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Wang:Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Ando:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment.