Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of approximately 2 percent. A pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4. A mutation was identified in the α-synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred and in three unrelated families of Greek origin with autosomal dominant inheritance for the PD phenotype. This finding of a specific molecular alteration associated with PD will facilitate the detailed understanding of the pathophysiology of the disorder.

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/ matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34 + cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

CM, Langston JW (1998a) Absence of mutations in the coding region of the alpha-synuclein gene in pathologically proven Parkinson's disease. Neurology 50:1136-1137 Chan P, Tanner CM, Jiang X, Langston JW (1998b) Failure to find the alpha-synuclein gene missense mutation (G209A) in 100 patients with younger onset Parkinson's disease. Neurology 50:513-514 Letters to the Editor

In patients with HTPR after PCI, prasugrel is more effective compared with high clopidogrel in reducing platelet reactivity, particularly in CYP2C19*2 carriers. Genotyping guidance might be helpful only in case an increased clopidogrel maintenance dose is considered. (Prasugrel Versus High Dose Clopidogrel in Clopidogrel Resistant Patients Post Percutaneous Coronary Intervention (PCI); NCT01109784).

The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

No abstract

Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34 + haematopoietic cells, but only transiently.To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34 + cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34 + /eGFP + cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP + -colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells.The design and use of extrachromosomal vectors, suitable for efficient and stable transfection of haematopoietic progenitor cells, is an important goal for the gene therapy of haemoglobinopathies. The development of extrachromosomal vectors has mainly been driven by the need to address the safety issue of gene therapy vectors, in particular, the problem of insertional mutagenesis 1 , and involves vectors such as self-replicating stable episomes 2 , pFARs-plasmids free of antibiotic resistance markers 3 , and minicircle DNA plasmid derivatives lacking a bacterial backbone 4 . The presence of the scaffold/matrix attachment region (S/MAR) also confers long-term mitotic stability to integration-deficient lentiviral, episomal vectors 5,6 ; however, that of the truncated S/MAR does not improve episomal retention 7 .Key issues in the development of episomal vectors are currently the establishment in the host nucleus 8,9 , the transgene expression 10,11 and the delivery in progenitor cells 10 . The prototype episomal vector pEPI-1 2 does not code for any viral protein, and it contains the S/MAR from the 5′ end of the human β -interferon gene 2 , an element that facilitates the vector's nuclear retention.The S/MARs are AT rich chromosomal elements that play a role in chromatin boundary formation 12 and bind to SAF-A protein 13 , mediating the tethering of pEPI-1 plasmid to the nuclear matrix. A prerequisite for

This study may help to inform clinical trials and provide the basis for studies of disease modifiers in genetic synucleinopathy cohorts. © 2016 International Parkinson and Movement Disorder Society.