Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.

Background and Purpose-Because fibroblast growth factor 2 is a mitogen for central nervous system stem cells, we explored whether long-term fibroblast growth factor 2 delivery to the brain can improve functional outcome and induce cortical neurogenesis after ischemia. Methods-Rats underwent permanent distal middle cerebral artery occlusion resulting in an ischemic injury limited to the cortex. We used an adeno-associated virus transfection system to induce long-term fibroblast growth factor 2 expression and monitored behavioral and histological changes. Results-Treatment increased the number of proliferating cells and improved motor behavior. Neurogenesis continued throughout 90 days after the ischemia, and the occurrence of newly generated cells with characteristics of neural precursors and immature neurons was most evident 90 days after treatment. Conclusions-Focal cortical ischemia elicits an ongoing neurogenic response that can be enhanced with fibroblast growth factor 2 leading to improved functional outcome. (Stroke. 2007;38:153-161.)

Tisagenlecleucel (Kymriah; Novartis Pharmaceuticals) is a CD19-directed genetically modified autologous T-cell immunotherapy. On August 30, 2017, the FDA approved tisagenlecleucel for treatment of patients up to 25 years of age with B-cell precursor acute lymphoblastic leukemia (ALL) that is refractory in second or later relapse. Approval was based on the complete remission (CR) rate, durability of CR, and minimal residual disease (MRD) <0.01% in a cohort of 63 children and young adults with relapsed or refractory ALL treated on a single-arm trial (CCTL019B2202). Treatment consisted of fludarabine and cyclophosphamide followed 2 to 14 days later by a single dose of tisagenlecleucel. The CR rate was 63% (95% confidence interval, 50%-75%), and all CRs had MRD <0.01%. With a median follow-up of 4.8 months, the median duration of response was not reached. Cytokine release syndrome (79%) and neurologic events (65%) were serious toxicities reported in the trial. With implementation of a Risk Evaluation and Mitigation Strategy, the benefit-risk profile was considered acceptable for this patient population with such resistant ALL. A study of safety with 15 years of follow-up is required as a condition of the approval. See related commentary by Geyer, p. 1133 Nonclinical Pharmacology and Toxicology Nonclinical safety studies were conducted with lentivirustransduced T cells prepared from healthy donors and patients in

Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotidedirected site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with >90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.

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R obust, accurate, and consistent testing methodologies are critical to the development of safe, highquality adeno-associated virus (AAV) vectors necessary to advance AAV-related clinical studies. AAV reference standard materials (AAV RSMs) can help ensure product safety by controlling the consistency of assays used to characterize AAV vectors used for gene therapy trials, especially critical lot release assays used for dose and potency measurements. Two AAV RSMs have recently been developed. 1,2 FDA encourages the use of the RSMs as benchmarking tools for qualifying inhouse reference materials and controls, and for demonstrating that assay methods are appropriately controlled. During investigational new drug and biologic license application review, the FDA evaluates the data submitted to support the suitability of assay methods. However, the FDA does not prescriptively define standardized assay methodology across the field or require that the values assigned to the rAAV2 or rAAV8 RSMs be duplicated during validation studies for in-house methods.We note that well-characterized and properly utilized AAV reference materials also have the potential to allow for the control and comparison of preclinical and clinical data across different laboratories. However, for greater utility of the RSMs, FDA recommends that the variability in the methods used to characterize the AAV RSMs 1,2 must be more thoroughly understood. References 1. Ayuso E, Blouin V, Lock M, et al. Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther 2014;25: 977-987. 2. Lock M, Mcgorray S, Auricchio A, et al. Characterization of a recombinant adeno-associated virus type 2 reference standard material.

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The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st (ϳ95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.Sendai virus is a prototypic paramyxovirus that replicates exclusively in the cytoplasm. The single strand negative-sense RNA genome of the Sendai virus encodes at least six genes, np, p, m, f, hn, and l (1). The p gene is transcribed into two polycistronic mRNAs, P/C and V/C (2). The polycistronic P/C mRNA is translated to synthesize the P, C, CЈ, Y1, and Y2 proteins from independent start sites in two overlapping reading frames. Although the C protein is expressed at levels comparable with P in infected cells (3), it is a minor component of virions (4). The other proteins, CЈ, Y1, and Y2 are expressed at relatively low levels in virus-infected cells (3). Although the P protein is required for viral transcription and replication (1, 5), the functional significance of the C proteins is not precisely defined. A recent study has suggested that the C protein may be involved in the regulation of viral transcription (6). To define further the functions of the P and C proteins, we have developed ribozymes to block the P/C mRNA expression in virusinfected cells.Ribozymes are RNA molecules with self-cleaving enzymatic activities (7,8). Ribozymes can be designed to act in trans to specifically cleave virtually any RNA molecule (9), making them particularly attractive as antiviral agents (10 -14) and as tools for studying gene function (15, 16). However, the factors that influence ribozyme and substrate interactions in vivo are not well defined. In many in vivo studies, very high ribozyme: substrate ratios were necessary ...