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Androgens stimulate neuronal differentiation of PC12 cells by nontranscriptional action of the endogenous androgen receptor (AR). AR is also required for NGF-induced neuritogenesis of PC12. Androgens or NGF trigger AR association with filamin and TrkA, TrkA interaction with PI3-K δ, and activation of PI3-K δ and Rac., Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in 24 patients out of 34 (71%) was predicted by the type of mutations. Preexisting Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.

Enzyme replacement therapy (ERT) is the current standard of care for Mucopolysaccharidosis type VI (MPS VI) that is caused by deficiency of arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans (GAGs). However, ERT is associated with inconvenient multiple and costly administrations and fails to ameliorate cardiac, visual and bone abnormalities.To overcome ERT limitations, we developed a successful gene therapy approach based on a single administration of AAV2/8 that targets liver of MPS VI animal models. Importantly, we showed that a single systemic administration of AAV2/8 at high doses (2×10e12 gc/kg) is at least as effective as the current ERT therapeutic regimen based on weekly infusions of recombinant human ARSB (rhARSB). If this translates to humans, gene therapy could replace ERT for MPS VI. However, the administration of high doses of AAV2/8 requires a challenging and costly production process, and results in cell-mediated immune responses that eliminate transduced hepatocytes. Similarly, weekly ERT infusions are costly and require high patient compliance.We therefore evaluated in a mouse model of MPS VI whether the combination of low doses of AAV2/8 at 6×10e11 or 2×10e11 gc/kg with a rarified ERT schedule (1mg/kg once a month) may be as effective as the single treatments at high doses or frequent regimen.Significant increase of ARSB activity was found in liver of all treated mice. Detectable but low activity was variably observed in spleen and kidney and was associated with significant reduction of tissue GAGs, regardless of treatment and ARSB activity levels, similarly to what observed in mice treated with high doses of AAV2/8 or weekly ERT. This supports previous data indicating that low enzymatic levels improve MPS VI visceral phenotype. Evaluation of GAG storage in myocardium and heart valves is in progress.Urinary GAG, which are a sensitive biomarker of systemic clearance of lysosomal storage and, thus of therapeutic efficacy, are slightly reduced in mice treated with either monthly ERT or 2×10e11 gc/kg of AAV2/8. The reduction is more consistent at 6×10e11 gc/kg. Importantly, urinary GAG decreased more in mice receiving the combined therapy than in those receiving single treatments. In particular, urinary GAG reduction in mice treated with both 6×10e11 gc/kg of AAV2/8 and monthly ERT was comparable to that obtained following administration of either high doses of AAV2/8, i.e 2×10e12 gc/kg, or weekly ERT.The data collected so far show similar efficacy between low-dose gene therapy combined with rarified ERT and the corresponding single treatments at high doses or frequent regimen. This should increase the safety and reduce the risks and costs associated with both therapeutic approaches.

Hereditary spastic paraplegia (HSP) is characterized by the specific retrograde degeneration of the longest axons in the central nervous system, the corticospinal tracts. The gene most frequently involved in autosomal dominant cases of this disease, SPG4, encodes spastin, an ATPase belonging to the AAA family. AAA proteins are thought to exert their function by the energy-dependent rearrangement of protein complexes. The composite function of these proteins is directed by their binding to regulatory factors and adaptor proteins that target their activity into specific pathways in vivo. We previously found that overexpressed spastin interacts dynamically with microtubules and displays microtubule-severing activity. Here, we demonstrate that spastin is enriched in cell regions containing dynamic microtubules. During cell division spastin is found in the spindle pole, the central spindle and the midbody, whereas in immortalized motoneurons it is enriched in the distal axon and the branching points. Furthermore, spastin interacts with the centrosomal protein NA14, and co-fractionates with gamma-tubulin, a centrosomal marker. Deletion of the region required for binding to NA14 disrupts spastin interaction with microtubules, suggesting that NA14 may be an important adaptor to target spastin activity at the centrosome. These data strongly argue that spastin plays a role in cytoskeletal rearrangements and dynamics, and provide an attractive explanation for the degeneration of motor axons in HSP.

Enzyme replacement therapy (ERT) has become the standard of care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSD manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI, we compared the efficacy of a single intravascular administration of an adeno-associated viral vector targeting liver to weekly infusions of human recombinant enzyme at the same doses used in mucopolysaccharidosis VI patients. While gene therapy results in increased and stable levels of circulating enzyme up to 1 year after vector administration, ERT has typical peak-and-drop serum kinetics. Both therapies similarly reduced glycosaminoglycan levels in urine and tissues including heart valves and myocardium, with gene therapy improving skeletal skull abnormalities slightly better, although not significantly, than ERT. Both therapies seem to similarly improve animal motor performance, with gene therapy possibly associated with less animal distress. Thus, a single vector administration that converts liver into a factory organ for systemic secretion of therapeutic proteins is at least as effective as ERT in a mouse model of LSD, potentially eliminating problems with compliance and costs. Only testing in humans will prove whether this holds true in a clinical setting.

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and the ability to differentiate into specific cell types. We performed the first genome-wide analysis of the mouse ES cell transcriptome using ∼250,000 gene trap sequence tags deposited in public databases. We unveiled >8000 novel transcripts, mostly non-coding, and >1000 novel alternative and often tissue-specific exons of known genes. Experimental verification of the expression of these genes and exons by RT-PCR yielded a 70% validation rate. A novel non-coding transcript within the set studied showed a highly specific pattern of expression by in situ hybridization. Our analysis also shows that the genome presents gene trapping hotspots, which correspond to 383 known and 87 novel genes. These “hypertrapped” genes show minimal overlap with previously published expression profiles of ES cells; however, we prove by real-time PCR that they are highly expressed in this cell type, thus potentially contributing to the phenotype of ES cells. Although gene trapping was initially devised as an insertional mutagenesis technique, our study demonstrates its impact on the discovery of a substantial and unprecedented portion of the transcriptome.

Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Pre-existing immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for Mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder due to arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. To the best of our knowledge, four mutations had not been previously described. These include: one missense (c.1178 A>G p.H393R) and three frameshift mutations [883-884duplTT (p.F295FfsX42), c.1036delG (p.E346SfsX11), c.1475delC (pP492LfsX80)] predicted to result in truncated proteins. The detection of ARSB protein in twenty-four patients out of 34 (71%) was predicted by the type of mutations. Pre-existing Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.

Background: All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development. Results: We used a unique combination of tools to obtain regional global-local alignments of orthologous loci. This approach takes into account shuffling of regulatory regions that are likely to occur over evolutionary distances greater than those separating mammalian genomes. This approach revealed one order of magnitude more vertebrate conserved elements than was previously reported in over 2,000 genes, including a high number of genes found in the membrane and extracellular regions. Our analysis revealed that 72% of the elements identified have undergone shuffling. We tested the ability of the elements identified to enhance transcription in zebrafish embryos and compared their activity with a set of control fragments. We found that more than 80% of the elements tested were able to enhance transcription significantly, prevalently in a tissuerestricted manner corresponding to the expression domain of the neighboring gene. Conclusion: Our work elucidates the importance of shuffling in the detection of cis-regulatory elements. It also elucidates how similarities across the vertebrate lineage, which go well beyond development, can be explained not only within the realm of coding genes but also in that of the sequences that ultimately govern their expression.