Recent Patents on Endocrine, Metabolic & Immune Drug Discovery, 2012
During the last 20 years, transgenic constructs based on adeno associated virus (AAV) have been t... more During the last 20 years, transgenic constructs based on adeno associated virus (AAV) have been tested in disease models and proved their efficacy to revert a wide range of pathologies without major side effects. Based on these results, up to 20 clinical trials have been set up to prove therapeutic effect of AAV vectors on neurodegenerative diseases, retinopathies and neuromuscular diseases, among others. It has been shown that AAV vectors support localized long-term, gene expression in the central nervous system, and that restoration of visual function can be achieved in Leber's congenital amaurosis retinopathy. The clinical trials also highlighted new challenges for AAV mediated gene transfer, such as the circumvention of T-cell response to transduced cells. Currently, miniaturized and codon-optimized transgenes, exon skipping cassettes, novel tissue-specific promoters and vector chimeras with tissue-selective tropism are being tested to improve the efficiency and safety of transgene delivery, as required to meet pharmaceutical industry standards. The aim of this review is to revise the latest patents and news on AAV vectors, in order to summarize the state of the art and the potential issues that still need to be faced by pharmaceutical companies for successful gene transfer and commercialization of AAV-based drugs.
Proceedings of the National Academy of Sciences, 2008
Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes ( GJB2 and GJB6 , respectively... more Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes ( GJB2 and GJB6 , respectively) that are found within 50 kb in the same complex deafness locus, DFNB1. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. To explore connexin coregulation, we manipulated gene expression using the bovine adeno-associated virus. Overexpression of Cx30 in the Cx30 KO mouse by transduction with bovine adeno-associated virus restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca 2+ signals. Ablation of Cx26 by transduction of Cx26 loxP/loxP cultures with a Cre recombinase vector caused concurrent downregulation of Cx30 and impaired intercellular communication. The coordinated regulation of Cx26 and Cx30 expression appears to occur as a result of signal...
In this study, we demonstrate that the in vitro interactions between a CD56neg/CD16pos (CD56neg) ... more In this study, we demonstrate that the in vitro interactions between a CD56neg/CD16pos (CD56neg) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1–infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK–DC activation and maturation as well as a defect in the NK cell–mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56neg NK cell subset, largely accounts for the ...
Proceedings of the National Academy of Sciences, 2007
Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened ... more Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.
In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, lim... more In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.
Recent Patents on Endocrine, Metabolic & Immune Drug Discovery, 2012
During the last 20 years, transgenic constructs based on adeno associated virus (AAV) have been t... more During the last 20 years, transgenic constructs based on adeno associated virus (AAV) have been tested in disease models and proved their efficacy to revert a wide range of pathologies without major side effects. Based on these results, up to 20 clinical trials have been set up to prove therapeutic effect of AAV vectors on neurodegenerative diseases, retinopathies and neuromuscular diseases, among others. It has been shown that AAV vectors support localized long-term, gene expression in the central nervous system, and that restoration of visual function can be achieved in Leber's congenital amaurosis retinopathy. The clinical trials also highlighted new challenges for AAV mediated gene transfer, such as the circumvention of T-cell response to transduced cells. Currently, miniaturized and codon-optimized transgenes, exon skipping cassettes, novel tissue-specific promoters and vector chimeras with tissue-selective tropism are being tested to improve the efficiency and safety of transgene delivery, as required to meet pharmaceutical industry standards. The aim of this review is to revise the latest patents and news on AAV vectors, in order to summarize the state of the art and the potential issues that still need to be faced by pharmaceutical companies for successful gene transfer and commercialization of AAV-based drugs.
Proceedings of the National Academy of Sciences, 2008
Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes ( GJB2 and GJB6 , respectively... more Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes ( GJB2 and GJB6 , respectively) that are found within 50 kb in the same complex deafness locus, DFNB1. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. To explore connexin coregulation, we manipulated gene expression using the bovine adeno-associated virus. Overexpression of Cx30 in the Cx30 KO mouse by transduction with bovine adeno-associated virus restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca 2+ signals. Ablation of Cx26 by transduction of Cx26 loxP/loxP cultures with a Cre recombinase vector caused concurrent downregulation of Cx30 and impaired intercellular communication. The coordinated regulation of Cx26 and Cx30 expression appears to occur as a result of signal...
In this study, we demonstrate that the in vitro interactions between a CD56neg/CD16pos (CD56neg) ... more In this study, we demonstrate that the in vitro interactions between a CD56neg/CD16pos (CD56neg) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1–infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK–DC activation and maturation as well as a defect in the NK cell–mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56neg NK cell subset, largely accounts for the ...
Proceedings of the National Academy of Sciences, 2007
Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened ... more Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.
In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, lim... more In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.
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Papers by S. Ortolano