Biomedical subjects

Alberto Auricchio

Publications and source records attributed to Alberto Auricchio.

30 records · Page 2Linked to original sources

PubMed2004

Pharmacological regulation of the insulin receptor signaling pathway mimics insulin action in cells transduced with viral vectors.

Diabetes mellitus derives from either insulin deficiency (type I) or resistance (type II). Homozygous mutations in the insulin receptor (IR) gene cause the rare leprechaunism and Rabson-Mendenhall syndromes, severe forms of hyperinsulinemic insulin resistance for which no therapy is currently available. Systems have been developed that allow protein-protein interactions to be brought under the control of small-molecule dimerizer drugs. As a potential tool to rescue glucose homeostasis at will in both insulin and insulin receptor deficiencies, we developed a recombinant chimeric insulin receptor (LFv2IRE) that can be homodimerized and activated by the small-molecule dimerizer AP20187. In HepG2 cells transduced with adeno-associated viral (AAV) vectors encoding LFv2IRE, AP20187 induces LFv2IRE homodimerization and transphosphorylation minutes after drug administration, resulting in the phosphorylation of a canonical substrate of the insulin receptor tyrosine kinase, IRS-1. AP20187 activation of LFv2IRE is dependent on the dose of drug and the amount of chimeric receptor expressed in AAV-transduced cells. Finally, AP20187-dependent activation of LFv2IRE results in insulin-like effects, such as induction of glycogen synthase activity and cellular proliferation. In vivo LFv2IRE transduction of insulin target tissues followed by AP20187 dosing may represent a therapeutic strategy to be tested in animal models of insulin resistance due to insulin receptor deficiency or of type I diabetes. This system may also represent a useful tool to dissect in vivo the independent contribution of insulin target tissues to hormone action.

Blotting, Western↗

PubMed2004

PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum.

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

Animals↗

PubMed2004

The microphthalmia transcription factor (Mitf) controls expression of the ocular albinism type 1 gene: link between melanin synthesis and melanosome biogenesis.

Melanogenesis is the process that regulates skin and eye pigmentation. Albinism, a genetic disease causing pigmentation defects and visual disorders, is caused by mutations in genes controlling either melanin synthesis or melanosome biogenesis. Here we show that a common transcriptional control regulates both of these processes. We performed an analysis of the regulatory region of Oa1, the murine homolog of the gene that is mutated in the X-linked form of ocular albinism, as Oa1's function affects melanosome biogenesis. We demonstrated that Oa1 is a target of Mitf and that this regulatory mechanism is conserved in the human gene. Tissue-specific control of Oa1 transcription lies within a region of 617 bp that contains the E-box bound by Mitf. Finally, we took advantage of a virus-based system to assess tissue specificity in vivo. To this end, a small fragment of the Oa1 promoter was cloned in front of a reporter gene in an adeno-associated virus. After we injected this virus into the subretinal space, we observed reporter gene expression specifically in the retinal pigment epithelium, confirming the cell-specific expression of the Oa1 promoter in the eye. The results obtained with this viral system are a preamble to the development of new gene delivery approaches for the treatment of retinal pigment epithelium defects.

Animals↗

PubMed2004

Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport.

In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients' disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

In vivo quantitative noninvasive imaging of gene transfer by single-photon emission computerized tomography.

Systems aimed at detecting gene expression noninvasively in vivo are desirable for evaluating the outcome of gene transfer in clinical trials. Several approaches have been exploited using magnetic resonance imaging and spectroscopy ((31)P MRS), positron emission tomography (PET), single-photon emission tomography (SPECT), and detection of bioluminescent signals. An ideal system is based on transfer of a marker gene, the activity of which can be detected against a background from the target tissue without interfering with normal physiology or eliciting an immune response. The majority of approaches described to date use genes encoding a nonmammalian protein that can elicit immune responses or a transmembrane receptor as a marker gene whose ectopic expression may cause aberrant signaling in the target cell through binding to endogenous ligands. The dopamine transporter (DAT) is normally expressed at high levels, mainly in the dopaminergic neurons of the central nervous system. We previously synthesized a radioactive ligand, [(99m)Tc]TRODAT-1, that binds with high affinity to the dopamine transporter, allowing for SPECT imaging of the striatum in normal control subjects and individuals affected with Parkinson's disease. Here we describe a strategy to monitor gene transfer based on adeno-associated viral vector (AAV)-mediated transduction of DAT in murine muscle followed by [(99m)Tc]TRODAT-1 imaging by SPECT of cells expressing the transgene. We show that quantitative, noninvasive imaging of gene transfer is successfully achieved in vivo, using a single-photon computed tomography camera. This system employs a reporter gene encoding a mammalian protein that is absent in most tissues, has no enzymatic activity, and does not activate intracellular pathways. This should be useful to monitor gene transfer in the settings of gene therapy.

Animals↗

PubMed2003

Pseudotyped AAV vectors for constitutive and regulated gene expression in the eye.

Since the first reports describing the injection of recombinant adeno-associated viral (AAV) vectors in the murine eye, the advancement of the field has been enormous resulting in the correction of several animal models of retinal diseases. The recent development of "pseudotyped" AAV vectors with transduction characteristics that best fit the correction of specific retinal disease phenotypes and of sophisticated systems for tight regulation of gene expression expands on the potentiality of this delivery system for the eye.

Animals↗

PubMed2003

Adeno-associated viral vectors for retinal gene transfer.

Vectors derived from adeno-associated viruses (AAV) represent a promising tool for retinal gene transfer in pre-clinical and clinical settings. AAV vectors efficiently transduce dividing and non-dividing cells, escape cellular immunity and result in long-non-term transduction. In addition, they may be targeted to specific retinal cell types by taking advantage of surface proteins from various AAV serotypes thus limiting transfer of therapeutic genes to those cells requiring correction. This review will provide an overview of the properties of AAV vectors followed by a detailed report of their use in retinal gene transfer for mendelian and non-mendelian disorders.

Animals↗

PubMed2003

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction.

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.

Animals↗

PubMed2003

An in vivo doxycycline-controlled expression system for functional studies of the retina.

PURPOSE: Transgenic mice were developed that express tetracycline-controlled transactivator 1 (tTA1) specifically in photoreceptor cells. In these mice the transcription of the gene of interest can be easily inactivated in the retina in a short time frame. METHODS: A construct was prepared containing tTA1 under control of the murine rhodopsin regulatory region. This construct was used for the generation of transgenic mice. In situ hybridization was performed to study the distribution of the transactivator in the retina. The activity of the transactivator was analyzed by mating the lines with a luciferase reporter transgenic mouse. tTA1 activity and doxycycline's ability to block it were analyzed by luciferase assay. The effects of tTA1 on the retina were assessed by histology and electrophysiology. RESULTS: Two transgenic lines were developed that specifically express tTA1 in photoreceptor cells. The time course of transgene expression replicated transcription of endogenous rhodopsin. tTA1 was not toxic to the retina. Transactivator activity was blocked readily by doxycycline. CONCLUSIONS: An expression system for photoreceptor cells was generated to drive transcription in a cell-specific and time-controllable manner. This system is suitable for the study of factors involved in retinal biology and of mutant forms of genes involved in retinal diseases.

Animals↗

PubMed2002

Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye.

The control, over time and space, of the levels of therapeutic proteins is crucial for successful retinal gene therapy. We tested the ability of adeno-associated viral vectors (AAV) delivered intraocularly to release a secreted protein (erythropoietin (Epo) used as a marker) in the eye, either constitutively or in a pharmacologically regulated manner using the dimerizer-inducible transcriptional regulatory system. Following delivery of a constitutively expressing vector to the intravitreal or subretinal space of nude rats, Epo protein was detected in both the anterior chamber and vitreous fluids. A dual-vector system inducible by the dimerizer rapamycin and expressing Epo was administered into the subretinal space in an attempt to achieve pharmacologic control of trangene expression in the eye. Before induction with rapamycin, the intraocular Epo level was negligible. However, following a systemic administration of rapamycin, Epo was detected in the anterior chamber, peaking on day 3 and returning to baseline 2-3 weeks after withdrawal of the drug. Peak-induced Epo in the anterior chamber was proportional to the dose of rapamycin and was not detected in serum. Similar results were obtained following subretinal administration of the vectors in one nonhuman primate. The rapamycin inducible system promises to be useful for developing gene therapies for inherited retinal degeneration and ocular neovascularization.

Animals↗

PubMed2002

Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents.

Neovascularization characterizes diabetic retinopathy and choroidal neovascularization associated with age-related macular degeneration, the most common causes of severe visual loss in the developed world. Gene transfer to the eye using adeno-associated viral (AAV) vectors is a promising new treatment for inherited and acquired ocular diseases. We used an AAV vector with rapid onset and high levels of gene expression in the retina to deliver three anti-angiogenic factors (pigment epithelium-derived factor, tissue inhibitor of metalloproteinase-3, and endostatin) to the eyes of mice in a mouse model of retinopathy of prematurity. All three vectors inhibited ischemia-induced neovascularization.

Angiogenesis Inhibitors↗

PubMed2002

Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins.

This study evaluates the use of vectors based on adeno-associated viruses (AAVs) to noninvasively deliver genes to airway epithelial cells as a means for achieving systemic administration of therapeutic proteins. We intranasally delivered AAV vectors to mice in which the same AAV2 genome encoding a cellular marker was packaged in capsids from AAV1, 2, or 5 (AAV2/1, AAV2/2, or AAV2/5, respectively). Gene expression levels achieved in both airways and alveoli were higher with AAV2/5 than with AAV2/1 and were undetectable with AAV2/2. The same set of vectors encoding a secreted therapeutic protein, erythropoietin (Epo), under the control of a lung-specific promoter (CC10) was intranasally delivered to mice, resulting in polycythemia with the highest levels of serum Epo obtained with AAV2/5 vectors. After a single intranasal administration of this vector, secretion of Epo was documented for 150 days. Similarly, intranasal administration of an AAV2/5-CC10-factor IX vector resulted in secretion of functional recombinant protein in the bloodstream of hemophiliac, factor IX-deficient mice. In addition, we demonstrate successful readministration of AAV2/5 to the lung 5 months after the first delivery of the same vector. In conclusion, we show that intranasal administration of AAV vectors results in efficient gene transfer to the lung only when the vector contains the AAV5 capsid and that this noninvasive route of administration results in sustained secretion of therapeutic proteins in the bloodstream.

Administration, Intranasal↗